Encyclopedia of Anti Doping 2000 2015 PDF

(always under enlargement and re-editing)
ENCYCLOPEDIA
OF ANTI-DOPING
IN AN ERA OF
EVIDENCED BASED
MEDICINE
Scientific literature made available

from January 2000 through December 2015
Selected and edited by
Åke Andrén-Sandberg
From the Department of Surgery, Karolinska Institutet at Karolinska

University Hospital, Huddinge, S-141 86 Stockholm, Sweden
EDITOR’S PREFACE (AND SEARCH ALGORITHM)
The scientific literature also in small medical fields like doping is today enormous – and it is
not possible to keep updated unless making strong and focused efforts. The present review
is an attempt to make it easier for those fighting doping to keep updated. From the beginning
it was foremost an effort to make the reviewer updated, but hopefully it can be used also of
others with the same interest, i.e. those who believe in fighting doping with the aim to
eradicate it – or at least make doping so difficult that nobady can succed with it. However, it
should be empahzised that this compliation of data will still is a personal review, which
means that the selection of presented articles have been up to the reviewer, and probable
another author might have made at least some other choices.
The definition of “doping” has varied over the use, not least because it is difficult to combine
what really is enhancing performance in sport from a theoretical point of view with what is
practical – caffeine, children’s medicine when they have caught a cold, and alcohol are good
example of this. Moreover, there is an ongoing evoluation all the time which gives new
possibilities but also new problems, e.g. gene doping. Today the used definition of “doping” is
very anti-intellectual: “What WADA put on the list of forbidden substances and methods is
doping”. In this book the limitis is more generous, as not only the WADA lit is used as an
inclusion criterium, but also not forbidden but often discussed substances and methods are
covered. The biggest part of the discussed items is dietary supplements, but there are also
other substances that might or might not be clasifie as doping subtances. Unfortunately the
borders of doping are ill defined – and with times it is even more difficult to define. This is
why this book has defined anti-doping from the athlete point of view: the aim of this
encyclopedia is to include what is discussed as performance enhancing substances and
methods, irrespectively if they are working or not.
So, why bother to compile a lot of articles and tease serious individuals interested in sports
and sports medicine to waste time on even a superficially look through these texts? The
answer to that is that even if each abstract of an article can be rightly criticised the total body
of abstracts tell a more important story. This body tells something on what the present issues
are in the part of sports medicine that deals with doping, what it is worked with scientifically
on these issues in today, and what the thoughts of the researchers were when they wrote
their reports. It also tells something where the edge of our knowledge is situated presently,
and from that we can better understand what we can expect in the nearest future. So, a
recommendation may be to read each paragraph with considerable scepticism, but enjoy
them all together as new bricks in our scientific wall and hopefully find that together with the
next bricks we will have a chance of reaching the next – higher – level that is more easy to
understand. It is all the knowledge together that counts much, much more than every single
article.
Moreover, as interested in clinical anti-doping I am proud tog breg that I am interested in a

field of medicine that can be included under the heading of evidence based medicine. The
anti-dopers are not just beliving and guessing; we can base our recommendations on
scientific facts!
Another reason to collect articles is that you can get a review not only of what has been said
but akso who said it. This means that if somebody is interested he or she can look for the
authors in the reference list and with that as a starting point find out who are the authorities in
a certain field of interest – and then maybe take contact or at least look up what more has
been written of this or these authors.
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There must be made some limitations, otherwise a review in this form should not be possible
to write due to lack of time and lack of brain capacity, and probably not possible to read
either. Regarding the limitations, first of all the writing here will be based on the abstracts of
the articles in almost all cases for practical reasons. This is also in line with the aim of the
review: not to report all what has been published, but rather to give an introductional sample
that hopefully will make the reader eager to read the whole article or articles: this should be
regarded as “a tast of anti-doping in the last years”.
A second limitation is that most of the selections has been made through PubMed; a few
other sources have also been scrutinized, but then more occasional and not systematically.
The medical subheadings (MeSHs) always used are (but some more have been used
occasionally):
Doping, testing, medical history, biological passports, inadvertent doping, mass

spectrometry, liquid chromatography, gas chromatography, isoelectric focusing,
anabolic androgenic substances, rhabdomyolysis, designer drugs, AICAR,
testosterone, nandrolone, dehydroepiandrosterone (DHEA), stanozolol, boldenone,
boldione, bolandiol, methyltestosterone, methylnortestosterone, adrenosterone,
androstenediol, androstenedione, hydroxyandrostenedione, trenbolone, metandienon,
finasterid, norbolethone, phytoecdysteroids, designer steroids, aromatase inhibitors,
exemestane, dehydrogenase inhibitor, selective androgen receptor modulator (SARM),
estrogens, oral contraceptives, aromatase inhibitors, selective estrogen receptor
modulator (SERM), toremiphene, raloxiphene, anti-estrogens, tamoxifen, blood doping,
red blood cells, total hemoglobin mass, reticulocytes, erythropoietin (EPO), darpoietin,
NESP, epoetin delta, hematide, perfluorocarbon, hemoglobin-based oxygen carriers
(HBOCs), hemopure, plasma volume expanders, dextran, hydroxyethyl starch,
hydroxyethyl starch (HES), glucose solution, saline solution, Ringer lactate solution,
albumin, plasma protein fraction, gelatins, alpha-keratose, pullulan, levan, acetyl
starch, polyvinylpyrrolidone, glycerol, polyethylene glycol, platelet-rich plasma, growth
hormone, insulin-like growth factor i (IGF-1), insulin, human chorionic gonadotropin,
clomiphene, luteinizing hormone, mechanical growth factors, AGRP, caffeine,
theobromine, ephedrine, pseudoephedrine, methylephedrine, modafinil,
methylphenidate, amphetamine, methamphetamine, methylamphetamine (MDMA),
mephentermine, metamfepramone , mephedrone, tuaminoheptane, mesocarb,
sydnocarb, strychnine, befluorex, sibutramine, beta2 adrenergic agonists, asthma in
sports, exercise-induced bronchoconstriction (EIB), salbutamol, terbutaline, procaterol,
clenbuterol, methoxyphenamine, beta-blockers, angiotensin converting enzyme (ACE)
inhibitors, diuretics, thiazides, mannitol, epitestosterone, desmopressin,
glucocorticosteroids, ACTH, sildenafil, morphine, cannabis, cocain, ecstasy, gamma-
aminobutyric acid (GABA), gamma-butyrolactone (GBL), gamma hydroxybutyrate
(GHB), beta-hydroxy-beta-methylbutyrate (HMB), gamma-oryzanol and ferulic acid,
alcohol, nicotine, non-steroidal anti-inflammatory drugs (NSAIDs), peroxisome
proliferator-activated receptor, xenon, nutritional supplementation, dehydration ,
exercise-associated hyponatremia, bicarbonate, carbohydrates, mouth rinse,
carbohydrate gel, milk, ribose, galactose, fructose, energy beverages, sport drinks,
Red bull, proteins, branched-chain amino acids (BCAA), arginine, ornithine, leucine,
tryptophan, taurin, cystein, cystine, theanine, glutathione, glutamine, alanine,
methionine, carnosine, citrulline, asparagines, aspartame, glycine, alpha-ketoglutarate,
alpha-ketoisocaproate, whey protein, fat, medium-chain triglycerides, fish oil, omega-3
fatty acid, calcium, copper, boron, chromium, zinc, magnesium, selenium, vitamins, N-
Acetylcysteine, antioxidants, creatine, carnitine, melatonin, methylhexaneamine,
gluthatione, lecithin, inoleic acid, gamma-linolenic acid, gonjugated linoleic acid, leptin,
3
inosine, glucosamine, dietary nitrates, methylsulphonylmethane, melamine, probiotics,
green tea, ginseng, ginko, garlic, macroalgae, quercetin, Teribulus terrestris, Arnica,
Rhodiola rosea, Cissus quadrangularis, Cordyceps sinensis, bee pollen, Actovigin,
colostrum, gene doping, medical ethics
most often combined with Sport, Sports, Exercise or Performace
This MeSHs will lead to a lack of some articles that might be of interest, e.g. in doping
physiology, doping biology and social aspects of doping, but the border has to be set
somewhere.
Yet another limitation is that this is a clinical oriented review and the term human has been
used in the search algorithm. Therefore almost all “preclinical” articles have been neglected;
i.e. molecular biology, cell lines studies and whole animal studies are not included except
exceptionally (when the authors could not resist the temptation to review also them). This is
not because the preclinical issues are not interesting, but because they are so numerous,
and because it is much more difficult to evaluate the importance of them.
Note that one aim of this collection of articles has been to file them in a way that the editor
and reader can find reports on the same issues close to one another. However, rather often
an article may be classified under more than one heading. In these cases the editor has had
to make a choise; if it was the best choise possible will be up the reader to judge.
Another “problem” is how to evaluate the quality of the reports. In the best of worlds the
reports with highest quality should be given more space, and those with low quality should
maybe just get a short note. However, this is an extremely difficult task and this books editor
has almost surrendered; there is almost no quality control, except for those that have an
outstandingly poor quality – they have been omitted ruthlessness. However, most of the
quality control has been left to the readers,
When presenting quarterly reviews, annual reports and this encyclopedia I sometimes am
criticized for just rewriting what others have just written. I can just agree with them; the only
thing I have done is to gather what others have worked with nothing more, nothing less. The
only thing I have contributed with is that I have made other’s work more available and easier
to compare with what others have done. Maybe it is not som much, but I am proud of what I
have done.
I am sometimes also asked why it is included so much on dietary supplements, as many of

them in without effect and “unscientific.” The reason to include them is , however, easy: they
are present out there among the atheletes and are compared to the doping agents if the
antidopers want it or not. Also, one must admit that the border between was is allowed and
what is not is not always logic or semantically right. For example water, carbohydrates may
be important for athletic performance in some cases, and if too much is given it might neven
be harmful, i.e. they might fulfil the criteria for doping agents. However, the most important
reason to include them is to make them “less mysterious” – if there is any science on the
dietary supplements it would be nice to spread that also to them who onsider to use them. In
most cases useless supplements will lead to a drain of money from the pocket of the
athletes, in a few cases the dietary supplements will be harmful, and in most cases they are
only irrelevant. However … some of them can be of value in the right dose and right setting
(and there is science behind the statemants on their value)
All of the text – including tables etc – has been written i Microsoft Words, as this is the most
spread mode for communicating with computers. This means that all words and all tables
2
can be search throughout the text; for example the references can most easily be looked for
this way. This is also why some of the reference figures have 0 or 00 as the first figures.
Also, there is a “Content in the book” but no pages. This is because it is ment to be a book
on a computer, and if so different persons and computers make there formatation different.
Also, the book is growing every third month, which makes impractibel to give pages, and
then change them four times a year would be a waste of time. Please insted use the content-
part for selecting the words for the “search” mode – that is how it was planned to be used.
Lastly, the language has not been checked by someone who is English spooken. Instead the
text is written by a persons who at his very best is speaking Swinglish. The written language
could have been presented more properly by a real translater, but the cost for that luxury has
been too high. Accept therefore that there is a place also for “bad English” in science as long
as it can be understood. Maybe some of the sentences or words make you smile a little, but
remember than that my Swedish probably still is much better than your English …
This encyclopedia shall belooked upon as a “living” book. This means that it will continuously
be added new articles to as they are published and get available – and I hope this can be
contimued for the foreseeable future. Then I re-edit it by putting articles in a place where they
better fit, put in new subheading etc. The most difficult thing is then to compress the text and
take away some of what is said twice, three times or more. I love every word, and cry a little
bit inside when I have to omitt a sentence here or there, just as if they were my old friends.
Despite that, the compression is in the end necessary and will be done. Gradually, but not
without tears. However, the reference list will never have any references taken away, as the
meaning of the encyclopedia is to direct the reader to the full articles.
There are chapters that are more difficult to handle regarding re-editing than others. The
worst part is by far those that deal with laboratory techniques. The author have difficulties in
understanding the issues and even more difficult to classifiy the different techniques. Also,
the chapter on ethical questions is difficult to handle, but for another reason: ethic has so
little facts and som much of it needs a lot of words – unless you try to describe ethics in black
and white (which this reviewer refuses to do). So, for the moment you have to stand with
these drawbacks, or help the reviewer to come up with something better.
This means that the plan is to follow up with quarterly reports during coming years (it is then
the quarter when the review was made available through PubMed that counts, not the month
it was actually published) and then to include the reports from each quarter in an annual
report that is then taken into this encyclopedia – which then is gradually re-edited. This also
means that there is probably always a later version by the autor than that you have in your
hand now. However, if you send me a mail I am happy to send you that latest (not the last)
version.
Is this all that has been published on doing? No, certainly not, there is much more out there.
However, what is gathered in this book is probably a good deal of what has been published
as full articles (no abstracts included), and hopefully all that has been of real importance. If
someone find more that should be included, please let me know. If we are of the some
opinion I will of course include it as soon as possible.
It is probable that my reporting system can be presented better than what is done here is, but
how this is done is not so easy for the author, who like the way he has done it. So, welcome
with comments, criticism, or cheers – but if they fail to appear, the next quarters, the next
annual report, and the encyclopedia will have the same dispositions as the present. This
means that also you have a responsibility for the future!
2
Welcome to contact!
Åke Andrén-Sandberg
Department of Surgery, “Gastrocentrum”

Karolinska University Hospital at Huddinge
SE-141 86 Stockholm, Sweden
ake.andrensandberg@gmail.com
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CONTENT
EDITOR’S PREFACE (AND SEARCH ALGORITHM)
ABBREVIATION
BACKGROUND FOR TODAY’S DOPING PROBLEMS

Definitions of doping
Relevant policy documents
A background for understanding performance-enhancing drugs in sports
Judging cheaters in different domains
Overviews
Little symptoms and signs
Socio-psychological background to doping
Elevate health risks in young athletes
Risk for drug dependence
To move borders …
Much to win
Ergogenic potential of AAS
Precursors of testosterone
Testosterone/epitestosterone (T/E ratio)
Isotope ratio mass spectrometry (IRMS)
Synthetic androgens
Androgen bioassays
Indirect androgen doping
Estrogen blockers
Androgen precursors
Gonadotropins
Boosting in paraolympics
In vitro models to study effects and mechanisms in doping
Combinations with nutritional supplements
Possible lack of effects of doping
Cognitive doping
Brain stimulation techniques
What can be done with brain stimulation?
Can it be detected? What are the risks?
Ethical issues
Effect of international results during the anti-doping era
World records in running as indication for doping in the elite
Prediction of further improvements (year 2002)
100 meter and 5000 meter
100 meter in Olympic Games
Influence on world records in running by doping and anti-doping testing
Is doping-free sport a Utopia?
Arguments for allowing doping
Drug abuse
Sponsors
The dilemmas of rational calculators
Fair play
Diffuse borders
Economical incentives
4
Drugs available
DOPING AND ANTI-DOPING HISTORY

Semantics
Overviews
Some specified dates in doping and anti-doping history
Tom Simpson 1967
Ancient history of doping and anti-doping
The first marathon
History of sports medicine in ancient Greece
Early (modern) history
Women in sports
The first doping in the Olympics
The Olympics 1976-1992
The Olympic Games in Athens
Football
Soviet and East Germany
Biking
IAAF banned first
Testing
Blood doping
Ben Johnson
History of testosterone and some other anabolic steroids
Detection of testosterone
The testes as a medicinal product: organotherapy
Testis transplantation
Scientific exploration of the testes and their endocrine function
The homestretch to arrive at testosterone as a chemical entity
Development of testosterone preparations
Transdermal testosterone
The era of anabolic steroids
Detection of abuse
Background to T/E ratio
Availability of drugs
Androgen receptors
US professionals
Anabolic steroid prodrugs in the US
Designer drugs
BALCO
History of nandrolone
History of aromatase inhibitors
History of 5-alpha-reductase inhibitors enzymes
History of blood doping
Early testing strategies for blood doping
History of dectection of recombinant erythropoietin and derivates
Background to the athlete biological passport
History of doping with growth hormone
Testing for growth hormonde
History of human chorion gonadotropin
History of doping with insulin
Insulin physiology
History of doping with caffeine
History of doping with ephedrine
5
History of doping with beta2-agonists
History of doping with morphine
History of doping with cannabis
History of doping with xenon
History of doping with alcohol
History of doping with gamma-Hydroxybutyric acid (GHB)
History of doping with ecstacy
History of biomarker approach regarding doping
History of nutritional supplements
History of caffeine-containing nutritional supplements
History of doping with creatine
History of carnosine
History of gene doping
History of athlete biological passport
Doping in the Olympics
The ancient Olympic games
Olympic nationalism
The first Olympic tests
Doping during the modern Olympics
The Olympics in medical journals
Canada
Formation of the IOC Medical Commission
Formation of the World Anti-Doping Agency
Formation of Court of Arbitration (CAS)
Out-of-competition testing
History of therapeutic use exemptions (TUE)
Anorchia
Aplastic anemia
Congenital adrenal hyperplasia (CAH) and hypogonadism
Stimulation medication
Danazol
Glucocorticoids and diuretics
Beta-blocker
Recognising the concept of TUEs
Recent Olympic Games
Legislation
Courtroom, economics and anti-doping
USA
Industrialized doping
Laboratory testing
Anabolic steroids
Adverse findings 2005-2011 from the Doping Control Laboratory of Athens
Addendum to the “Prohibited list” in 2011
The 2005 WADA list
The 2011 WADA list
The 2012 WADA list
The 2013 WADA list
Category 0
Monitoring program
The 2014 WADA list
Erythropoesis-stimulating agents
Amendment to the 2014 Prohibited List
Implementation of the World Anti-Doping Code 2015
6
Non-approved substances
Important persons in anti-doping
Richard W Pound
Arne Ljungqvist
ORGANISATION OF ANTI-DOPING
An international background to the anti-doping movement
Indirect evidentc of doping effects: world records
World Anti-Doping Agency (WADA)
Background
National antidoping organizations (NADOs)
Pre-WADA history
The code
Definitions
Fundamental of the doping tests
Overview of prohibited drugs
The prohibited list
The 2015 Prohibited list
Sanctions of violations
Forensic intelligence
Anti-doping rules
The rules
Strict liability rule
The whereabouts rule
Separation of power
Privacy
Random testing
Education and research
Laboratory testing
Out-of-competition anti-doping laboratory's analytical menu
In-competition testing programme
Therapeutic use exemption (TUE)
Abbreviated TUE
Standard TUE
Therapeutic use exemptions (TUEs) at the Olympic Games
Prevalence of use of TUEs in asthmatics
Prioritation in anti-doping
Strategy to reduce illicit drug
Australian football
Sports supplements from Australian point of view
Agreements with the pharmaceutical industry
Doping controls in practice
International Sport Federations in the protection of the athlete's health
During the Olympics
Biomarkers
"Exercisenomics"
Multi-class and multi-analyte test methods
Statistics
Negative tests despite later confessed doping
Legislations
Brazil
Organization in football
7
UEFA
FIFA
FIFA Medical Assessment and Research Centre (F‐MARC)
FIFA's approach to doping in football.
Future challenges
Costs of the anti-doping organization
Where-abouts
Sports federations
Cost reduction proposals
Narrowing the gap between doped and anti-doping controllers
WADA-accredited doping laboratories
Some antidoping organization
United Nations Educational, Scientific and Cultural Organization (UNESCO)
The World Anti-Doping Agency (WADA)
United States Anti-Doping Agency (USADA)
Statistics
The Sochi Olympics 2014
Laboratory equipment
EPIDEMIOLOGY OF DOPING
Scientific considerations of determing prevalence of doping
Available evidence
Estimated number of unreported cases
Doping in the community
The types and patterns of PED use
Epidemiological confounding factors and false consencus effect (FCE)
Elites
Use of drugs during the Olympics
Age of onset
Student athletes
Adolescents
Versus those not participating in sports
Disableds in sports
Issues in paraolympics
Gym users
State-based sports institute
Pharmaco-epidemiology of anabolic steroids
Comparison with a prison population
Drug Information Database
Global lifetime prevalence rate
Different countries
Sweden
Denmark
Norway
Finland
The Netherlands
UK
Greece
Italy
France
Belgium
Germany
Spain
8
Serbia
Croatia
Poland
Europe, combined countries
Kuwait
Iraq
Israel
Jordan
Iran
Korea
USA
Australia
Brazil
Cameroon
Ghana
Ivory coast
Different sports
Prevalence of use depending on type of sports
Professional ballet dancers
Track and field
Football
Tennis
Table tennis
Combined racket sports
Biking
Bodybuilding
Dancesport
Recreational sports
Prevalence of use by fitness centre members
THEORETICAL ASPECTS ON DOPING-TESTING

Overview
Theories on laboratory testing
Homo economicus: pay-offs and sanctions
Aim of anti-doping
The Goldman dilemma
Sports drug testing – an analyst's perspective
Indirect evidence of hormone abuse
Forensic intelligence in anti-doping
Non-analytical testing
Colored illicite tablets
Human mitochondrial DNA analysis
Determining the authenticity of athlete urine in doping control
Theories on doping in sports
Doping detection
Understanding the basics of testing for banned substances
Doping prevention
Explaining the doping behavior
Multiple drug use
The types and patterns of performance-enhancing drug use
Combination of anabolic steroids and other substances
Association of performance-enhancing drug use with other high-risk behaviors
Recombinant proteins
9
Co-operation in drug testing
Methodology for investigation of doping in the society
Alternative matrices
A support vector machine
Performance profiling
Controls at random
Errors in drug testing
Incongruity of data
Gather data to reveal true extent of doping in sport
Contextual modulation of androgen effects on agonistic interactions
Theoretical testing
Self-reporting
Testing as a way of decreasing intent for doping
Fatigue as a limit for test performance
Psychometric testing in doping control in sport
Implicit Association Test
Economical aspects
Chemoinformatics-based classification of prohibited substances
Need of excretion studies
Chemical and physical manipulations of doping tests
Designer drugs in Japan
Testing efficiency
Anti-doping research opportunities
Economy
Placebo
Medical history of placebo
Effect of placebo on runnig results
Placebo effect of carbohydrate feedings during a 40-km cycling time trial
Powerless placebo
Lack of effect of flavor
Ethical issues
Concluding remarks on placebo
Measurement uncertainty in anti-doping quantitative analysis
Statistical aspects
False negatives
High true prevalence
Preanalytical variability
Doping prevalence at drug testing
Wald test
Bayesian statistics
Decision limit (CCalpha) and detection capability (CCbeta)
Prediction of future doping
ATHLETE BIOLOGICAL PASSPORT (ABP)

Overviews and background
Legal aspects
Limitations of earlier anti-doping strategies
Individualized statistics
Personalized monitoring of biomarkers for doping
Technical specifications necessary
Still an interpretation
An international tool
Three complementary items of ABP
10
Hematological parameters
Hematocrit
Hemoglobine mass
Variability of serum markers of erythropoiesis during intense exercise
Plasma osmolality
Animal studies
Biking
Possible variables
Example of the haematological modules
How does the haematological module work?
Haematological markers and athlete's information for the ABP
Technical documents associated to the haematological module
Microdoses
Erythropoietin
Steroid profile for athlete biological passport
Endogenous factors, general considerations
Ageing and endogenous steroid synthesis
Gender effects, circadian variations and physical activity
Metabolism, genetics and interindividual variation
The steroidal markers
UGT2B17 gene deletion influence on Athletes Biological Passport
Effect of Ramadan
Impact of genetics and oral contraceptives on the steroid profile in female
athletes
Longitudinal steroid profiling
Korean values of urinary levels of testosterone and epitestosterone
The growth hormone module
Potential confounding factors
Plasma osmolality
Stability of athlete blood passport parameters during air freight
Gastroenteritis
Oral contraceptives
Influence of transport and time on blood variables
High altitude training
Impact of altitude training on haematological parameters
Hyperhydration
Heterogeneous factors
The analytical procedure
Result management: the role of the APMU
The expert review
Also for research
Law issues
Software
Passports in practice
Erythropoesis stimulating agents
Testing during cycle tournament
Positive cases in biking
Practical experience with athletics, cycling, football,swimming and biathlon
UCI
FIFA/UEFA
FINA
IAAF
Biathlon
11
Future modules and integration
General scheme of the athlete biological passport (ABP)
Key points for the implementation of the ABP
Database and sharing of information
Continuous evaluation of the ABP
SOCIO-MEDICAL, SOCIAL AND PSYCHOLOGICAL ASPECTS ON DOPING

Semantics in doping
Political and economical aspects of anti-doping
Social and socio-medical issues
An integrative model of doping use with adolescent athletes
Doping in sport: a test of the strength-energy model
Social and psychology characteristics of users of anabolic steroids
Moral disengagement and associated processes
Psychological background
The performance enhancement attitude scale
Cerebral correlates of automatic associations of performance enhancing
substances
Athletes confessions on doping
Elite amateur cyclists' perspectives on drug use and professional cycling
Subjective effects
Expectation of the doped
Rejuvenation
Interaction between athletes and coaches
Sports addiction
Attitudes towards doping with regard to achievement goals
Motivational and social cognitive predictors of doping
Young athletes' awareness and monitoring of anti-doping in daily life
Motives for use
Motivation for anti-doping
Measurement of attitudes
Beliefs about the causes of success in sports
Elite athletes' attitudes, beliefs, and knowledge
Knowledge of anabolic steroids among gym users in Jamaica
Associations of physical activity and sport with at-risk substance use in young
men
Attitude of elite cyclists on doping
Age of onset of performance-enhancing drug use
Young athlets attitudes towards doping
Attitudes on reporting of whereabouts
Judgments of the fairness of using performance enhancing drugs
Other athletes’ attitudes regarding doping
Attitudes against dopers
Dissatisfaction with body imaging as a reason for doping
Muscle dysmorphia as an addiction
Use of placebo-induced performance enhancement in sports
Polypharmacy
Concomittant use of other substances
Neuropharmacy addiction
Connexion to use of dietary supplement
Complementary and alternative medicine (CAM)
Connexion to eating habits
Concomittant symptoms and signs
12
Performance versus recreational drugs
Health promotning effects of sports
Lifestyle alteration
Prediction of later life-style
Risk factors for doping
Psychiatric comorbidity diseases
Sponsoring of sports
Information to dopers
Medical practitioners' knowledge, attitudes and beliefs
Anabolic steroid users' attitudes towards physicians
Contagiousness of doping
Economical aspects
Importance of sports medicine as a medical speciality
Impact of national programs
Staffing protocols in high-performance sport
Law issues
Doping + violence
Knowledge in different countries
Illicit drugs around an Olympic game
US position stand on androgen and human growth hormone use
Characteristics and behaviors of older male anabolic steroid users
Body builders
Influence of religion on use of doping
The adolescent athlete
Persons influencing adolescents’ doping
Attitudes towards doping of high school athletes
Attitudes and behaviors among male adolescents on doping
Availability of illegitimate drugs in the society
Norway 2011-2014
Denmark 2002-2003
Germany 2012-2013
Switzerland 2013-2014
Spain
Brazil 2007-2014
Internet drug availability
Psychoactive drugs in the society
Organised crime and drugs in sport
Legitimate use of drugs in sports
Over-the-counter medicine
Doping agents as medical treatment
Pharmacists
Compared to general practitioners
Pharmacy students
Qatar
A cancer risk of doping?
Dependence in clinical practice
Treatment of drug addicts
Educational programs
Social perceptions
Medical risks with illegal drugs
Increased mortality in former dopers
Doping and the respiratory system
13
Information on doping
Internet
Users' sources of information and medical advice of doping substances
Telephone hot-line
Media medicine
Factors contributing to the limited appreciation of the adverse effects of doping
Harm reduction
An integrated approach
Difficulty accessing health services for dopers?
Doping: proposing a functional analysis based on the color yellow
US Army: stimulants, anabolic hormones, and blood doping
Protesting against things which have not yet happened
Different countries
Spanish football
Elite Scottish athletes' attitudes towards doping
Street market in Senegal
Uganda
UNINTENTIONAL DOPING
Why preventing unintentional doping is important?
Protection of athletes from inadvertent doping with anabolic agents
How to help responsible athletes prevent unintentional doping?
Black market products and potential doping agents
In Germany
Black market anabolic steroids
Estradiol
Estradiol and its metabolites in meat
Clenbuterol
In pork liver
In calf hair
Various living tissues for monitoring clenbuterol abuse in food-producing cattle
Dexamethasone
Sibutramine
Melamine
Methylhexaneamine
Illicit blue tablets containing anabolic androgen steroids
Counterfeit products
Doping through dietary supplement use, general aspects
Non reported contents in dietary supplements
Assessment and management of the risk in Australia
Anabolic steroids in nutritional supplements
Dietary supplements containing prohibited anabolic agents
Anabolic steroids detected in 23/24 bodybuilding dietary supplements
Stable carbon isotope ratio profiling of illicit testosterone preparations
Other laboratory techniques
Dietary supplements contaminated with prohormones
Dietary supplements with containing-agonists
Clenbuterol
Dietary supplements containing prohibited peptide hormones
Dietary supplements containing caffeine (in unknown dosage)
With ephedrine
With creatine
Dietary supplements containing 2-ethylamino-1-phenylbutane
14
Zeranol
Boar meat
Musk pod
Designer drugs
Emerging drugs
Counterfeit drugs
Doping substances contaminating food
Meat
Anabolic steroids in bovine liver
Anabolic steroids in bovine bile
Doping substances in urine
DETECTION OF DOPING AGENTS IN ENVIRONMENT, FOOD AND FOOD

SUPPLEMENTS
Hormones in international meat production
Steroid growth promoters from beef cattle feedyards
Influence on reproduction
Influence on resistance to antibiotics
Analytical strategies
Potential gene expression biomarker signature
Revised EU criteria for the confirmation of anabolic steroids in meat
Metabolomics
Screening for hormone residues in drug residues
The Netherlands
Anabolic steroids
Nandrolone
Trenbolone
Anabolic steroids in dietary supplements
Metenolone
Methenolone acetate in a veal calf
Detection of anabolic residues in implantation sites in cattle
Plasma steroid variations in bull calves
Sex steroid levels in urine of cattle of different ages
Eating non-castrated male pork induce increase of nandrolone in urine
Estrogenic endocrine disruptors
Clostebol through sexual intercourse
Sexual behavior of fish after trenbolone
In milk
Growth hormone
Growth promoters given to livestock
Beta-agonists in pork
Clebuterol
Clenbuterol in muscle
Clenbuterol in pig retina
Clenbuterol in milk
Acute intoxication
Caffeine
Cannabis
Hemp-containing food
Cocaine
Metamphetamine
Poppy seed-containing food
Herbal coca tea
15
Ephedra
Codeine
Famprofazone
Screening of residues in food
Egg
Bovine
Swine
Fish
Screening of anabolic substances in solid nutritional supplements
Non-labelled anabolic androgenic steroids in nutritional supplements
Detection of doping agents in drinking- and wastewater
Doping substances in general
Anabolic steroids, including testosterone
Amphetamine-like psychoactive substances
Ketamine and mephedrone
Drugs of abuse, cytostatic drugs and iodinated contrast media in tap water
France
Italy
Czech Republic
Influence on frog
Stability of illicit drugs in sewers and wastewater samples
Androgenic and estrogenic activity in water bodies receiving cattle feedlot
effluent
Rests in other parts of nature
Anonymous pooled urine
Beef palatability
Laboratory techniques
In meat
In faeces
Liquid chromatography tandem mass spectrometry
High- and low-resolution mass spectrometry
Laboratory testing of fluids
PREVENTION OF DOPING
Epidemiological issues
A conceptual framework for achieving performance enhancing drug
Culturally sensitive measures
Drug prevention programs
Education
The risk of being caught for doping
Economic incentives
Sponsors
Fines
Laboratory costs
Self-reporting
Mass media potential responsability
Prevention versus incentives for the use of performance-enhancing substances
Assessment of physical and sports aptitudes, and prevention of doping
A novel antidoping and medical care delivery model in Nanjing 2014
Athletes Targeting Healthy Exercise and Nutrition Alternatives (ATHENA)
Prevention of doping amoung children and adolescents
Rationale for a statement on performance-enhancing substances and youth
Current definitions of performance-enhancing substances
16
Strategies for preventing use of performance-enhancing substances
Identification of the young person using performance-enhancing substances
Innovation leading to improved adolescent health
Recommendations
Funded research
A Swedish health promotion programme to prevent misuse of anabolic steroids
An American anti-doping program
SAFE (Safe and Fair Events)
Harm minimisation – by the sports doctor
The FIFA program
Sport-specific risk assessment
Prevalence measurement
Sport-specific test distribution plans
Storage and reanalysis
Analytical challenges
Psychological approach to optimise most deterrent effect
Data management system: ADAMS
Education
Research needs and necessary advances
Inadvertent doping
Management and ethics: biological data
The FIFA Antidoping program during the World Cup in Brazil 2014
Telephone counseling
National policy against doping
Sweden
Switzerland
Spain
Brazil
USA
OVERVIEWS OF GENERAL LABORATORY TECHNIQUES

Olympic laboratories
Practical testing in Brazil
Transportation
Quality of doping testing
Blood sampling and blood samples handling
Handling of urine
Reference materials: freeze-dried urine samples
Stability of doping substances in urine
Diluted urine
Urinary screening
Direct injection of urine
Effects of exercise on the urinary proteome
A purified enzyme for the mild hydrolysis of steroid sulfates
Proteases in doping control analysis
Forensic toxicology
Testing in famous cases
Katrin Krabbe et al
Lance Armstrong
Parallel investigations of saliva and urine
17
Effects of sample storage condition on salivary hormones
Screening methods
A general analytical platform and strategy
Automated sample preparation
Multi-analyte testing
”Alternative” specimens
Drug identification
Blood tests
Laboratory report interpretation
Accuracy of testing
Chromatography
Mass spectrometry
Drug confirmation by mass spectrometry
Small peptides
Isotope ratio mass spectrometry or carbon isotope ratio
Vacuum MALDI-linear ion trap mass spectrometry
Benchtop quadrupole-orbitrap hybrid mass spectrometry
Chromatographic-mass spectrometry
Miniaturized competitive immunoassays on a protein chip
Liquid chromatography
Hydrophilic interaction liquid chromatography
Ultra-high-performance liquid chromatography
Fast liquid chromatographic/mass spectrometric screening
Ultrahigh pressure liquid chromatography-(tandem) mass spectrometry
Multi-targeted liquid chromatography-mass spectrometry screening procedure
Metabolite-based liquid chromatography-mass spectrometry
High-resolution/accurate-mass LC-MS
With information dependent acquisition
Liquid chromatography/time-of-flight mass spectrometry
High performance liquid chromatography retention time of small molecules
Nano-liquid chromatography/benchtop quadrupole orbitrap tandem-mass
spectrometry
Liquid and gas chromatography time-of-flight mass spectrometry
Gas chromatography
Two-dimensional gas chromatography
Gas chromatography-microchip atmospheric pressure photoionization-mass
spectrometry
Gas chromatography-positive chemical ionization triple quadrupole mass
spectrometry
Gas chromatography-mass spectrometry
Gas chromatography-combustion-IRMS (GC-C-IRMS).
Gas chromatography-triple quadrupole mass spectrometry
Gas chromatography-QqQ-MS
Carbon isotope (CIR)-based analyses
IRMS (isotope ratio mass spectrometry)
Isoelectric focusing
Electrospray ionization
Micellar electrokinetic capillary chromatography and electrospray mass
spectrometry
Capillary electrophoresis
Capillary electrophoresis time-of-flight mass spectrometry
18
Full-capillary sample injection combined with sweeping CE stacking
Hyphenated mass spectrometric techniques
siRNA
Mass spectrometric detection of siRNA
High-resolution liquid chromatography-time-of-flight mass spectrometry
Ultra-high-pressure liquid chromatography-quadrupole time-of-flight mass spectrometry
Isotachophoresis sample stacking
Polar organic chemical integrative samplers (POCIS)
Fourier-transform infrared spectroscopy
Microwave assisted extraction
Ultrasound and microwave
Liquid/liquid extraction
"Dilute-and-inject" multi-target screening assay for highly polar doping agents
Phase I and phase II intact urinary metabolites
Two step derivatization
Adsorption to metallic plasmonic nanoparticles
Surface plasmon resonance
Isotope ratio mass spectrometry
ETD and CID tandem mass spectrometry
Protein chips
Small molecular analysis
Sample preparation techniques
Chromatographic approaches
Mass spectrometry detection
Peptide analysis
Small peptide hormones
Solid-phase extraction of small biologically active peptides on cartridges
Immunoaffinity purification and LC-HRMS/MS for peptide hormones
Affinity-based biosensors
SIRT1-activating drugs
Bioassay-guided fractionation
Unknown fusion proteins
In vitro studies
Mammalian reporter gene bioassays
Transcriptome analysis
Compound-specific isotope analysis (CSIA)
Dual-color bioluminescent bioreporter
Two-dimensional gas chromatography with heart-cutting
RNA sequencing
DNA typing
Urinary steroid sulfate metabolites
The urine marker test
Hydrogen isotope ratio of urinary steroids
Dried blood spots (DBS)
A new hyphenated mass spectrometry
mRNA transcripts
Deuterium/hydrogen ratio
Quantitative structure-retention relationships
Solid phase extraction (SPE) procedure
Solid-phase microextraction
Yeast analysis
Yeast transactivation system
Yeast and mammalian cell-based androgen bioassays
19
Laser desorption
Non-target metabolomics
Composite metabonomic data sets
FCMIA
Lack of influence of NSAIDs
In hair
Hair color as a potential biasing factor in hair analysis
Trace elements
Children
Hair and saliva
In saliva
Finger nails
In sweat
In oral fluid
Designer drugs
Virtual screening
Artificial networks
Appearance and Performance Enhancing Drug Use Schedule (APEDUS)
ANABOLIC ANDROGENIC SUBSTANCES

Overviews
Practical explanations (definitions) of anabolic steroids
Research activity
Limitations of research on the effects of AAS in athletes
Common dosage of anabolic steroids in dopers
A variety of substances
Differences between polydrug regimens and single drug administration
Clinical use
Hereditary angioedema
Cystic fibrosis
Reduced risk of atrophy after tendon rupture
Effect of AAS after repair of tendons
Rehabilitation of hip fractures
Effects of androgen deprivation
Andropause and somatopause
Abuse
Methods of abuse
Abuse dosage
Recognizing steroid abuse
Different actions due to ways of administration
Different anabolic androgenic steroids with different specific actions
Training euphoria due to anabolic steroids
Possible use in children and adolescents
Pubertal androgen therapy in boys
Clinical effects
Prevalence of adolescent anabolic-androgenic steroid use
Use of testosterone precursors by adolescents
Use in women
Prevalence
Effects
Steroid precursores (androstenedione and dehydroepiandrosterone)
Anabolic steroids in young females
Pregnancy
20
Estrogen antagonists
Genetic influence
Covariation with BMI
UGT2B17 gene deletion
Doping tests and genetic confounders
UDP-glucuronosyltransferase and UGT2B17 genotype
Genetic polymorphism
Association with renal disease and gene polymorphism
Chemistry
Steroid hormones
Testosterone and its modifications
General metabolism
Steroids from musk deer
Influence of pharmacological interventions on the serum and urine steroid
profile
Physiology
Complexities of androgen action
Effect on glucocorticoid receptors
Mechanism of action
Anabolic effects
Effects on the brain
Neuroendocrine effects
Chemical structure versus function
Effect on human skeletal muscle
Effect on bone tissue
Endogenous steroids
Androgen disposition and genetic variation
Steroid hormone binding globulin (SHBG)
Hydroxysteroid dehydrogenase
Modulation of follistatin and myostatin propeptide
Chromosomal damage
Androgen receptors
Selectivity of ligand action on androgen receptors
Androgen receptor polymorphism
Antagonists of the androgen receptor
Physiological and clinical effects and side effects
Muscle characteristics in bodybuilders
Mesenchymal pluripotent cell as the target of androgen action
Anabolic steroids and antioxidant vitamins on ethanol-induced tissue injury
Variation in phase II metabolism of sex steroids
Anabolic steroids effect on other drugs of abuse
Decreased oxygen consumption
Androgen replacement therapy (ART)
Hormonal contraception in the male
Commonest AASs in use worldwide, according to main effect
Molecular function
Androgen metabolism
21
Exogenous factors
Drugs and medication
Ethanol and tea
Environment and bacterial contamination
Physiological cellular effects of androgens
Role of satellite cells in anabolic steroid-induced muscle growth
Episodical secretion
Circadian rhythm
Effect on older men
Effects on andropause
Muscle atrophy after androgen deprivation
Erythropoesis
Exercise-induced low testosterone levels
Androgen receptors after exercise
Modulation of training on anabolic steroids values
Endogenous steroids
Effect of smoking
Influence of alcohol on steroid metabolism
Effects of dietary components on testosterone metabolism
Testosterone plus an ornithine decarboxylase inhibitor
Salivary hormones
Salivary testosterone
Effects on cognitive functions
Effects on training
Effects according to MRI techniques
Morphology versus performance after use of anabolic steroids
Effect of training status and exercise mode on endogenous steroid hormones in
men
Effects on training in males
Effects on training in females
Effects of resistance exercise
Effects on stress
Effect of anabolic steroids on endurance capacity
Effect of anabolic steroids on muscle growth
Effects on muscles and tendons
Effect of exercise of serum sex steroid levels
Effect of acute exercise on muscular sex steroidogenesis
Effect of chronic exercise on muscular sex steroidogenesis
Effect of sex steroid hormone administration on muscle sex steroidogenesis
Effect of AAS on bodyweight
Effect of muscle oxygenation during resistance exercise
Effects of AAS on lean body mass
Effects on recovery after exercise of anabolic steroids
Effect of AAS and physical activity on the hypothalamic-pituitary-gonadal axis
Effects on immunological function
Influence on endurance
Influence on strength
Influence of sprint
Influence of bodybuilders’ fasting periods
Flywheel ergometer workouts
Watching a previous victory
22
Effects of magnesium supplementation
Effects of training on salivary levels
Effects of androgens on IGF-1
Influence on GABA of anabolic steroids
Interactions with opioids
Induction of nitric acid
Lack of influence of NSAID
Body composition changes after withdrawal of anabolic steroids
Different effects on different parts of the body
Effect of anabolic steroids on hematological variables
Long-time effects of anabolic steroids
Multi-parametric steroid profiling
Aspects on anabolic steroids in different sports
Use in Brazil
Prevalence of missuse
Dependence
Overview of detection of AAS abuse
Ratio between testosterone and epitestosterone
Comparative safety evaluation of SARM and anabolic androgenic steroids
Testosterone treatment of females to men
Male hormonal contraception
Altered gonadal steroidogenesis in critical illness
Anabolic steroids and opioids
Changes in androgenic steroid profile due to urine contamination by microorganisms
Anabolic steroids in pre-hibernating Arctic ground squirrels
Specific laboratory techniques for anabolic steroids
Internal standard
Interlaboratory comparisons
Prolongation of the detection window for exogenous AAS
Doping control and analytical factors
Bayesian based screening
Purity certified reference materials
Stacking method of repetitive large volume sample injection
Endogenous steroids
ELISA
Gas chromatography/mass spectrometry
Gas chromatography/tandem mass spectrometry
Solid-phase extraction purification of steroid sulfates
Fully automated solid phase extraction and LC-MS-MS
Liquid chromatography-tandem mass spectrometry method (LC-MS/MS)
Ultra-liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS)
Ultra-high-performance supercritical-fluid chromatography hyphenated tandem
MS
Direct measurement of urinary testosterone and epitestosterone conjugates
Ligand and structure-based virtual screening
Dried blood spot
3-Oxo-steroidal agents
Stable isotope dilution liquid chromatography electrospray ionization
Microflow tile technology and LC-MS/MS
Effects of sample storage condition steroid hormones in saliva
23
IRMS
Gas chromatography coupled to IRMS
Gas chromatographic/time-of-flight mass-spectrometric
HPLC-MS/MS
Enzymatic hydrolysis of conjugated steroid metabolites
HPLC using atmospheric pressure chemical ionisation
Quantification of neurosteroids in rat plasma and brain
Bar adsorptive microextraction (BAmyE)
LC-silver ion coordination ionspray/triple-quadrupole mass spectrometry
Dilute-and-shoot liquid chromatography-high resolution mass spectrometry
Carbon isotope ratio (CIR)
Prediction of metabolic pattern of new derivatives of AAS
Mass spectometry
GCxGC-TOFMS
UHPLC-HRMS
Single-stage-Orbitrap-MS
Full-capillary sample injection combined with a sweeping CE stacking method
Oxidizing adulterants’ effect on the steroid profile of human urine
Electrospray ionization tandem mass spectrometry (ESI-MS/MS)
Glucuronoconjugated metabolites
Relative retention times
Variability in the 13C/12C ratios
Principal components analysis
Androgen receptors assay
Solvent and solid-phase extraction of natural and synthetic anabolic
High-performance liquid chromatography-ion trap mass spectroscopy
Sensor chip preparation and assay construction for immunobiosensor
Androgen bioassays
Monoclonal antibodies
Protein assays
Enzyme-immunoassay kit
Triptorelin test
Serum inhibin B as a potential marker of testosterone doping
Designer drugs
Molecularly imprinted polymer filaments (MIPFs)
Leidenfrost phenomenon assisted thermal desorption
Alternative methodologies for steroid quantification
Proteomics and steroidomics
Screening hybridomas antigen microarray
Urinary steroids
Liquid chromatographic/mass spectrometric screening method
Friedel-Crafts acylation
Micellar liquid chromatography
Liquid-phase microextraction sample clean-up
In saliva
In hair
Faecal analyses
In food
Musk extracts
A user-friendly library
24
Reference values from South America
Experimental
Clinical guidelines for detection of exposure of doping with anabolic steroids
History
Physical examination
Management
Laboratory abnormalities in anabolic-androgenic steroid users
Markers for anabolic steroids
AICAR
Activity on carboanhydrases
Purchase over the Internet
Anabolic steroid use and condome use
Case reports
Experimental
GABA type A receptors
Effect of subcutaneous testosterone on emotionality
Effect of testosterone in castrated guinea pigs
Apoptosis and NOS2 (nitric-oxide synthase 2)
Androgen-induced cardiac autonomic dysfunction
Interaction of testosterone with cocaine
An opioide-like dependence
Effect on tissue glycogen
Lack of effect on exploratory-based anxiety
Submaximal training and anabolic androgenic steroids administration
Effect of anabolic steroids of rat cardiomyocytes and adipocytes
Modulate adolescent steroid-induced aggression in hamsters
Steroids impair set-shifting and reversal learning in male rats
Androgen deficiency exacerbates high-fat diet-induced metabolic alterations in
male mice
SIDE EFFECTS OF TESTOSTERONE AND OTHER ANABOLIC STEROIDS

Overviews
Explanatory models
Experimental
Self-reported adverse effects
Short-term side effects
One year of abuse
Anesthesia risk
Different effects of different anabolic steroids
Toxicokinetics
Impurities in illicit samples of anabolic steroids
Mortality
Anabolic steroids’ impact on the cardiovascular system
Overview
A national population-based cohort study
Metabolic syndrome
Coronary artery calcifications
Myocardial infarction
Heart failure due to anabolic-androgenic steroids
Cardiac arrhythmias and abnormal electrocardiography
Maximal heart rate
Heart rate recovery
Cardiac structure and functioning
25
Myocardial fibrosis caused of anabolic steroids
Androgenic anabolic steroids and arterial structure and function
Left ventricular myocardial dysfunction and cardiac hypertrophy
Cardiac (autonomic) dysfunction
Vascular reactivity
Dyslipidemia
Impaired exercise-induced cardioprotection of antioxidant enzymes
Hyperhomocysteinemia
Sudden death
Cardiovascular risk in older men on testosterone
Long-term risk
Abnormal plasma lipoprotein
Trombocyte function
Hypercoagulability
Arterial thrombosis
Thrombosis
Pulmonary embolism
Endothelial cells
Increased iIntima-media thickness
Aortic elasticity
Arterial hypertension
Inflammation, oxidative stress, and vascular functioning
Other vascular effects
Experimental
Cardiovascular effects of low androgens
Summaries of effects of anabolic steroids on the heart
Increased risk of diabetes
Effects on the brain, cognition abnormalities and psychiatric effects
Neurotoxicity
Brain trauma
Brain development
Cerebral infarction
Calcification of the basal ganglia matter of the cerebellum (Fahr's disease)
Brain nerve growth factor
Decreased memory
Behavioural manifestations
Long-term effects of pubertal steroid exposure on aggressive behaviors.
The relationship between serum testosterone levels and mental state/behavior
Sex and exercise interact to alter steroid-induced anxiety-like behaviors
Mania
Effects on GABA
Rewarding systems
Monocygotic twins
Sleeping pattern
Other psychologic and psychiatric effects
Lack of energy in hypogonodanal men
Agression and violence
Association with criminality
Suicide risk
Addiction
Withdrawal effects
Treating psychiatric effects of steroid use
26
Cognitive deficits
Spontaneous subdural haematoma
Cerebral oedema
Randomized trial on psychiatry of supraphysiological doses of anabolic steroids
Forensic experiences of use of anabolic steroids
Effects of alcohol intake, defensive behaviors and brain opioid peptides in the
rat
Aggression after altering anterior hypothalamic-arginine vasopressin expression
Increased vasopressin V1A receptor binding
Liver changes due to sex hormones (anabolic steroids and oral contraceptives)
Overview
Aminotransferase elevations: hepatitis or rhabdomyolysis?
Metabolism of anabolic steroids in the liver
Enzyme elevations
Cholestatic liver disease
Non-alcoholic fatty liver disease (NAFLD)
Peliosis
Hepatocellular adenoma and carcinoma
Hepatocellular necrosis.
Spontaneous hepatic rupture
Hepatitis
Hepatotoxicity
Hypertensive encephalopathy
Nonobstructive sinusoidal dilatation of the liver
Hepatoprotective effects of silymarin in androgenic-anabolic steroid-induced
liver damage
Anabolic steroids and reproductive system and male infertility
Dose-dependent effects of testosterone on sexual function
Pituitary side effects of anabolic steroids
Pathophysiology of infertility: Feedback suppression
Infertility and the molecular biology of the androgen receptor
Idiopathic male hypogonadotropic hypogonadism (IMHH)
Physiology of reproduction-endocrinology
Anabolic steroid-induced hypogonadism in young men
Azospermi
Histopathlogy
Impact on semen quality
Aneuploidies and ultrastructural changes in spermatozoa
Sexual orientation and anabolic-androgenic steroids in adolescent boys
Spontaneous corpus cavernosum abscess
Testicular tumours
Prostate
Experimental
Case report
Adverse effects and treatment recommendations
Reproductive-endocrine effects in women
Other female-specific side effects
Hirsutism and alopecia
Deepening of the voice
Risk of breast cancer
Skin
Acne
Hirshutism
27
Androgenic alopecia (baldness)
Effects on pancreas
Effects on the immune system
Infection risks after injections of anabolic steroids
Tetanus
Injection policies
Gluteal mass
Sepsis
Pyomyositis
Tuberculosis
Invasive fungal rhinosinusitis
Bone
Effect on bone density
Influence on muscle and tendon injury and injury healing
Pectoralis major ruptures
Muscle healing in power-lifters
Tendon adaptation
Compartment syndrome
Rhabdomyolysis
Effects on kidneys
Collapsing glomerulopathy
Acute kidney injury
End-stage renal disease in a bodybuilder
Gynecomastia
Androgen and estrogen receptors in gynecomastia
Treatment
Hyperplastic changes and receptor status in the breast
Other side effects
Thyroidal effects
Adrenal effect (on cortisol production)
Hematological
Hypercalcemia
Persistent hiccups
Effect on inflammation
In basket ball players
Side effects in elderly
Dental health
Effect on gingival tissues
Toxicity
Genotoxicity (cancer risks)
Fatal events
Multiple organ failure
Side effects of topical anabolic steroids
Influence on reaction on pain
Anabolic steroids combindes with other abuses
Testosterone and opiate use
Anabolic-androgenic steroids and heroin use
Reversibility of the effects in former anabolic-androgenic steroid abusers
Long-term effects on social-medicine demography
Swedish data
Increased mortality in former users of anabolic steroids
Tour de France (1947-2012)
Finland
28
TESTOSTERONE
Theoretical, overviewing, aspects
Metabolites
Influence of exercise on growth hormone and testosterone in prepubertal boys
Normal values
Problems in evaluating serum testosterone values
Time of day is of importance for testosterone levels
Plasma and urinary markers of oral testosterone
Detection of injected testosterone in blood
Candida albicans in urine can produce testosterone
Normal production
Synthetic derivatives
Half life
Testosterone/epitestosterone concentration ratio (T/E)
Free testosterone/cortisol ratio
Time-course of testosterone action
Stability in the urine
Effects of anabolic precursors on serum testosterone concentrations
Testosterone concentrations are blunted after resistance exercise in men
Stress from exercise in the below sea level environment
Effect of rowing
Biphasic dose responses to prolactin
Seasonal variations
Ethnic differences in steroid-related diseases
Urinary levels of testosterone and epitestosterone in a Korean male population
Status of lean elite athlets
Levels in male Olympics
Associations between dehydration and testosterone at weight loss before
competition
Effect of calorie restriction on testosterone in training
Effects of meal form and composition on plasma testosterone
Effect of sexual activity on testosterone levels
Testosterone levels during recovery
Older men
Women
Testosterone after use of chlorinated swimming pools
Testosterone prohormones
Salivary testosterone (and testosterone to cortisol ratio)
Testosterone deficiency produced by castration
Medical history
Biological action
Hypothalamic-pituitary-gonadal (HPG) axis
Brain
Mood
Heart
Tendon
Muscle
Breast
Liver
Fat and fat metabolism
Effect of testosterone on muscle protein synthesis
29
Effect of different doses of testosterone on lipid profile
Testosterone and insulin sensitivity
Effects of testosterone supplementation on inflammation markers
Testosterone effects on coagulation and fibrinolytic factors
Serum testosterone and physiological effects
Testosterone deficiency
Opioid-induced androgen deficiency (OPIAD)
Long-term effects of testosterone
Effect of magnesium on testosterone levels
Testosterone and motivation to compete
Psychological influence on testosterone levels
Influence on testosterone levels by sports environment
Influence of psychosocial environment
Testosterone concentrations and playing position in professional basketballers
Differences between winners and losers
Home versus away competition: effect on psychophysiological variables
Spectators
No effect of red color
Influence of stress on testosterone (and other anabolics) levels
Hormone profile in men
Hormone profile in women
Plasma testosterone and work-related neck and shoulder disorders
Influence of fasting (Ramadan)
Circadian rhytm
Genetic influence (polymorphism)
Influence of exercise on testosterone levels
Effect of training and recovery on testosterone levels
In trained and in not-trained
Effects of hard exercise
Effect of endurance training
Effect of resistance training
Effect of different types of training
Effect of different short-time types of exercise
Order effects of combined strength and endurance training on testosterone
Explosive performances
Testosterone response to acute resistance exercise in obese versus lean
Active recovery versus passive recovery
Effect of mood changes
Influence of red color
Long rest interval promotes durable testosterone responses
Pre-game free testosterone concentrations and outcome
Pre-game testosterone level: home advantage
Effect of testosterone on myoblasts
Testosterone levels after concussions
Overreaching
Increased physical activity increases testosterone in obese men
Females
Effects in a young female
Gender differences in testosterone and cortisol response to competition
Adolecents
Effect of soccer
Effect of golf
Low testosterone in basket players
30
Biking
Rowing
Response to marathon running
Testosterone to cortisol ratio after exercise
Saliva testosterone during competition
Effect of amount of training on testosterone levels
Responses to different intensities of exercise
Effect of high-intensity training
Response to three football matches in a week
Response to 164-km road cycling in a hot environment
Responses to a competitive 5,000 m race
Effect at orthopedic ligament surgery
Rehabilitation and testosterone
Testosterone and metabolic syndrome
Effects on immune system
Testosterone and age
Aging athletes
Older men
Old versus young individuals
Testosterone dose-response relationships in healthy young men
Effects of long flights
Influence of space flights
Effects of diet on testosterone metabolites
Influence of zinc
In hypogonadal men
Hypogonadal symptoms in young men associated with low serum total
testosterone
Dietary adjuncts for improving testosterone levels in hypogonadal males
Testosterone level as an indicator of gender
Testosterone supplementation
Topical preparations
Transdermal preparations
Gels and solutions
Transdermal testosterone on bone and muscle in older men
Hypertension as a complication of topical testosterone therapy
Subcutaneous injections
Effect of postexercise ethanol ingestion
Testosterone and cocaine
Effect on cocaine’s vascular effects
Interaction with NSAIDs
Influence on gastric ulcer precursors
Side effects
Cardiovascular disease
Diabetes
Salivary hormones and IgA in relation to physical performance in football
Salivary testosterone and anxiety in winners and losers in international judo
Blunting of exercise-induced salivary testosterone in elite-level triathletes
31
Circadian salivary testosterone
Responses to different rugby training exercise protocols
A 10-day training camp blunts salivary testosterone in elite level triathletes
Salivary steroid hormones profile, and physical effort over a 3-week stage race
Rugby
Football
Testosterone metabolite and neurosteroid 3alpha-androstanediol
IRMS
Gas chromatography/combustion/isotope ratio mass spectrometry
(GC/C/IRMS)
Mass spectrometry of steroid glucuronide conjugates
Testosterone versus epitestosterone
Testosterone and epitestosterone in human urine analysed by liquid
chromatography
Gas chromatography-combustion-isotope ratio mass spectrometry
TLC-densitometry method
Mobility spectrometry separations
Analysis of the transcriptome
Metabolomics
UHPLC for glucuronides resistant to enzymatic hydrolysis
In hair
Experimental
Testosterone undecanoate
Intracerebroventricular administration
Influence on the brain
Testosterone self-administration in male hamsters
Testosterone self-administration in female hamsters
A comparative study between man and rat regarding testosterone response to
exercise
Dependence in hamsters
Androgen receptor immunoreactivity of male rat cervical motor neurons
Testosterone attenuates amphetamine-induced locomotion in male rats
Decision-making
NANDROLONE
Frequency of abuse of nandrolone in sports
Physiology
Effects on mitochondria
Effect on dynorfin A in the brain
Metabolism and metabolites
Stimulation on testosterone biosynthesis in Leydig cells
Synthesis and biological evaluation of nandrolone-bodipy conjugates
Aromatisation
Nandrolone excresion
Endogeneous nandrolone
Microbial transformation of nandrolone
Body composition and anthropometry after nandrolone
Effects on CNS
Genetics
Protein disulphide isomerase (PDI)
Diagnostic metabolites
32
Nandrolone metabolites in antidoping control
Nandrolone and metabolites in urine from sedentary persons and sportsmen
Urinary nandrolone metabolites are regulated by human chorionic gonadotropin
Effects of the menstrual cycle
Influence on hypothalamic-pituitary-adrenal axis
Trauma and hypoglycaemic stress
Norandrosterone before and after submaximal standardized exercise
Effect of training
Effect on healing of muscle injuries
Positive effect on nerve regeneration
Effect of exercise on excresion
Side effects
Effect on male fertility
Cardiotoxic effects
Nandrolone-induced aggressive behavior
Harmfull effects on brain axons
Harmful effects on learning capacity
Effect on hypertension
Effect on failing heart
Effect on peripheral nerve injury
Effect on growth hormone
Effect of small doses on stress response
Aortic adaptations to exercise
Nandrolone-mediated testosterone reduction during alcohol intoxication
Impact of nandrolone on biosynthesis of steroids
Effects of nandrolone on recovery of denervated muscle
Mineral cofactors and herbal products
Genotoxic effects
Upregulation of aromatase expression
Endogenous or exogenous origin of nandrolone
Significance of 19-norandrosterone in athletes' urine
Formation of 19-norsteroids of endogenous steroids in stored urine samples
Liquid chromatography/tandem mass spectrometry
Cyclic, differential pulse and square-wave voltammetry
Gas chromatography-tandem mass spectrometry
Chemiluminescent
Molecular imprinted polymer coated QCM
In hair
Experimental
Heart effects
No effect on IGF-1 mRNA
Effect the content of substance P in the rat brain
Effect on opioid peptides in the rat brain
Down-regulation of delta opioid receptor mRNA by nandrolone
Effect on GABAA receptor-mediated currents in the rat forebrain
Effects of nandrolone decanoate on cocaine-induced kindling in male rats
Effect on regenerating skeletal muscles in the rat
Effect on heat shock protein (HSP)
Cardiopulmonary reflex and cardiac cytokines
Activation of calcineurin-NFAT signaling
Lack of nandrolone rewarding effects in adolescents
33
Effects on redox status in exercised rats
Testosterone suspension
Influence of nandrolone decanoate on serum lipids and liver enzymes in rats
Effects of chronic administration of nandrolone decanoate on redox status in
rats
Role of atrial natriuretic peptide (ANP)
Influence on serum FSH, LH and testosterone and hematological parameters in
rats
Enhanced vasoconstriction and reduced vasorelaxation
Effects of nandrolone decanoate on the neuromuscular junction
Increased dopamine transporter density in the male rat brain
Remodeling and strength in human supraspinatus bioartificial tendons
Long-term effects on dominance in a competitive situation
Effects on emotional behavior and monoaminergic neurotransmission in
adulthood
Proliferation and adhesion of myoblasts
Nandrolone and oxidative stress
Effect on the pleasurable properties of cocaine
Ineffectiveness for stimulating skeletal muscle mass
Increase of extent of apoptotic changes affecting fertility
Deterimental effect on rotator cuff tear
Effect on liver regeneration
Amphetamine-induced aggression enhanced by nandrolone
Effect on neural stem cell proliferation
Effects on agression
Reduced volume of testis and length of seminiferous tubules
Effects on the ovaries
Long-time effects on dominance (psychological effects)
Modulation of cell cycle regulation in functionally overloaded rat soleus muscle
Effect of sub-chronic nandrolone on dopaminergic and serotonergic systems
Effects on substance P
Effects on phenylephrine
Cardiac autonomic dysfunction
Homocysteine
Decreased glucogenesis
Nandrolone induces learning and memory impairments in rat
Combined effect of nandrolone and growth hormone on the brain function
Dysfunction of the reward pathway in rats
Metamizol and morphine-induced analgesia and tolerance/dependence in rats
Reduction of hypothalamic proopiomelanocortin mRNA levels
Effects on sarcoplasmic reticulum Ca2+ ATPase function
Prevention of immobilized musles’ atrophy
Induction of alterations of serotonergic 5HT1B and 5HT2 receptors in the male
rat brain
Enhancment of hypothalamic biogenic amines in rats
Differential post-effects in slow- and fast-twitch skeletal muscles
Dopaminergic effects after chronic treatment with nandrolone
Combined treatment with nandrolone and cocaine
Unweighting-induced functional changes in rat soleus muscle
Activation of calcineurin-NFAT signaling
Brain-derived neurotrophic factor (BDNF)
Density of dopamine receptors in the male rat brain
Influence on muscle fibres distribution
34
BOLDENONE, BOLDIONE AND BOLANDIOL
Overviews
Boldenone potency compared with other anabolic steroids
Effect on reproductive functions
Possible endogenous origin
Urinary samples contaminated with faecal boldenone
Metabolism study of boldenone in human urine
Influence of renal function
Methylstenbolone
Metabolism of 1-ene-steroids
Stenbolone
Quinbolone
Boldione
Experimental
Liquid chromatography-tandem mass spectrometry
Conjugated and unconjugated
In cattle
DEHYDROEPIANDROSTERONE (DHEA)
Why is DHEA prohibited?
Dehydroepiandrosterone (DHEA) versus dehydroepiandrosterone sulfate (DHEA-S)
Medical uses
Physiology
Synthesis and expression
Metabolism
Protein synthesis as a result of DHEA
Correlation between DHEA and bioavailable testosterone
Molecular and cellular mechanism of DHEA
Effects of DHEA on body composition, bone metabolism, and skin
Influence on mood
Role of DHEA in sexual function
Excretion
Normal values related to age
Influence of menstrual cycle
In women
Dehydroepiandrosterone sulfate
Neuroprotective-neurotrophic effect of endogenous dehydroepiandrosterone
sulfate
Use in athletes
Dehydroepiandrosterone supplementation in healthy men
In sedentary and physically trained aged men
At adrenarche
Decrease with age
Metabolism
Efficacy in performance enhancement
Exercise and DHEA in skeletal muscle
Effects of walking training
Combination of DHEA supplementation and exercise training on metabolism
Dependence on exercise intensity and training level
Submaximal and maximal acute exercise: DHEA
Submaximal and maximal acute exercise: DHEA-S
35
Supramaximal and strength exercise: DHEA
Supramaximal and strength exercise: DHEA-S
Chronic physical exercise, basal values: DHEA
Chronic physical exercise, basal values: DHEA-S
Physical exercise response: DHEA
Physical exercise response: DHEA-S
Response of muscle sex steroidogenesis to resistance exercise in human
Effects on the cardiovascular system
Effects on asthma
Effect on female bone
DHEA metabolites activate estrogen receptors
DHEA in vascular disease
Neuroprotective-neurotrophic effect of DHEA-S
Enhancement of the antidepressant effect of cocaine
Side effects
Mania
Impact on hepatocarcinogenesis
Diurnal secretion
Dietary supplements with DHEA
In swimmers
In judo
DHEA rather than testosterone shows saliva androgen responses to exercise
In hair
Current medical recommendations
Futurum regarding DHEA
Regarding muscles
STANOZOLOL
Cardiac effects
Metabolism
Drug-drug interaction
Excretion
Specific binding protein for the anabolic steroids stanozolol and danazol
Side effects
Effect on the liver
Pubertal stanozolol administration on reproductive and aggressive behaviors
Hypokalemia
Drug interactions
16beta-hydroxystanozolol in urine and faeces
Experimental
High-intensity exercise modifies the effects of stanozolol on brain oxidative
stress
Exercise modifies the effects of stanozolol on brain oxidative stress in rats
OTHER SPECIFIED ANABOLIC ANDROGENIC STEROIDS

Oxandrolone
Metabolism
Given to prepubertal boys
Neuroregenerative effect of: a case report
Microbial transformation
36
Oxymestrone
Oxymetholone
Other OXO-compounds
Transdermal application
Megestrol
Megastol and dronabinol
Methyltestosterone
Biomarkers
Cerebrospinal fluid and behavioral changes after methyltestosterone
Side effects
17alpha-methyltestosterone
Desoxymethyltestosterone (DMT)
Metabolism
Methandrostenolone
Methenolone
Mestrolone
Epitestosterone
Antiandrogenic activity of epitestosterone in male mice in vivo
Madol
Methylnortestosterone
Testing
Dihydrotestosterone (DHT, androstanolone)
Synergistic effect of DHT and IGF-1 hyperstimulation
DHT and estrogen regulation of rat brain androgen-receptor immunoreactivity
Deposterone
Dromastanolone
Adrenosterone
Hydroxyandrosterone and hydroxyetiocholanolone
Androstenediol
Norandrostenedione and norandrostenediol
Metabolism of 1-androstenediol and 1-androstenedione
Chronic androstenediol-herbal supplementation in 30 to 58 year old men
3alpha-Androstanediol
Androstenedione
Androstenedione does not stimulate muscle protein anabolism
Androstenedione gives no elevation of testosterone
Elevation of estradiol
Androstadiene-17beta-ol-3-one
6alpha-Hydroxy-androstenedione
Hydroxyandrostenedione
Androst-4-en-3-one-based steroids
nor-4-Androstene-3,17-dione
Norandrostenedione
Dehydrochloromethyltestosterone
Danazol
Fluoxymesterone
Desoxymethyltestosterone
Keto-androgens
Clostebol
Turinabol
37
Trenbolone
Trenbolone acetate metabolite biotransformation under aerobic conditions
Experimentally
Tibolone
1-Testosterone
Metabolism
Methenolone
Metandienone (Dianabol)
Metabolism
Laboratory tests
Mestranolon
Norbolethone
Mepitotestane
Dimethazine
Ecdysteroids
Phytosterols
Phytoecdysteroids
17-hydroxyandrosta-3,5-diene ("Syntrax Tetrabol")
Tetrahydropyranyl ether (THP)
Designer steroids
Non-targeted approach
Indirect approach
Tetrahydrogestrinone (THG)
YK11
Delta6-methyltestosterone
Methylstenbolone
Methyl-1-testosterone (M1T)
5-alpha-Reductase inhibitors
Finasteride
Dehydrogenase inhibitor
Transsexuality
SELECTIVE ANDROGEN RECEPTOR MODULATORS (SARMs)

Background
Biotransformation
Steroidal ligands and their clinical applications
Arylpropionamide-based SARMs
SARMs produced by fungus
Ligand efficiency of selective androgen receptor modulators (SARMs)
Comparative safety evaluation of SARMs and AASs
Early biomarkers of response to SARMs
No effect of locally implantes SARMs
Treatment of various muscle-wasting diseases
A SARM drug candidate sold via the Internet
Andarine
New SARM compounds
ESTROGENS AND FEMALE SPORTS

Ethnic variations
38
Serum androgen levels in elite female athletes
Endogenous anabolic hormone and endurance versus resistance exercise
46, XY
Androgen alterations during pregnancy
Urinary estrogens and androgens during pregnancy
Estrogen replacement influence on physical performance in female hypogonadism
Estrogen compared to estrogen plus progesterone on the exercise electrocardiogram
Responses to resistance versus endurance exercise in premenopausal females
Endogenous versus exogenous estrogens
Sex-hormone binding globulin
Testosterone
Prolonged aerob exercise
Urinary steroids in exercise
Salivary testosterone in exercise
Hyperandrosteronism in women
Hyperandrogenism in female athletes with functional hypothalamic amenorrhea
Ethics
Androgen metabolism in women with infertility and hypoandrogenism
Effects of menstrual cycle
Luteal phase deficiency
Influence of carbohydrates on menstrual cycle
Performance during the menstrual cycle
Cycling time trial performance during different phases of the menstrual cycle
Effects of exercise on the female reproductive system and sex hormones
Exercise and estrogen make fat cells "fit"
Effects of strength training
Comparison of baseline free testosterone between elite and non-elite female
athletes
Effect of stretching
Effect of estrogen usage on eccentric exercise-induced damage in rat testes
Gender differences in injury during top-level international athletics championships
Effect of estrogens on muscle regeneration
Influence of estradiol on muscle damage and leg strength
Sex hormones and anterior cruciate ligament rupture in females
Estrogen on muscle damage biomarkers following prolonged aerobic exercise
Effect of a periodized training program in female basketball players
Muscle sympathetic nerve activity and systemic hemodynamics in young women
Influence of IGF-1
Influence of age on muscle mass in women
Bone anabolics
Maximal force and tremor changes across the menstrual cycle
Gonadotropin-releasing hormone
Pregnenolone
Gestrinone
Estrogen blockers
Effects in men
Caffeine ingestion enhances perceptual responses in females
Nutrition in female endurance runners
The effect of epitestosterone on estrogen biosynthesis in vitro
Influence on growth hormone
Oral contraceptives
Influence of oral contraceptives on serum testosterone values
39
Effect of antiandrogens in hormonal contraception
Prior eccentric exercise on heavy-intensity cycling: the role of oral
contraceptives
Oral contraceptive use and exercise-induced muscle damage and recovery
Effect of bone mass
Effects on performance of oral conceptives
Oral contraceptive use effect on maximum force production in women
Influence of oral conceptives on tendons and ligament
Heart rate variability across the menstrual cycle in young women on
contraceptives
Blood pressure
Effects of acute resistance exercise on hormonal and cytokines changes
Maximal fat oxidation is affected by oral contraceptive use in young healthy
women
Exercise per se masks oral contraceptive-induced postprandial lipid
mobilization
Impaired sleep female athletes using oral contraceptive steroids after coffee
Glucose tolerance during in women with oral contraceptives
Oral contraceptive use and intermittent exercise performance and metabolism
Effect on the salivary testosterone and cortisol responses to training
Salivary SIgA responses to acute moderate-vigorous exercise
No effect on knee ligament damage with heavy exercise
Impact of genetics and hormonal contraceptives on steroid profile in female
athletes
Experimental
Menopaus
Hormone replacement therapy
Influence of SERMs on steroid metabolism
Folinic acid supplementation
Experimental
The modulation of androgen metabolism by estradiol
Estrogen effect on leucine and carbohydrate oxidation and lipid oxidation
Short-term 17-beta-estradiol administration to young males
Selective estrogen receptor modulator (SERMs)
Effect on metabolic homeostasis
Safety overview
Emerging SERMs
Tamoxifen
Toremiphene
Raloxiphene
AROMATASE INHIBITORS
Overview
Aromatase excess syndrome
Exemestane
Anastrozole and exemestane
Formestan
Metabolism 4-OH-androstenedione (formastane)
Letrozole
Androst-4-ene-3,6,17-trione (6-OXO)
Metabolism of keto and hydroxy steroids
40
4-OH-testosterone
6-Oxo-androstenedione
7-Keto-dehydroepiandrosterone
Oxabolone
Testolactone
Aminoglutethimide
Interactions of antioestrogens and aromatase inhibitors
Experimental
BLOOD DOPING
Overviews
Prevalence of blood doping
Biochemical markers of hypoxia
Exercise hemorheology
Which is the "normal" hematocrit from an hemorheologist's viewpoint?
Theoretical aspects on energy transfer in the body
Determinants for performance from the blood point of view
Hemoglobine mass and physical performance
Physiological effects of transfusions on aerob performances
Physiological effects of transfusions on anaerob performances
Erythropoesis
Production and destruction of red blood cells
Erythropoiesis after high altitude acclimatization
Hemolysis due to running
Effects of training in juniors
Reference ranges in elite athletes for markers of altered erythropoiesis
Red blood cells
Variation of red blood cell distribution width and mean platelet volume
Plasma volume
Effects of iron on performance
Effect of iron on performance
Testing of blood
Freezing red blood cells
Effect of endurance training on erythrocyte deformability
Water exchange lifetime value across red blood cell membrane
Hemoglobin and hematocrit
Changes with age
Within-subject hemoglobin variation in elite athletes
Rapid body mass loss affects erythropoiesis and hemolysis
Seasonal variations of hemoglobin
Regulation of red blood cell mass
Circadian rhythm
Stability of hemoglobin under testing conditions
Statistical models for blood cell survival
Preanalytical mixing of whole-blood specimens
Hemoconcentration
Paradox of hematocrit
Changes in hemoglobin values in elite cross-country
Competitive cycling and hematocrit
Blood parameters during endurance exercice
Hematological indices and iron status in athletes of various sports
During flights
Total hemoglobin mass
41
Blood volume in elite athletes of different disciplines
Within-subject variation in hemoglobin mass
Influence of exercise
Abnormal hematologic profiles in elite cross-country skiers
Effects of injury and illness on haemoglobin mass
Hemoglobin mass and peak oxygen uptake in female altitude residents
A stable parameter
Influence of travelling
Influence of erythropoietin
Laboratory technique
Reticulocytes
Sports anemia
Influence of erythropoietin
Blood drawing
Measurment
Diurnal variation
Biological variability
Kinetics of reticulocytes production in humans
Reported reticulocyte values in athletes
Gender effects on reticulyctes
Stability of reticulocytes
Effects of exercise on reticulocytes
Reticulocytes in doping
Reticulocyte as potential discriminators of recombinant human erythropoietin
abuse
Cation transport and cell volume changes in maturing rat reticulocytes
Neocytes and neocytolysis
Polycytemia
Blood conservation and transfusions
Blood transfusions in general
Autologous transfusion
Non-autologous transfusion
Adverse events
Plasma markers for testing
Screening for homologous blood transfusions
Screening for autologous blood transfusions
mRNA
Leucocytes
Subpopulations of RBCs
Membrane proteins
Plasticiser
A fast automated screening method for the detection of blood transfusion
DNA-based method for detecting homologous blood doping
Response to foreign erythrocytes
Effects of blood withdrawal and reinfusion on biomarkers
Laboratory problems
Robustness of measurements after storage of blood
Reticulocytes
Whole blood transfusion
Detection of homologous blood transfusion
Capillary electrophoretic separation
Intravascular hemolysis
Evaluation of blood parameters
42
Erythropoietin (EPO)
Overview
Renal erythropoietin-producing cells
Physiology
Pharmacokinetic and pharmacodynamic considerations
Increase in erythropoietin after an ultramarathon run at moderate altitude
Plasma-volume contraction and induced hypoxaemia modulate erythropoietin
Different ways of action
Erythropoietin and the heart
A neuroprotective agent
Neovascularization
Psychological effects
Influence of N-acetylcysteinin
Interactions with androgens
Erythropoietin and muscle disease
Effects on performance with longer duration than effect on erythropoietin
Effects of exercise on erythropoietin molecules
Effect on performance
Effects of cycling?
Serum levels of sTfR and cycling performance
Haematological abnormalities
Effects of erythropoietin on serum hepcidin and serum iron bioavailability
Soluble transferrin receptor as an indirect biomarker of erythropoietin
Cross reactivity between human erythropoietin antibody and horse
erythropoietin
Erythropoietin increases lactate influx into erythrocytes
New biomarkers
Prolonged erythropoietin treatment does not impact gene expression in muscle
Gender difference in platelet aggregation and reactivity induced by
erythropoietin
Erythropoietin and diabetes mellitus
Chronic erythropoietin treatment improves diet-induced glucose intolerance in
rats
Erythropoietin enhances whole body lipid oxidation during prolonged exercise
Erythropoietin does not reduce plasma lactate, H+, and K+ during intense
exercise
EPO-Fc protein
Theoretical aspects of detection blood doping with erytropoietin
Micro dosing
Excessive dosing
Enzymatic desialylation
Side effects of erythropoietin
Hepcidin as a marker
MicroRNAs
Confounding factors for erythropoietin detection
Lack of influence on GH-IGF axis or makers of bone turnover
Glycosylated erythropoietins
Erythropoiesis-stimulating agents (ESAs)
Available erytropoietins
CERA, a continuous erythropoietin receptor activator
Darpoietin/NESP
43
Epoetin delta (Dynepo®)
Hematide®
Non-EPO-related erythropoiesis stimulating agents
Synthetic peptide-based EPO receptor agonist
EPO-Fc fusion protein
New erythropoietin-like drug principles
Adverse effects of erythropoietin
Novel erythropoietin doping strategies
Detection rate
Detection of missuse of erythropoietin in competitive sports and laboratory
testing
Effects outside the bone marrow
Economy
Experimental
Cobalt salt as erythropoietic agent
Cobalt in the human body
Biokinetics model of cobalt in the human body
Medical uses of cobalt and cobalt chloride
Cobalt activation of matrix metalloproteinases
Blood substitutes, in general
Myo-inositol trispyrophosphate (ITPP)
Perfluorocarbon
Perfluorodecalin-filled poly(n-butyl-cyanoacrylate) nanocapsules
Side effects
Efaproxiral
Ex vivo erythrocyte generation
Irradiation of red blood cells
Enhancement of oxygen transfer
2,3 DPG
Hypoxia-inducible factor (HIF)
Skeletal muscle hypoxia-inducible factor-1 and exercise
Hemoglobin-based oxygen carriers (HBOCs)
Objective comparison between cellular and acellular HBOCs
Redox concerns in the use of acellular hemoglobin-based therapeutic oxygen
carriers
Allosteric modulators of hemoglobin
Hemoglobin measurements in samples containing HBOC
Pharmacokinetics and mechanisms of plasma removal
New products
Hemopure®
Hematide/Peginesatide
Hypoxia-inducible factor (HIF) stabilizers
Phtalates as indicators of blood doping
Granulocyte colony-stimulating factor (G-CSF)
Products based on new biotechnology and with possible use for blood doping
Other blood products
Actovegin
Intravenous fluid use in athletes
Discussion on dangerous dehydration in sports
Oral versus intravenous fluids
44
Exertional muscle cramp prevention
Preexercise fluid requirements
IV volume expanders
Rehydration
Plasma volume expanders
Dextran and hydroxyethyl starch
Hydroxyethyl starch (HES)
Glucose solution
Saline solution
Ringer lactate (or acetate) solution
Hypertonic crystalloid solution
Albumin
Plasma protein fraction
Plasma
Gelatins
alpha-Keratose
Combined solutions (hypertonic crystalloid/colloid)
Pullulan
Levan
Acetyl starch
Polyvinylpyrrolidone
Mannitol
Glycerol
Polyethylene glycol
Newer plasma expander
Intravenous rehydration
Hyponatremia
CREATED HYPOXIA (“HIGH ALTITUDE TRAINING”)

Introduction
Not only for individuals but also for teams?
Different ways of altitude training
Nomenclature
Definitions of levels of altitude
State of research on altitude training
Theoretical aspects on energy transfer in the body
Impact of the ATP systems
The body’s measurement of low oxygen
Short- and long-term influence of hypoxia on muscles
Alteration of the red blood cell during high-altitude stay
Erythropoesis
Production and destruction of red blood cells
Effect on erythropoietin
Effects of hypobaric hypoxia on erythropoiesis
Biochemical markers of hypoxia
Physiological mechanisms to explain effects of altitude training
Overview
Cellular mechanisms
Impact of high altitude on cardiovascular system
Impact of high altitude on ventilation adaptation and gas exchange
Impact of high altitude on blood and blood volume
Impact of high altitude on skeletal muscle
Effect on hematological values of hard exercise
45
Similar hemoglobin mass response in hypobaric and normobaric hypoxia in
athletes
Impact of altitude training on haematological parameters
Pathophysiology
Intermittent hypoxic training
Moderate exercise blunts oxidative stress induced by normobaric hypoxia
Hydration and nutrition
Hemoglobine mass and physical performance
Correlation between Hbmass and altitude training result
Training at high altitude
A combination of altitude and time
Sprint training in hypoxia (RSH)
Team sports
Living high, training low
Hypoxic tents
Short-term normobaric hypoxia exposure
Randomized studies
Immunosuppression
Recovery after training at high altitude
Problems with sleep at high altitudes
Effect of intermittent hypoxic training
Intermittent normobaric hypoxia
Cycling
Effects on laboratory parameters of intermittent hypoxic training
Swimming
Effect on serum CK, LDH and ALT
Influence on the Athlete Biological Passport
Training while at altitude
A two-week traditional altitude training
A meta-analysis
Effect on the erythrocytes
A three-week traditional altitude training
A four-week traditional altitude training
Hematological adaptations
Practical recommendations regarding start of training at high altitude
Individual responses
Variability of Hbmass in altitude training
Position statement on hypoxic training
For which team sports might altitude training be relevant?
Which altitude training intervention should be recommended?
How should the effects of altitude training be evaluated?
What is the optimal altitude dose to be used?
Special considerations for team athletes
Possible impact of reduced air resistance
The ethics of hypoxic training
A complex situation – not just a question of what is natural and what is not
Continuing development of technology
Cheating
Like in former East Germany?
Australian Football League (AFL)
No connection with drug abuse
Consensus in ethics and “the spirit of sport”
Performance-enhancing technologies that do not require athlete effort and skill?
46
Differentiation between the logic and goals of human practices
A philosophic point of view
The ethical dilemmas for WADA
PLATELET-RICH PLASMA
History of platlet rich plasma
Current general classification
Platelets in PRP
From terminology to biological mechanisms
Cochrane review (2015)
A randomized study
Rotator cuff
In epicondylalgia
PPR was not better than autologous whole blood for tennis elbow
Tendopathies
Patellopathy
Plantar fasciopathy
Hamstrings
In acute hamstring muscle injury
The knee
In osteoartritis
Achilles tendon
Muscle injuries
In muscle rupture with haematoma
In rats
Acute ankle sprains
A case report
Inguinal disruption
Experimentally
Systemic effects of locally injected platelet rich plasma in a rat model
GROWTH HORMONE
Overviews
Olympic jitters at power drug theft
Various isoforms
Isoforms of growth hormone after administration of recombinant human GH
Does hGH enhance athletic performance?
What are the metabolic effects of GH that make it attractive as a drug of abuse?
Clinical pharmacology
Growth hormone counteracts established steroid catabolism
Recombinant human growth hormone
Epidemiology and demographic factors of abuse
GH-values in the elite
Adolescents
Growth hormone during puberty
Growth hormone supplementation in adolescent athletes
GH-values in training young girls
Children
Neuroregulation of growth hormone during exercise in children
Women
Prevalence
Side effects
Effect of aging
47
Physiology
Molecular and metabolic mechanisms
Promotors of secretion
Influence of nutrition
Influence of testosterone on GH
Influence of gluthathione on GH
Immunofunctional and traditional growth hormone
Effects on circulation
Fett-substrate metabolism
Glucose-substrate metabolism
Effect on energy expenditure
Effects on muscle mass and strength.
Body fat, extracellular water, and thermoregulation.
Interactions with thyroid hormones and sex steroids
Hypothalamo-pituitary-adrenal (HPA) axis
Effect of stress on the growth hormone-hypothalamus axis
Growth hormones secretagouges
The GH-IGF-I axis and exercise in normal subjects
Supraphysiological GH and exercise performance
Influence of training on GH response
Influence of carbohydrates and proteins
Influence of different hypoxic conditions on GH
Influence on glucose homeostasis
Influence on cardiovascular system
Growth hormone binding protein
Effect on IGFBP-4 and -5
Effect on collagene
Influence of alcohol
Effects on other variables
Effects on respiration
Effect on mitochondrial function and viability of blood mononuclear cells
Increased mortality risk
Side effects
Acromegaly
Hypoglycemia
Clinical use
Growth hormone deficiency
Medical indications for hGH
Delivery methods
Growth hormone therapy in adults
Use in weightlifters
Biomarkers for use (other than IGF-1)
GH2000
Intra-individual variability
Effect of training of the analyse results
Theories for detection of doping with growth hormones
Challenges of detecting GH abuse
Isoform approach to detection abuse
Possible ethnic differences
Pharmacokinetics and pharmacodynamics of GH
Metabolism of GH
GH proteoforms
48
C-terminal fragment of human growth hormone
Effect on red blood cells
Positive association with estrogens in men
Negative effects on collagene synthesis
Idiopathic adult growth hormone deficiency
Growth hormone replacement in adults with a fixed low dose
Effect on memory
Gene expression in peripheral blood
GH-receptor antagonists
Effect on the mitochondrial function of peripheral blood mononuclear cells
Growth hormone releasing peptides
Synthetic growth hormone releasers detected in seized drugs
Legal framework
Effect of dietary supplements on GH levels
Effects of hypertemia on GH levels
Effects of exercise on GH levels
Physiologically elevated hormone levels in training
Effects of resistance training
High intensity versus high volume edurance training
Effect of resistance training on GH responses in young and older men
Influence of prolonged continuous exercise on hormone responses
Cycling with electrical stimulation of antagonist muscles increases plasma GH
Effects in immobilisation
GH and luteinizing hormone (LH) at caloric and sleep restriction
Prior endurance exercise attenuates growth hormone response
GH release during acute aerobic exercise
Growth hormone responses to sub-maximal and sprint exercise
Respons to field sports
Responses after eccentric bench press exercise in resistance-trained men
Influence of strength and work on exercise-induced plasma growth hormone
isoforms
Effect of hydration on exercise-induced growth hormone response
Effects of GH on performace in sports
Effect of high dose growth hormone on anabolic effects
Endurance
Resistance exercise
Sprint
Muscle strength
With and without carbohydrate + protein
Slow eccentric exercise velocity
Tendons
Meta-analysis
In women
Football
Repeated bouts of aerobic exercise
Sleep deprivation
Effects on muscle fiber type and diameter in moderately frail older people
Impact of injuries
Boxers
Horse growth hormones
Detection of different brands
Case reports
Growth hormone in saliva
49
Relation between GH and IGF-1
Radio receptor method
The isoform methods
With nano-technology
Stability of GH during testing procedures
microRNA as a biomarker
Electrophoresis and mass spectrometry
Immunoaffinity purification
Immunoassays
Freeze-thaw cycling
C-terminal fragment og hGH
Biological basis
Intra- and inter-laboratory validation
Tests for GH deficiency
Ethics of use of growth hormone in sports
Experimentally
Growth hormone release in rats in response to a single bout of treadmill
exercise
GH misuse in the future
INSULIN-LIKE GROWTH FACTOR I (IGF-1)

Polymorphism
Physiology
IGF-1 receptor polymorphism and athletic performance
Molecular biology
Regulation of insulin-like growth factor-I in skeletal muscle and muscle cells
Higher levels in winners than loosers
IGF binding protein-3 concentrations in children
IGF-1 response to acute resistance exercise in obese versus lean physically
active
IGF in women
Urinary IGF
In peripubertal females
Effects of training
Resistance exercise
IGF-I and androgen receptor in three rat skeletal muscles
Exercise and circulating insulin-like growth factor I
IGF-1 in the postcompetition setting
Training with calorie restriction and sleep deprivation
In Kenyans
Influence of endurance
Negative energy balance for the IGF-I response to exercise training
Effect of basketball
Effect of hard cycling
IGF-I receptor 275124A>C (rs1464430) polymorphism and athletic performance
Circulation IGF-1 and fitness
Effect of diet and exercise
Modulating the effects of nandrolone
Childhood obesity: the GH-IGF-I connection
50
Degeneration of ageing skeletal muscle: a central role for IGF-1 signalling
Effect of creatine supplementation
IGF-binding protein
Local administration
IGF-1 in deer antler
Markers for IGF-1
Circulating IGF-I mediates effects of exercise on the brain
Protective effects of physical exercise against brain insults
Relationships between blood viscosity and IGF-I status in athletes
Aerobic capacity is not independently related to circulating IGF-1
Serum IGF-I is higher in gymnasts than runners
Interlaboratory agreement of insulin-like growth factor 1 concentrations
Selected reaction monitoring (SRM) LC-MS/MS
Detection
Variation in measurements
In saliva
Experimental
Total parenteral nutrition to rats
INSULIN
Principal actions of insulin related to sports and exercise
Actions of insulin
Insulin and sport
Effects of protein and amino acids on insulin secretion
Ingestion of protein hydrolysate increases postexercise plasma insulin
responses
Vascular resistance and cardiac output
Sympathetic nervous system activation
Insulin-mediated capillary recruitment
Evidence for two vascular flow routes in muscle
Mechanisms for the vascular actions of insulin
Mechanism of capillary recruitment
Influence of blood flow on glucose uptake
Insulin resistance
Interstitial insulin levels
Exercise enhancement of insulin sensitivity
Effects of acute, heavy-resistance exercise on urinary peptide hormone
Insulin physiology
Is insulin a performance-enhancing drug?
Contraction signalling of muscle glucose uptake
Postoperative hypoglycemia
Disagreement between available indices regarding insulin in sports
In weightlifting
Amino acid‐stimulated insulin secretion
Effects of leucine on post‐exercise muscle protein synthesis
Protein hydrolysates
Whey
Effects of insulinotropic nutritional mixtures on insulin secretion
Effects of insulinotropic nutritional mixtures on post‐exercise muscle anabolism
Protein shake
Effects of post‐exercise hyperinsulinaemia on fat oxidation and de novo
lipogenesis
51
Exercise and newer insulins
Improved insulin action following short-term exercise training
Oral antidiabetics
Anti‐inflammatory effects of insulin
A case report
Metabolism of human insulin after subcutaneous administration
A possible means to uncover insulin misuse
Side effects
OTHER DEFINED PEPTIDE HORMONES

Gonadotropins
Human chorionic gonadotropin
Chorionic gonadotrophin (CG) and luteinizing hormone (LH)
Clomiphene
Luteinizing hormone
Natural agonists
Prolactine
Secretagouges: gonadotrophin-releasing hormone
GnRH
Leuprolide
LH releasing hormone
CGRP (calcitonin-gene-related peptide)
Myostatin
Postexercise plasma myostatin concentration after resistance training
Myostatin-neutralizing antibodies in plasma and serum
Muscle hyperthrophy
Effect of psycological stress
Effect of training
Experimental
Mechanical growth factors
Stretching
Antibodies against human MGF E-peptide
Fibroblast growth factor (FGF) and mechano growth factor (MGF)
E peptide
AGRP
CAFFEINE
Overviews
Ergogenic claims
Meta-analysis
History of anti-doping versus caffeine
Use of caffeine by athletes
UK
Spain
Belgium
Canada
Use as a flavor for consuming behavior
Emergency medicine residents' use
Pharmacology of caffeine
52
Absorption of caffeine
Pharmacokinetics
Pharmacokinetic interactions between dietary caffeine and medications
Mode of action
Methylxanthine
Molecular effects
Effects on adenosine receptors
Mobilization of intracellular calcium
Adrenaline-induced effects
Stimulation of CNS
Mental effects
Inhibiton of the phosphodiesterase
Increased post-exercise muscle glycogen accumulation (neoglycogenesis)
Hypoalgesia
Effect on lipolysis
Effects on muscles
No effects on endothelial function
Metabolism of caffeine
Fate in the liver
Factors influencing serum caffeine concentrations
Dependence on time of the day of caffeine’s effects
Time for abstention from intaken caffeine
Dosing of caffeine
Sources of caffeine
Amount per serving
Bioavailability of coffee
Effect versus dosage
Duration of effect
Optimum time to exercise after caffeine ingestion
Divided doses or bolus?
Caffeinated versus decaffeinated coffee
Caffeine versus anhydrous caffeine
Turkish coffee
Users versus non-users
Low doses of caffeine
Lethal dosage
Mouth-rinse
Prior coffee consumption impact on subsequent effect of anhydrous caffeine
Caffeine levels before and after the removal of caffeine from the doping list
Effects on exercise of caffeine
Overview
Meta-anaylsis of the effects of caffeine ingestion on exercise testing
Confounding factors
Effect of caffeine on repeated sprints in team-sport athletes
Effects on short term performance (endurance)
Effects of caffeine on endurance
Effects of caffeine on vertical jump height
Effects on strength
Effects on torque and muscle activity during resistance exercise men
Effects on skill performance
Effects of performance of chronic use of caffeine
Specific sports-related investigations
Running
53
Biking
Football
Basketball
Volleyball
Tennis
Badminton
Jiu-jitsu
Rugby
Basket
Weight-lifting
Rowing
Iron-man
Field hockey
Taekwondo
Paraplegic and tetraplegic compared to able-bodied individuals
Golf
Effect on sedentary men
Physiological effects of caffeine on the nervous system (central and peripheral)
Effects of caffeine on the brain
Caffeine's ergogenic effects on neuromuscular and perceptual factors
Caffeine lowers threshold for exercise-induced beta-endorphin and cortisol
release
Caffeine in central fatigue
Thermogenesis and lipolysis
Ratings of perceived exertion
Impact of caffeine on pain perception
Influence on circadian rhythms
Pre-existent expectancy regarding the effects of the caffeine
Dose-dependent neuromuscular effects
Effects of caffeine on arousal
Effect on alertness
Morning coffee
Effect on insomnia, nervousness, and activeness
Caffeine’s effect on autonomic nervous activity
Physical performance during 24 h of active wakefulness
Moderated effect of caffeine on anxiety
Psychological effects of caffeine
Experimental: performance-enhancing task considerations
Caffeine effects on glucose homeostasis
Caffeine’s effect of inhibition of glycogen phosphorylase
Caffeine increase of exogenous carbohydrate oxidation during exercise
Impact on glycogen accumulation
Caffeine at low muscle glycogen availability
Caffeine in combination with carbohydrate supplement
Caffeinated mouth-rinse
Caffeine’s effect on energy expenditure
Caffeine’s effect on individuals with negative energy balance
Impact on postexercise oxygen consumption
Caffeine ingestion increases estimated glycolytic metabolism
Caffeine as a lipolytic food component
Effect of caffeine on renal functions
Effect of caffeine on hydration
Diuretic effects
54
Effect on urea formation
Ad libitum caffeinated carbohydrate-electrolyte solution
Impact of caffeine on immunological factors
Impact on the inflammatory response
Effect of caffeine ingestion on lymphocyte counts
Effect on NK cells
Immunoendocrine effects
Impact of caffeine on the heart
Caffeine’s effect on cardiac blood flow
Caffeine decreased exercise-induced myocardial flow reserve
Effects of caffeine on linear and nonlinear measures of heart rate variability
Other physiological effects of caffeine
Gastrointestinal function during exercise with caffeine
Impact on ventilation
Impact on sweating
Effect of caffeine on delayed onset muscle soreness
Lack of effect on oxidative stress
Effects on performance after a fat meal
Duration of coffee- and exercise-induced changes in the fatty acid profile
Caffeine improves performance at high altitude
Influence of caffeine, cold and exercise on multiple choice reaction time
Effect on liver during training
Possible health-promoting effects of caffeine
Specific diagnoses
Caffeine in exercise-induced bronchoconstriction
Caffeine in diabetics
Possible adverse effects of caffeine on health
Addiction
Withdrawal effects
Cerebral effects
Cardiovascular problems
Core body temperature
Increased diuresis
Respiratory system
Muscle fatigue
Effect on sleep
Interference with females and female hormones
Interference with alcohol consumption
Effects in children
Athletes’ knowledge of effects of caffeine
Urinary caffeine levels
Combination of pharmacological substances with caffeine
Combination with ephedrine
Caffeine with and without pseudoephedrine
Caffeine versus theobromine
Combination with synephrine
Combination with creatinine
Combination with albuterol
Combination with sodium bicarbonate
Combination with sodium citrate
Combination with carbohydrates
Combination with epigallocatechin
Caffeine with phosphatidylserine
55
Combination with amitryptilin
Combination with amino acids
Combination with taurine
Combination with carnitine
Acceleration of caffeine metabolism of tobacco and cannabis
Combination with ecstasy (methylenedioxymethamphetamine, MDMA)
Combination with green tea and cayenne powder
Combination with sodium phosphate
Impact on delayed-onset muscle soreness
Influence of caffeine on biomonitoring data
Effect of smoking and caffeine
Impact on testosterone levels
Impact on potassium levels
Impact on glutamine acid levels
Impact on creatinine levels
Impact on sex-hormone binding globulin levels
Impact on hematological variables
Stability studies of caffeine in urine
Different sources of caffeine in sports, alternatives to coffee and beverage
Caffeinated “energy shots”
Caffeinated chewing gum
Caffeinated beverages
Caffein gel
Time-release caffeine containing supplement
Guarana
Practical recommendations the International Society of Sports Nutrition
Experimental
Inhibition of adenosine receptor agonist-induced decreases in motor
performance
Inhibitory respiratory responses to progesterone in newborn rats treated with
caffeine
Caffeine combined with carnitine and choline decreases body fat and serum
leptin
Increases in VO2max and metabolic markers of fat oxidation by caffeine
The changing landscape of caffeine research
OTHER STIMULANTS
Stimulants, general aspects
Pharmaceutical cognitive enhancement
Misuse of prescription stimulant medication in a sample of college students
Exercise outcomes in prevalent users of stimulant medications
Ergongenic effects
Theobromine
Theobromine and theophylline
Ephedrine
Chemical composition of various Ephedra species
Herbal preparation of ephedra
Some problem cases regarding doping and ephedrine
Use in US colleges
Use in US Air force
Use in Taiwan
56
Use in women
Use in different sports
Men's online accounts of ephedrine use
Legal affairs
Pharmacology
Thresholds for doping
Physiological effects
Efficay and safety
Elimination
Single nasal and oral doses of ephedrine in healthy subjects
Side effects
Value of case reports
Ephedrine in sport
Effects on performance
Ephedra-containing dietary supplements
Mixture of ephedrine and pseudoephedrine
Weight reduction
Pseudoephedrine
Dose-response in cycling
Influence of preexercise food intake
Pseudoephedrine and circadian rhythm interaction on neuromuscular
performance
Kounis syndrome
Methylephedrine
Synephrine
Prenylamine
Octopamine
Dopamine
Phenylethylamine
Trimetazidine
Modafinil
Effects of modafinil in healthy people
Cognitive enhancing effects of modafinil in healthy volunteers
Clinical pharmacokinetic profile of modafinil
Off-label uses of modafinil
Combined with cocaine
Combined with cannabis
Adrafinil
Armodafinil
Experimental
Ethical considerations
Methadon
Pharmacokinetics
Methylphenidate (MPH)
Combination with aerobic exercise
Transdermal
Experimental
Amphetamine
Precursor compounds to amphetamine and methamphetamine
Attention deficit hyperactivity disorder (ADHD)
57
Physiologic effects of amphetamine
Side effects
Neurotoxicity
New amphetamine-like drugs
Amphetamine in sport
In hair
Famprofazone
Methamphetamine
Amphetamine-related drugs neurotoxicity
Melatonin-attenuated methamphetamine-induced neurotoxicity
Cardiac complications
Outcomes of exercise as post-residential treatment care
Methamphetamine and the hypothalamic-pituitary-adrenal axis
Treatment of dependence
L-methamphetamine
2-ethylamino-1-phenylbutane
Methylamphetamine
beta-Methylphenethylamine
Mephentermine
Metamfepramone
Cathinones
Synthetic cathinones
Driving under the influence of synthetic cathinones
Thermal degradation of synthetic cathinones
Chloromethcathinone (clephedrone)
Designer psychostimulants
N,N-dimethyl-2-phenylpropan-1-amine (NN-DMPPA)
Laboratory testing
Methylenedioxypyrovalerone (MDPV)
Sudden death
Mephedrone
Pharmacological evaluation
Long-term effects
A fatal case
Deaths of individuals aged 16-24 years after using mephedrone
Experimental
Tuaminoheptane
Isometheptene
Sydnocarb®/mesocarb
Strychnine
Benfluorex
Sympathomimetic in general
Informed decision-making on sympathomimetic use
Noradrenergic reuptake inhibitor
SIBUTRAMINE
Cardiovascular safety pharmacology of sibutramine
Side effects
58
Experimental
Fibrous formation in rats
BETA2 ADRENERGIC AGONISTS

Impact of WADA regulations
Current anti-doping regulations (2015)
Finland
Therapy or doping?
Improves swim ergometer sprint performance?
Which sport leads to asthma?
Is swimming beneficial or detrimental for asthma?
Environmental issues
Doping efficiency of beta2-agonists
Physiology
Asthma and sympatomimetica
Definition of asthma in sports
Extended diagnostic criteria
Diagnosis of asthma
How should bronchial hyperreactivity be defined?
Testing of asthma in atheletes
Predictive values of tests
Exercise induced arterial hypoxaemia
Asthma in sports
Hard training as a cause of bronchial hyperreactivity
Prevalence of asthma in sports
Increased bronchial parasympathetic tone in elite cross-country skiers
In elite swimmers
In canoe- and kayak athletes
Misdiagnosis of exercise-induced bronchoconstriction in professional soccer
players
Use of antiasthmatic medicine in athletes
Effect on the heart during training
Effect on recovery after muscle damage
Seasonal allergy and seasonal decrements in athletic performance
Allergic rhinitis in athletes
During Olympic games
Prevalence of use beta-2-agonists
Exercise-induced bronchoconstriction (EIB)
Two distinct phenotypes of asthma in elite
Children
Pathophysiological background
Prevalence of exercise-induced bronchoconstriction
Diagnosis
Eucapnic voluntary hyperpnea
In children
Management of asthma and bronchoconstriction in athletes
Nonpharmacologic measures
Pharmacotherapy
beta-Agonists versus glucocorticoids
Comparison of blood and urin levels between routes of administration
Ergogenic effects
In chronic pulmonary disease
In non-asthmatics
59
Muscle hypertrophy in men
Meta-analysis of beta2-agonists effects
Designer beta2-agonists
Pharmacokinetics and pharmacodynamics of drugs administered by aerosol
Salbutamol (albuterol)
Discrimination of prohibited oral use of salbutamol from authorized inhaled
Effect on muscle strength and endurance performance in nonasthmatic men
Genetic polymorphism of salbutamol metabolizing enzyme
Effects of short-term oral salbutamol administration on exercise endurance
Effect of salbutamol on performance
Neuromuscular function
After intense exercise in endurance athletes
Renal elimination
Acute effects on muscles
Influence of exercise and dehydration
Side effects
Salbutamol and caffeine in combination
Toxicological effects of clenbuterol in human and animal
Clenbuterol and epinephrine for insulin-stimulated muscle glucose uptake
Long-term use of high dose salbutamol
Case report
Experimental
Terbutaline
High-dose inhaled terbutaline and muscle strength
Enhancements in muscle force and power output during maximal exercise
Increase of power output and glycolysis during sprinting in men
Urine and serum concentrations of inhaled and oral terbutaline
Formaterol
Formoterol via Turbuhaler gave better protection than terbutaline
Procaterol
Clenbuterol
Pharmacokinetics
Protective effects against dexamethasone-induced muscle atrophy
Induction of IGF and myostatin
Clenbuterol in food
Clenbuterol in hair
Clenbuterol and heart problems
Supression of bacterial phagocytosis
Salbutamol and clenbuterol
Rhabdomyolysis
Acute clenbuterol overdose
Toxicological effects of clenbuterol in human and animal
Clenbuterol and epinephrine for insulin-stimulated muscle glucose uptake
Experimental
Illegal in cattle
In horses
Olodaterol and vilanterol
Methoxyphenamine
Experimental
60
BETA1-BLOCKERS
Essential tremor
Essential tremor according to their response to beta-blockers
Molecularly imprinted solid-phase extraction
On-line molecularly imprinted solid-phase extraction coupled to LS/MS/MS
ANGIOTENSIN CONVERTING ENZYME (ACE) INHIBITORS AND ANGIOTENSIN II TYPE

RECEPTOR ANTAGONISTS
Telmisartan
Effect on physical and cognitive performance
I/D polymorphism
Genotype and physical performance
Risk factor for wheat-dependent exercise-induced anaphylaxis
Experimental
DIURETICS
Thiazides
Rapid weight loss
Furosemide
Chlorazanil
Mannitol
Mannitol as a challenge test to identify exercise-induced bronchoconstriction
Tripamide
Weight loss practices of college wrestlers
OTHER MASKING AGENTS

Epitestosterone
Proteases
Probenecid
Desmopressin
Pharmacokinetics
Other chemical and physical manipulation of doping tests
GLUCOCORTICOSTEROIDS
Overview
Glucocorticoid receptors
Stimulation of myocytes
Effect of hypercortisonemia on muscle proteins
Effects on exercise performance
Tissue sensitivity to glucocorticoids
Mechanisms of steroid impairment of growth
Adrenal insufficiency after use of corticosteroids
Corticosteroid-induced adrenal insufficiency in elite cyclists
ACTH
Tetracosactide (Synacthen®)
Synacthen(®) in dried blood spots for doping control analysis
Budesonide
Betamethasone
Methylprednisolone
Dexamethasone
61
Dexamethasone inhibits the stimulation of muscle protein synthesis
Triamcinolon acetonid
Single-dose local injection of triamcinolone acetonide
Cortisol value dururing an exercise season
Glucocorticoids oppose translational control by leucine in skeletal muscle
Effect of AAS on adrenocorticotropin hormone and CRF
Changes in cortisol and T/C ratio during an endurance competition and recovery
Hypothalamic-pituitary-adrenal axis activity during exercise
Prednisolone in human urine
Topical administration of prednisolone
Detection of ophthalmic topical corticosteroids as a positive doping test
Time-course of prednisone effects on hormonal and inflammatory responses
Effect of inhaled corticosteroids on bronchial asthma in athletes
Effect on muscles
Glucocorticoid-induced skeletal muscle atrophy
Effect of testosterone on glucocorticoid-induced muscle atrophy
Transfer factor
Use in football
Endogenous prednisolone
Pseudo-endogenous glucocorticoids
Microbial transformation of cortisol to prednisolone
Effect of oral glucocorticoid intake on autonomic cardiovascular control
Side effects
Heart
Side effects of local injections
Life-long treatment
Salivary cortisone tracks the plasma cortisol response to exercise
Prednisolone
Measurement in hair
Experimental
Beneficial effects of glucocorticoids in single and repeated swimming stress in
male rats
MELATONIN
Effect of melatonin on the longevity of mice
As marker for training
A marker for optimal training time
Treatment on circadian patterns in resistance-trained athletes
Ergogenic only during the wakefulness period
Effects on strenuous exercise
Experimental
Jet lag
Salivary
Effect on osteoarthritis
Experimental
Influence on plasma glucose
Combination with imipramine
MORPHINE
Decreased sensitivity after exercise
Codeine
62
Dermorphine
Fentanyl
Naloxone
Effect of nandrolone on acute morphine effects
Opioid receptors
Endogenous opioid peptides
Endogenous opioids and exercise
Efficacy of transdermal morphine for delayed onset muscle soreness
Dried blood spot desorption coupled to on-line SPE-LC-MS/MS
Experimental
Infusion of endorphins
Effects of treadmill running exercise during the adolescent period of life on
behavioral deficits
Swimming reduces the severity of physical and psychological dependence of
morphine
DOPAMINE
Dopamine and nitric oxide
RECREATIONAL DRUGS: CANNABINOIDS, COCAINE, ECSTASY AND GAMMA-

BUTYROLACTONE (GBL)
Cannabinoids
Overviews
Long-term use
Epidemiology of use of cannabis
Cannabinoid receptors
Synthetic cannabinoids
Mechanism of action
Cardiovascular system effects of marijuana
Medical use
Duration of detection in blood
Effects
Influence on psychomotor performance
Influence on respiration
Cannabis use in sports
Influence of nandrolone on cannabinoid dependence
Cannabis in urine
Addictive behaviour policy
Does cannabis have a doping effect?
Endogenous cannabinoid system
“Spice”
Occasional or frequent smokers
Association between cannabis and psychosis
Role of cannabinoids in adult neurogenesis
Khat
Side effects
Cocaine
Evironmental factors important for abuse
Effects
Exercise and withdrawal symptoms
63
Mechanism of cocaine-induced hyperthermia in humans
Concomitant use of testosterone and cocaine
Cardiovascular consequences of cocaine use
Metabolomics of cocaine
Cocaine and exercise
Cocaine in sport
DHEA-sulfate enhances the antidepressant effect of cocaine
Mate de Coca
Side effects
Experimental
Methylenedioxymethamphetamine (MDMA; Ecstasy)
Metabolites
Why all stimulant drugs are damaging to recreational users
Facilitation of fear extinction learning
Learning, memory, and executive function in new MDMA
Effect on body temperature
Cognitive and behavioural indices of change
Laboratory testing
Experimental
gamma-Aminobutyric acid (GABA)
Influence of anabolic steroids
gamma-Butyrolactone (GBL)
gamma-Butyrolactone and gamma-butyrolactone
gamma-Butyrolactone and 1,4-butanediol
gamma-Hydroxybutyrate (GHB)
Stability
Fatalities
Proposed mechanisms of action
Enhancing sexual desire and experience
Enhancing mood and prosocial behavior without affecting plasma testosterone
Metabolism and neuromodulatory properties
Positive aspect of GHB
Cardiovascular and respiratory effects
Central nervous system effects
GHB in obstetrics
Sexual enhancing effects of GHB
GHB use in alcohol and opiate withdrawal
Interaction with alcohol
Gamma-hydroxybutyrate withdrawal syndrome
Pharmacokinetics and pharmacodynamics
Intracerebral effects in animal models
Sleep and growth hormone effects
Side effects
Treatment of substance abuse
Mechanism for the sudden awakening in GHB
Toxicity
Other negative aspect of GHB
GHB abuse
GHB detection
In hair
beta-Hydroxy-beta-methylbutyrate (HMB)
64
Effect of HMB on muscular strength and body composition
Efficacy of a beta-hydroxy-beta-methylbutyrate on physical capacity
Synthesis of beta-hydroxy-beta-methylbutyrate
Delivery in the free acid and calcium salt forms
Metabolism
HMB safety
No effect on strength or body composition during resistance training in trained
men
Onset of blood lactate accumulation and VO2 peak
Creatine and beta-hydroxy-beta-methylbutyrate (HMB)
No influence the urinary testosterone to epitestosterone ratio
Effect on strength
Effect on resistance training
Effects on muscle damage after a prolonged run
Effects on skeletal muscle damage, protein breakdown, and recovery
Effects of training status
Duration of supplementation, dose, and timing
The effects on skeletal muscle hypertrophy in healthy untrained and trained
adults
HMB in athletes training in an energy restricted state
HMB supplementation in youth and adolescent populations
HMB supplementation in aging and masters athletes
HMB improves indices of aerobic performance, fat loss, and energy metabolism
Proposed mechanisms of action
Skeletal muscle regeneration
Skeletal muscle proteolysis
Practical implications and use of HMB in specific sports
HMB in young trained subjects
Comparison of availability and plasma clearance rates of free acid and calcium
salt
Mitochondrial biogenesis and skeletal muscle health
In American football
HMB in trained versus untrained subjects
HMB mixed with other molecules in young trained subjects
HMB mixed with other molecules in young untrained subjects
HBM reduce abdominal adiposity in healthy elderly men
Dose and safety of treatment
HMB versus glucocorticoids
Attenuation of muscle loss during sustained energy deficit
beta-hydroxy-beta-methylbutyrate combined with creatine
Influence of HMB on protein synthesis
Influence on free acids
With alpha-ketoisocaproic acid (KIC)
Subchronic toxicity
Laboratory testing
Experimental
Summarised aspects of HMB in sports
1,4-Butanediol (1,4-BD)
gamma-Oryzanol and ferulic acid
ALCOHOL
Epidemiology of drinking among athletes
Germany
65
France
USA
Motives and attitudes for alcohol use by atheletes
Mechanism of action
Studies on alcohol and performance
Practical effects of energy drinks on alcohol priming
Effect of alcohol intake on muscle glycogen storage after prolonged exercise
Randomised controlled trial of an alcohol management in community football clubs
Alcohol prevention in community football clubs
Effects of mixing caffeinated energy drinks with alcohol
Alcohol consumption and athlete identity
High-risk drinking among student-athletes
Alcohol consumption and insulin resistance in young adults
Effect of ostexercise ethanol intoxication on the neuroendocrine response
Effects of ethanol on glycogen metabolism
Hydration and thermoregulatory function
Nicotinic receptor signaling in alcohol abuse and alcoholism
Journalists and substance use
Effect of drinking on performance
Effects of acute alcohol consumption on neuromuscular function.
Effects of acute alcohol consumption on recovery after exercise
Effects on muscle the day after intake
Effects on brain’s white matter
The aftermath of alcohol use
Effects of alcohol on injury and incapacity
Influence of sex
Impact of alcohol on steroid profiles
Cardioprotective effects
Carbohydrate deficient transferrin (CDT)
In football
In rugby
Sport participation and alcohol and illicit drug use
Consumption in US college sports
Combined with sport drinks
Sponsorship
Advertisments in TV
Prevention
Alcohol control strategies at large sports events
Sport-based intervention for preventing alcohol use
Experimental
Running increases ethanol preference
NICOTINE
Mechanism of action
Cigarette smoke-induced changes of systemic inflammation and muscle structure
Prevention
Effects of smoking abstinence on movement regulation
Urinary protein biomarkers for tobacco smoking
Long-term survival
Snus (snuff)
Smokeless tobacco, sport and the heart
Medical risks
66
World wide consumption
Pharmacology
Pharmacokinetics
Cardiovascular effects
Haemodynamic effects at rest
Haemodynamic effects during exercise
Long-term effects
E-cigarettes
Detection
Effect of transdermal nicotine on information processing
Experimental
Attenuated vulnerability by self-administered nicotine in rats
XENON
NON-STEROIDAL ANTI-INFLAMMATORY DRUGS (NSAIDs)

Mechanism of action
Effect on delayed muscle soreness
Ibuprofen
Ketoprofen
Topical NSAID
How safe are NSAIDs?
Gastrointestinal
Cardiac
Renal and hypertension
Asthma
Pregnancy
Comparison with other analgesics
Effects on a stable PGF2alpha metabolite and morphological adaptations
Use in handicapped athletes
High risk use of OTC NSAIDs and ASA
Osteoartritis
In ultramarathon runners
Side effect
Recommendations
Experimental
Medical ethics
ACETAMINOPHEN
AICAR (5-AMINO-4-IMIDAZOLECARBOXYAMIDE RIBONUCLEOSIDE)
MELDONIUM (MILDRONATE®)
ANTICONVULSANTS
PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR

Genetics
Myofibrillar protein synthesis after concurrent exercise
Metabolomics of skeletal muscle of mice overexpressing PGC-1alpha
67
PGC-1alpha overexpression attenuates mitochondrial deterioration of skeletal muscle
Isoform structure and biological functions
Structural and dynamic mechanisms of partial agonism
Specific agonist
Peroxisome proliferator-activated receptor gamma coactivator-1alpha
Muscle wasting
Effect of lactate
Pioglitazone
Interaction with beta-adrenergic blocker
PHOSPHODIESTERASE INHIBITORS
At high altitudes
Experimental
THYROID HORMONES
Regulation of exercise-induced growth hormone release
Thyroid hormone infulence on growth hormone release
INTENTIONAL ACTIVATION OF AUTONOMIC DYSREFLEXIA ("BOOSTING")
INTERACTIVE EFFECTS OF PERFORMANCE-ENHANCING DRUGS AND SPORTS

INJURY
Extreme training
Influence on the brain
Tendons
NUTRITIONAL SUPPLEMENTS: GENERAL ASPECTS

Governance around sports supplementation
Role of sports medicine specialists
Authorities’ problems
Statements
Sports Dietitians Australia (SDA)
US and Canada
Legislation
Theoretical aspects
Possible ergogenic effects
Scientific nutrition strategy
Sportspeople with a varied and balanced diet do not need supplements
Attempts to measure effects of supplements
Methodologic issues in evaluating the role of dietary supplements
Measurements of enzyme levels
Molecular markers in dietary supplement research
Analysis of actual content in supplements
Quality assurance of dietary supplements
Research protocols
Perceptions of nutritional supplementation
Selected herbals and human exercise performance
Are there evidence for enhanced skeletal muscle hypertrophy by supplements
Motives for use of supplements
Attitudes towards dietary supplements
Stacking
For injury prevention
68
Effects of energy conditioning on food preferences and choice
Possible ergogenic effects
Glycogen recovery is not significantly different between fast food and
supplements
Effect of dietary supplements and vitamins on cognition
Nutritional interventions to augment resistance skeletal muscle hypertrophy
Adolescents
Education on nutrition supplement knowledge
Side effects of dietary supplements
Nutritional supplement screening
Appropriate regulations
Quantitation of use of supplements in sports
Combination with anabolic steroids
Use of nutritional supplements
Denmark
England
Germany
Spain
Greece
Italy
Slovenia
Poland
Singapore
Canada
USA
Jordan
Saudi Arabia
Oman
Brazil
Judo
Sailing
Football
Basketball
Track and field
Rowing
Master femal cyclists
Aquatic sport
Males and females
Use of nutritional supplements among master athletes
Use of dietary supplements among adolescents
Use of nutritional supplements of athletes with an impairment
Recovey after sports
Individualized nutrition as doping
Side effect
Cholestatic janundice
Contaminated supplements
Anabolic steroids
Ephedrine
Hepatotoxicity
How to deal with supplements in the field of practice
Recovery time after sports
Self-reported recovery
69
Practical nutritional recommendations
Adolescents’ nutrition
Self-regulation concepts
Excess of protein and fat
Female needs in nutrition and hydration
A triad
Energy need
Carbohydrate need
Protein need
Hydration during sports
Calcium
Iron
Creatine
Nutrition in the aged athletes
Dietary recommendation
Impact of dietary recommendations
Energy intake
For team sports
For power sports
For endurance sports
For young soccer players
For bodybuilders
Gastric emptying during running
Response to long distance running (energy needed)
Food provision during Olympic Games
General recommendations
Nutrition for sprinters
Nutritionj for runners
Nutrition for triathlon and marathon
Nutrition for football players
General recommendatons regarding the use of dietary supplements
Source of information
Recommendation of the International Society for Sports Nutrition
Energy need
Carbohydrate
Protein
Strategic eating and refuelling
Vitamins
Minerals
Water
Convenience supplements
Muscle building supplements
Creatine
Essential amino acids (EAA)
Beta-hydroxy beta-methylbutyrate (HMB)
Branched chain amino acids (BCAA)
alpha-Ketoglutarate (alpha-KG)
alpha-Ketoisocaproate (KIC)
Ecdysterones
Growth hormone releasing peptides (GHRP) and secretagogues
Ornithine-alpha-ketoglutarate (OKG)
Zinc/Magnesium aspartate (ZMA)
Glutamine
70
Isoflavones
Sulfo-polysaccharides (myostatin inhibitors)
Conjugated linoleic acids (CLA)
Gamma oryzanol (Ferulic acid)
Prohormones
Tribulus terrestris
Vanadyl sulfate (Vanadium)
Green tea extract
Phosphatidyl choline (Lecithin)
(DHEA) and 7-Keto DHEA
NUTRITIONAL SUPPLEMENTS: WATER, CARBOHYDRATE, PROTEIN, AND FAT

Hydration
Ergogenics of fluid and electrolyte balance
In adolescents
Relationship between muscle water and glycogen recovery after prolonged
exercise
Habitual total water intake and dimensions of mood
Effects on environment and persons around for drinking habits
Sweating
Hydration assessment
Dehydration
Hyperhydration
Muscle problems in hypohydration
Modifying factors for hypohydration
Diet
Fluid replacement before exercise
Fluid replacement during exercise
Fluid replacement after exercise
Fluid and electrolyte supplementation for exercise heat stress
Effect of fluid intake volume on 2-h running performances
Drinking pattern during different parts of activity
Different commercial available sport drinks
Dehydration despite favorable conditions for fluid intake
Gastric emptying
Fluid restriction increases GI permeability
Effects of hypohydration on performance
Drinking pattern during sports
Hydration in marathon
Water and carbohydrates
Water and electrolytes
Half-marathon performance
Alcoholic beverage for treatment of dehydration
Oral salt supplementation
Exercise-associated hyponatremia
Use of NSAID
Pathophysiology
Arginine vasopressin (AVP)
Sweating
Clinical features
Prevention of EAH
Therapy of EAH
Bicarbonate
71
Strategies to modulate intracellular and extracellular buffering capacity
Physiology
Acute versus chronic sodium bicarbonate ingestion and anaerobic work
Responses to sodium bicarbonate: a randomized double-blind study
Effect on exercise
Effect on symptoms from the stomach
Effect of alkalosis on plasma epinephrine
No effect of adding creatine to bicarbonate
Sodium bicarbonate and sodium citrate
Citrate
Sodium citrate
Hydroxycitric acid
Energy need
Meal frequency
Carbohydrates
Quality of dietary carbohydrates
Proposed performance mechanisms of CHO supplementation
Multiple transportable carbohydrates
Effects of carbohydrate loading
Effect of low-carbohydrate diet on the pattern of hormonal changes during
exercise
Carbohydrate supplementation in the heat
Manipulating carbohydrate availability between twice-daily sessions
Carbohydrate-protein supplements during exercise on endurance performance
Fructose-glucose composite carbohydrates and endurance performance
Isomaltulose
Isomaltulose versus fructose-maltodextrin
Metabolic responses to exercise after carbohydrate loads
Metabolic responses to high carbohydrate meals with different glycemic indices
Glucose homeostasis in athletes
Glycogen
Effect on perceived exertion
Effect on endurance exercise
Effect of different amounts of carbohydrate on endurance running capacity
Effect of pre-exercise carbohydrate-loading on endurance performance
Effect on marathon running
Effect on swimming
Effect on combat sports
Effect on tennis
Effect for jockeys
Effect on football
Effect on cycling
Effect on rugby referees
Effect on sleep onset
Mouth rinse with carbohydrate solutions
Influence on testosteone levels
Lack of effect of carbohydrates on saliva flow rate and immunoglobulin A.
Influence of carbohydrate ingestion on cytokine responses
Liquid carbohydrate
Intake recommendations
Can low carbohydrate high fat provide fuel for sport?
Catabolic effects of prolonged inactivity are offset by dietary supplementation
Carbohydrates and protein
72
Carbohydrates plus whey
Carbohydrates with fat
Milk carbohydrate and protein
Carbohydrates and caffeine
Carbohydrates and electrolytes
Influence of age and pubertal status on substrate utilization
Carbohydrate gel
Raisens
Fruits and vegetables
Cornstarch
Hydroxypropyl-distarch
Honey-sweetened beverage
Low-carbohydrate diets and performance
Carbohydrate loading effect of menstrual-cycle phase
Effects of carbohydrate beverage ingestion on the salivary IgA
Carbohydrates and immune fuction
Carbohydrate in young aldolescents
Glucose polymers
Carbohydrate effect on oxidative changes
Increased lactate (decreased lipolysis) due to carbohydrates
Lactate
Pyruvate
Sucrose
Ribose
Galactose
Galactose and fructose
Fructose
Trehalose, galactose and glucose
Multiple transportable carbohydrates
Maltose
Carbohydrates in tennis
Experimental
Milk
Milk protein concentrates
Influence of milk on somatotropic axis
Hydration
Effect of milk on the attenuation of exercise-induced muscle
Enzymatic milk protein hydrolysates
Milk protein versus beef protein
Carbohydrate- versus milk-based beverages on muscle damage and glycogen
Effect of milk on the attenuation of exercise-induced muscle damage
Iron stores
For rehydration
Postexercise milk consumption on whole body protein balance in youth
Milk phospholipids
Milk minerals
Fermented milk
Effects depending on exercise status
Milk protein-derived peptide inhibitors of angiotensin-I-converting enzyme
Lysine in selected milk-based products
In an elite sprint kayaker
Experimental
Chocolate milk
73
Flavanol content
Postexercise chocolate milk compared to water consumption
For cycling
Sports (energy) drinks
Use of sports drinks
Science on “sports drinks”
With caffeine
With carbohydrate, protein and antioxidants
With other nutrients
Chemical ingrediences
Cardiovascular effect
Neurological and psychological effect
Gastrointestinal and metabolic effects
Gastric emptying
Renal effects
Dental effects
Knowledge of suger content
Beneficial effects
Pain tolerance
Promotion of an acute positive energy balance?
Effect on exercise performance
Sport drinks versus light meal combat rations
Effect on muscle glycogen
Energy drinks and their role in energy expenditure and weight loss
Safety considerations
Consumption pattern
Perceptions about energy drinks
Attitudes regarding sport drinks
Self-reported hyperactivity and inattention symptoms
Effect of energy drinks on selected fine motor tasks
Energy drinks and traumatic brain injuries among adolescents
Together with mouthguards
Beverage after aerobic activity
In school children
Added calories in sports drinks
Effect of content of carbohydrates
Effect on teeth
Effect in football
Influence on blood pressure
Adverse effects
Provider education
Sweet, non-alcoholic beverages
Effectiveness of commercially available sports drinks
Effects of a large volume of carbohydrate-electrolyte solution on rehydration
Influence of a carbohydrate drink during desert training
Red bull
Canada
USA
Effects of cold drinks
In tennis
In children
Energy shots
Functional energy drinks
74
Proteins
Protein supplements
Whole body protein metabolism following resistance exercise
Peroxiredoxins (PRDXs)
Protein turnover
Protein for managing weight loss
Thermic effect of food and resting energy expenditure
Protein and muscle mass, strength, and aerobic and anaerobic power
Exercise and regulation of protein metabolism
Protein-specific appetite
Importance of satiety
Appetite and subsequent food intake after higher-protein meals
High-protein diet for reducing body fat
Muscle protein synthesis and breakdown
Alteration of dietary intake by protein supplement
Protein for managing weight loss
Timed ingestion of protein
High protein diet (3.4 g/kg/d) combined with a heavy resistance training
program
Protein supplementation does not alter intramuscular anabolic signaling
Clincial outcomes with controlled higher-protein diets of ≤1 year
Clinical outcomes with long-term higher-protein diets of ≥1 year
Protein ingestion before sleep increases muscle mass
Protein metabolism at exercise
Protein and athletic performance
Muscle strength and hypertrophy occur independently of protein
supplementation
Protein supplementation does not alter intramuscular anabolic signaling
Post-exercise dietary protein
Protein assessment
Protein rating scales
Protein efficiency ratio
Biological value
Net protein utilization
Protein digestibility corrected amino acid score
Protein quality rankings
Protein sources
Animal protein
Vegetable protein
Isoflavones
Dietary protein and protein supplements
Recommendated daily allowance in athelets
High protein feeding
Egg white protein
Hydrolyzed protein
Dietary protein and renal function
Dietary protein and bone
Protein intake and liver disease risk
Whey
Wheat germ
Soy-protein
Combination of sago and soy-protein
Caseine
75
Wheat gluten hydrolysate
Building muscles in fed and fasted state
In orienteering
Antioxidants and protein oxidation
Possible side effects
Protein drinks
Effect on endurance and muscles
The effects of protein intake timing in relation to strength training
Proteins before exercise
Protein-rich intake for recovery after exercise
Needs in older, training subjects
Peptides
Amino acids
Amino acids as growth hormone-releasing agents
Effect of training
Essential amino acids
Effects of amino acid derivatives on physical, mental and physiological activities
Amino acids during biking
Keto analogues and amino acid supplementation
Impact on muscle wasting
Fat metabolism stimulation
Amino acid and immune function
Branched-chain amino acids (BCAA)
Branched chain amino acids activate mRNA
Plasma lactate, GH and GH-binding protein levels following BCAA
supplementation
Effects on serum creatine kinase and lactate dehydrogenase after exercise
Branched-chain alpha-keto acid dehydrogenase (BCKDH)
Effect of carbohydrate ingestion on brain exchange of branched-chain amino
acids
BCAA plus arginine
In children
Branched-chain amino acid supplementation during bed rest
Immune response of long-distance athletes
Impaired function of macrophages is not ameliorated by BCAA supplementation
Branched-chain amino acid supplementation improves immunological variables
Branched-chain amino acid catabolism is a conserved regulator of ageing
Co-ingestion of carbohydrate with branched-chain amino acids and serum
IGF-1
No effect on muscle damage
In handball
Experimental
Neutral amino acids
Effects on muscle soreness
Alanine
L-alanine
beta-Alanine
Kinetics of beta-alanine ingestion
Ergogenic effects of beta-alanine
76
Supplementation strategies
beta-Alanine safety
Effects of beta-alanine on exercise performance
Increased gluconeogenesis from alanine after endurance training
Effects of 28-day beta-alanine supplementation on isokinetic exercise
Supplementation and military performance
Effect on physical fitness, brain plasticity, and behavioral performance in mice
High-intensity cycling performance and blood lactate in masters female cyclists
Alanine-glutamine dipeptide
Alanine ingestion increases muscle carnosine content
Alanine supplementation enhances human skeletal muscle relaxation speed
Exercise training and beta-alanine-induced muscle carnosine loading
In female cyclists
Combined with dietary epigallocatechin gallate
Alanine plus creatine
In football
In alpine skiing
Experimental
Position stand of the International Society of Sports Nutrition (ISSN)
Arginine
Arginine and exercise performance
Arginine and muscle protein synthesis
Arginine as a secretagogue
Arginine nutrition and cardiovascular function
Arginine enhances aerobic exercise capacity with augmented nitric oxide
production
Arginine may facilitate the effect of exercise by limiting somatostatin release
Arginine and growth hormone
Arginine and creatine
Arginine and nitric oxide
Combined with ornithine
Combined with ornithine and lysine
Combined with citrullin
Combined with arginine, ornithine and citrulline
Combined with HMB, and glutamine
Safety of long-term arginine
Cross-country skiing
Experimental
Aspartic acid
Aspartame
Aspartates
Aspartate and asparagine
Citrulline
Leucine
Effects of dietary leucine supplementation on exercise performance
Effects of different doses of leucine
Gender differences
Effects of acute creatine monohydrate supplementation on leucine kinetics
Changes in plasma and urinary taurine and amino acids in runners after a
marathon
Leucine fed dose effects on muscle protein synthesis after endurance exercise
4-hydroxyisoleucine
Experimental
77
Taurine
Effects on steroidogenesis, redox and inflammatory cascades
Experimental
Cystein and cysteine
Effect on bronchoconstriction following exercise
Cystine/theanine
Gluththione
Influence of glutathione on GH
Glutathione after different intensities of resistance exercise in trained men
Glutathione suppresses muscle fatigue induced by prolonged exercise
Combined with citrulline
Effect of moderate physical activity
Experimental
Glutamate
Glutathione and glutamate
Glutamine
Hypoglutaminemia
Mechanism of action
Route of administration
Dosing
Studies in intensive care units
Glutamine, athletes, and immunity
Glutamine, muscle function, and athletic performance
Effect of free glutamine on the rate of muscle glycogen resynthesis in man
Impact of glutamine supplementation on glucose homeostasis
No effect on salivary IgA
Plasma glutamine responses to high-intensity exercise
In weightlifting performance
Experimental
Methionine
Threonine
Tryptophan
L-tryptophan
Tyrosine
Effect of tyrosine ingestion on cognitive and physical performance
Valine
CBEX
Histidine-dipeptides
Carnosine (beta-alanyl-L-histidine)
Physiological background
Carnosine washout
Trained versus non-trained muscles
Influence on exercise performance
Beta-alanine is essential for carnosine levels
Carnosinase
Combined effect of carnosine and beta-alanine
With and without sodium bicarbonate
Effect of training
Experimental
Glycine
Glycine-arginine-alpha-ketoisocaproic acid
alpha-Ketoglutarate
alpha-Ketoisocaproate
78
Prolin
Amino acids and creatine
Fat
Biochemistry
Dietary needs and food sources
Resting metabolism
Exercise metabolism
Intensity: the “Crossover Concept”
Adaptation to a fat-rich diet: effects on endurance performance in humans
Effects of varying dietary fat on performance and metabolism in runners
Effect of fat adaptation on metabolism and performance during prolonged
cycling
Fat metabolism during high-intensity exercise in endurance-trained men
Dietary fat, ergogenesis and athletic recovery
Altering fatty acid availability does not impair prolonged, continuous running
Fat as a fuel for endurance exercise
Olive oil (Monounsaturated oils)
Diacylglycerols
Structured triacylglycerols
Medium-chain triglycerides (MCT)
Carbohydrates and fat
Effect of high-fat diet on sarcoplasmic reticulum Ca2+-ATPase
Maximal lipid oxidation
In cyclists
Omega-3 docosapentaenoic and other fish oils
Overview
Legal aspects
Biosynthesis
Biological functions of docosapentaenoic acid (DPA)
Mental health and cognitive function
Food sources of DPA and current intakes
Improvement of peripheral neuromuscular function
Omega-3 fatty acid
Omega-3 fatty acid supplementation improves aspects of neuromuscular
function
Omega-3 fatty acids and skeletal muscle health
Influence of the n-3/n-6 Ratio on Metabolic Health
Eicosapentaenoic acid (EPA) versus docosahexaenoic acid (DHA)
Supplementation does not improve glycaemic control or insulin sensitivity
Reduction of protein nitrosative damage in male athletes
Contamination with persistent organic pollutants reduces antioxidant capacity
In wheelchair basketball
Differentially modulated enzymatic anti-oxidant systems in skeletal muscle cells
In elite paddlers
Experimentally
Multi-ingredient performance supplements (MIPS)
Effects of a dietary supplement on golf drive
In swimmers
NUTRITIONAL SUPPLEMENTS: CREATINE

Introduction
79
Strategies of creatine supplementation
Increased body weight after creatine loading
A wide spectrum of indications
A safe substance
Creatine as nutritional supplementation and medicinal product
Theoretical aspects
Improving energy state with training
Improving buffer capacity with training
On the strength of research on creatine
Inconsistent results
Medical use of creatine
In neurological, aging and psychiatric diseases
No effect on blood pressure
Biochemistry of creatine
Creatine kinetics in healthy men and women
Relative importance of phosphocreatine (PCr)
Biological action of creatine
Creatine supplementation increases total body water
Plasma levels after exercise
Possible multiple mechanisms for effect of creatine
Effect on myogenic satellite cells
Amounts and anatomy of creatine
Muscle glycogen accumulation
Effect on muscle ATP
Phosphocreatine (PCr) action on cell membrane structures
ATP resynthesis
An antioxidant effect
Effect on myosin heavy chain
Uptake of oral creatine
Absorption of creatine supplied as a drink, in meat or in solid form
Pre-exercise oral creatine ingestion
Urinary excetion
Metabolism of creatine
Creatine kinas
Exogenic factors’ influence on muscle creatine levels
Oral creatine supplementation
Vegetarians
Effect of carbohydrate together with creatine to increase muscle levels
Effect of endurance running on creatine levels
On dosage of creatine
Increase of muscle water of creatine
Different preparations
Hyperhydrating supplements containing creatine
Time schedule of response
Effects of creatine supplementation before or after physical performance
Effect of low-dose, short-duration creatine supplement
Individual responses
Typical dosage of creatine by athletes
On the use of creatin supplementation in sports
Use of US youths
Use of creatine in high schools
Use in women
Use by military
80
Effect of creatine supplementation on exercise performance
Overview
A meta-analysis
Effects on ability to train
Positive effects in hot environments
Creatine supplementation affects muscle creatine during energy restriction
Creatine supplementation improves sprint performance in male sprinters
Short-term effects
Long term effects
Low-dose effects
Effects of creatine supplementation on isometric force-time curve
characteristics
Creatine supplementation and muscular adaptation to resistive overload
Upper storage limit
Physiological responses to short-term exercise in the heat after creatine loading
Minimal effect on electromyographic fatigue threshold
Effects of creatine supplementation on skeletal muscle hypertrophy
Creatine supplementation alters the response to a graded cycle ergometer test
Effects of oral creatine on muscular strength and body composition
Strength loss after eccentric contractions is unaffected by creatine
supplementation
Creatine monohydrate supplementation increases satellite cell mitotic
Effect of exogenous creatine supplementation on muscle PCr metabolism
Effect of creatine loading on neuromuscular fatigue threshold
Contractile properties, fatigue and recovery are not influenced by short-term
creatine
Increased enhances oxygen uptake during alternating intensity exercise
Sprint performance enhancement after one-week creatine supplementation
Effect of oral creatine supplementation on isokinetic torque production
Creatine supplementation and upper extremity anaerobic response in females
Effect of continuous low dose creatine on force, power, and total work
Acute creatine loading enhances human growth hormone secretion
Acute creatine loading increases fat-free mass
Creatine monohydrate enhances high-intensity exercise performance
No acute effects of short-term creatine muscle properties and sprint
performance
Creatine reduces human muscle PCr and pH decrements and Pi accumulation
Protein- and carbohydrate-induced augmentation of creatine retention
No effect of heavy resistance training and creatine supplementation on blood
lipids
Decreased range of motion
Little effect on maximal strength
Effects on jumping, sprinting or cycling
Resistance training
Power performance
Effects of creatine on anaerobic exercise
Similar ergogenic effect in sprinters and long-distance runners?
Endurance
Effect of creatine loading on oxygen uptake during a 1-km cycling time trial
Role of creatine supplementation in exercise-induced muscle damage
Effect on musculotendinous stiffness and performance
Effect on human muscle protein turnover at rest
Effect of creatine on plasma levels of pro-inflammatory cytokines
81
No effect on thermoregulation and isokinetic muscular performance
Creatine supplementation and lower limb strength performance
Creatine for endurance in female soccer players
No impact on upper-body anaerobic power in trained wrestlers
Effect of pre-exercise creatine ingestion on performance in healthy aging males
Creatine deficiency syndromes
Effects in specified sports
Football
Running
Swimming
Rugby
Handball
Wrestling
Tennis
Ice-hockey
Biking
Track and field
Squash
Rowing
In high school football players
Perceived effects in select division I collegiate athletes
Use of creatine by members of civilian and military health clubs
Effect of creatine in combination with other oral substances regarding effects
Combination of creatine with glucose
Combination of creatine with protein and carbohydrate supplementation
Combination of creatine-dextrose versus protein-dextrose
Combination of creatine and whey protein
Combination of creatine and beta-alanine
Combination of creatine and carbohydrate or cinnamon
Combination of creatine and bicarbonate
Combination of creatin and magnesium
Combination of creatine with ribose and glutamine
No effect of co-ingestion of betaine with creatine
No effect of co-ingestion of D-pinitol with creatine
Combination of creatine and conjugated linoleic acid
Combined with Russian tarragon
Cognitive effects of creatine
Effect of creatine after sleep deprivation
Possible side effects (and possible counter-action of side effects of creatine)
Overview
Few side effects overall
Increased body weight
Liver
Brain
Heart
Kidney
Diarrhoea
Muscle cramping
Cytotoxicity
Children
Chronic exposition to individuals with chronic disease
Safety levels
Effects on inflammatory markers
82
Neuroprotective
Creatine decreases plasma markers of adenine nucleotide degradation
Acute creatine supplementation in older men
No positive effect of creatine on muscle wasting in cortisone treatment
Experimental
Creatine loading and depletion on rat skeletal muscle contraction
Effect of creatine supplementation on cardiac muscle in rats
Decreased plasma lipid peroxidation and enhanced anaerobic performance in
rats
Guanidinoacetic acid (GAA), a precursor of creatine
NUTRITIONAL SUPPLEMENTS: TRACE ELEMENTS, VITAMINS AND OTHER

OXIDANTS
Minerals in general
Iron
Screening
Iron metabolism
Iron concentration increases after moderate endurance exercise
Iron supplementation for marathon runners
Determination of iron status
Different types of anemia
Prevalence of iron deficiency
Dietary iron
Dietary iron treatment methods
Therapy
Iron metabolism and bioavailability
Iron deficiency
Modifications of iron homeostasis in iron deficiency
Diagnosis of iron-deficient states
Iron deficiency anemia
Iron deficiency without anemia
Iron and the female athlete
Iron status and the acute post-exercise hepcidin response
Soluble transferrin receptor and iron-related parameters
Increased values of S-ferritine
In elite sportsmen during a period of professional competition
Iron and vitamin D status of female adolescent ballet dancers
Iron status markers are only transiently affected by a football game
Depleted iron stores in gymnasts
Iron status among Japanese collegiate elite female rhythmic gymnasts
Adaptation of iron requirement to hypoxic conditions at high altitude
Iron, folate and vitamin B12 status of Ethiopian professional runners
In physically active boys
Sweat iron
With nitric oxide
Combined energy and iron deficiency
Influence of antioxidants
In basketball
In weight-training exercise
In swimmers
In cycling
Mutations in the hemochromatosis HFE gene
Experimentally
83
Boron
Calcium
Increasing dietary calcium through supplements and dairy food on body weight
Calcium supplementation during prolonged hypokinesia
Calcium and phosphate regulation in cyclists
Supplementation
Cobalt
Copper
Chromium
Use in athletes
Effects of combined carbohydrate and chromium ingestion
Magnesium
Pathophysiology
Effects of magnesium supplementation on exercise performance
Magnesium and disturbances in carbohydrate metabolism
Experimental
Phosphorous
Selenium
Reference values
Vanadium
Zinc
Immediate effects of aerobic exercise on plasma/serum zinc levels
Determination of zinc status in humans
Associations between dietary iron and zinc intakes
Zinc and Magnesium
Zinc and copper
Zinc monomethionine aspartate
Zinc and copper
Phosphate
Dietary nitrate
Metabolism of dietary nitrates
Nitrate supplementation enhances the contractile properties of skeletal muscle
Postexercise muscle metabolic recovery
Nitrate enhances skeletal muscle fatty acid oxidation
Nitrate supplementation and human exercise performance
Effect of nitrate supplementation on muscle contraction in healthy adults
Acute dietary nitrate supplementation increases maximal cycling power in
athletes
Effect on oxygen consumption during submaximal exercise in hypobaric
hypoxia
Beetroot (betaine)
Experimental
Vitamins, in general
Safety, legality and ethicality
Does a 100-km walking affect indicators of vitamin status?
Effects on hemorheological alterations
Effect on delayed onset of muscle soreness (DOMS)
Vitamin B
Choline
Vitamin D
Metabolism of vitamin D
Vitamin D and muscle tissue
Vitamin D recommendations (intake and desirable levels)
84
Vitamin D status of athletes
Vitamin D and athletic performance
In swimmers
In females
Summer versus winter
Experimental
Vitamin A
Coenzyme Q10
Folate
Vitamin K
Vitamin C
Modest hyperoxia and oral vitamin C
Increased skeletal muscle blood flow and oxygen consumption
Exercise hyperaemia and reactive hyperaemia in healthy young men
Vitamin C for preventing and treating the common cold
Prolonged vitamin C supplementation and recovery from eccentric exercise.
Lack of effects on immune responses to prolonged exercise
Lack of effects on brain antioxidants
Vitamin C supplementation and recovery from demanding exercise
Effect of vitamin C on neutrophil function after high-intensity exercise
Muscle soreness and damage parameters after vitamin C supplementation
Vitamin C supplementation following ultramarathon running
Young females in Japan
Optimal vitamin C intake for athletes
Experimental
Vitamin C and E
Influence of vitamin C and vitamin E on redox signaling
Effect on exercise-induced reactive oxygen species (ROS)
Effects on expiratory flow rates at rest and during exercise
Effects on running
Side effects
Vitamin C, vitamin E, and beta-carotene
Vitamins A, C, and E
Vitamin E
Vitamin E regulates changes in tissue antioxidants induced by fish oil
Vitamin E intake and oxidative stress in response to acute exercise in females
Vitamin E supplementation attenuates leakage of enzymes following running
Multivitamins
Dietary antoxidants, in general
Redox interventions to increase exercise performance
Effects of antioxidant supplementation on exercise performance
Antioxidant supplementation in the heat and the cold
Combined antioxidant treatment effects on blood oxidative stress
Effect on erythrocytes
Effect on lymphocytes
Effect on plasma non-esterified fat acids
Effect on oxidative stress
Lack of effects on DNA-damage
Theoretical aspects on the cellular level
Influence of training
Redox state in athletes
Delayed-onset muscle damage
Antioxidants and muscle tissue damage
85
Antioxidants and muscle disuse
Antioxidant vitamin intake and mortality
Effect on muscle fatigue
Isoflavones
After exhaustion
Effects of antioxidant therapy in women exposed to eccentric exercise
Decreased oxygen pressure and an reactive oxygen and nitrogen species
In rugby players
In football
In elite alpine ski racers
Cardiac oxidative stress
Phlebodium decamanum
Adverse effects of antioxidants
In basketball
Experimental
Reservatrol
Reservatol and quercetin
A placebo-controlled, double blind study
N-Acetylcysteine
Intravenous
Experimental
Allopurinol
In soccer
Other antioxidants
Redox individuality
In femal runners
Congnitive performance
Flavonoids (including quercetin)
Astaxanthin
In handball
alpha-Tocopherol, ascorbic acid, and beta-carotene
Catechins
In chronic obstructive pulmonary disease
Experimental
Hydroxycitrat
Applephenon
NUTRITIONAL SUPPLEMENTS: OTHER DEFINED CHEMICAL SUBSTANCES AND

METHODS
Carnitine
Primary carnitine deficiency
Hypothalamic carnitine metabolism
Choline and carnitine in women
Effect on oxidative stress
Influence of insulin
Malonyl-CoA and carnitine
Effect on post-resistance-exercise (RE)
Carnitine acetyltransferase
Vegetarians
Side effects
Experimental
86
Methylhexanamine, 1,3-dimethylamylamine (DMAA)
Phenethylamine
Phenylbutane
N-methyl-D-aspartate receptor activator (NMDA)
Gluthatione
Choline bitartrate plus acetylcholine
Chondroitin/glucosamine
Lecithin
Linoleic acid
gamma-Linolenic acid
Conjugated linoleic acid
Dietary exposure to CLA
Pharmacokinetics
Effect on body composition
Animal in vivo research
Human in vivo research
Mechanisms of action of CLA
Effect of CLA on insulin resistance
Dosage and side effects
Leptin
Inosine
Glucosamine
“Fat burners”
L-arginine alpha-ketoglutarate (AAKG)
Blend supplements
Nutritional support to maintain proper immune status during intense training
Electrolytes
Dimethylglycine
Dihdroxyacetone phosphate and pyruvate
Methylsulphonylmethane
Melamine
In powdered milk
Nootkatone
Octacosanol and policosanol
Superoxygenated water
Non-pharmacological therapy
Acupuncture
Cryostimulation
Cooling
Sublingual, ergogenic spray
NUTRITIONAL SUPPLEMENTS: FROM PLANTS AND ANIMALS

Herbs
Risk-benefit profile of commonly used herbal therapies
Herbal drinks
Ergogenic theory
Kamishimotsuto
Herbal weight loss supplements
Prebiotics
Probiotics
87
Probiotic intervention studies in athletes
Poppy seeds
Bluberries
Wolfberry (goji berry)
Grapes
Tomato juice
Accumulation of large amounst of GABA
Green tea
Green tea and memory
Efficacy of green tea extract in two exercise models
Effects of green tea on the nutritional status of the exercise
Combined with carbohydrates
Improvement of antioxidant capacity in sedentary men
Epigallocatechin-3-gallate in high-fat-fed mice
Effect on exercise-induced oxidative stress parameters in male sprinters
Decaffeinated green tea
In hypertensive women
Black tea
Gluten
Ginger
Ginseng
Use in athletes
Effects of ginseng supplementation on supramaximal exercise performance
Siberian ginseng (Eleutherococcus senticosus)
American ginseng
Ginkgo biloba
Garlic
Macroalgae
Spirulina (microalgae)
Phosphatidylserine
Quercetin
Overview
Vitamin D and quercetin, alone and in combination
Soldiers
Mice
Experimental
Capsaicin
Curcumin
Experimental
Pineapple (bromelain)
Spinach
Pomegranate
Pheromones
Saffron
Delayed-onset muscle soreness
Pycnogenol
Kava kava (Kava)
St. Johns wort
Yucca
Teribulus terrestris
Inadvertent doping
Arnica
Rhodiola rosea
88
Lack of effect on marathon running
Cordyceps sinensis and Rhodiola rosea
Cissus quadrangularis
Withania somnifera
Hydroxycut
Echinacea
Angelica sinensis
Cordyceps sinensis
Cordyceps sinensis and Rhodiola rosea
Schisandra chinensis
Experimental
Eurycoma longifolia Jack
Citrus aurantium
Ghavoot
Cytoseira canariensis
Smilax (sarsaparilla)
Yerba maté
Yohimbine
Bee pollen
Honey
Royal Jelly
Phlogenzym and wobenzym
Cytochrome C
Glandulars
Lactobacillus casei Shirota
Colostrum
Effect on buffer capacity
Effect of the immunoglobulins
Effect on salivary IgA
In cycling
Fermented papaya
Ultrasonography-guided interventions
GENE DOPING
Overviews
The “natural gene selection”
Critique of the categorical preference for natural talent over doping
Is gene doping already a reality?
The science behind gene doping
Definitions and history
Gene transfer approach to therapy
Therapeutic gene transfer vectors introduced into human beings
Reversing the effects of transplanted genes
Methods of gene delivery
Medical uses of gene therapy
Is gene doping already a reality?
Animal use of gene doping
Vectors
Genes versus environmental influence
Gene therapy versus gene doping
Epigenetics
Evolving definitions
89
RNA interference
Genetic enhancement
In vivo gene doping
Ex vivo gene doping
Possibilities for gene doping
Gene-based doping for muscle function
Gene-based doping for oxygen delivery to exercising tissues
Small interfering RNA (siRNA)
Further possibilities of detecting gene doping
Doping targets
Glucose metabolism
Red blood cell activity/delivery
Skeletal muscle size, strength and endurance
Mechano-growth factor (MGF)
Vascular endothelial growth factor
Fibroblast growth factor
Preventing pain (endorphin and enkephalin)
alpha-Actinin 3
Peroxisome proliferator-activated receptor-delta
Cytosolic phosphoenolpyruvate carboxykinase
Gene doping with intracellular molecules
Options for gene doping
Gene doping targets
Genetic manipulation enhancers
Policy options for genetic enhancement
Candidate genes for athletic gene doping
Hematopoietic/vascular systems
Hypoxia inducible factors
Vascular endothelial growth factor
Actin-binding peptides
Angiotensin-converting enzyme
Insulin-like growth factor
Myostatin
Peroxisome proliferator-activated receptor delta
Endorphins
Erytropoietin gene transfer
Polymorphism
Sport as a case study
Genetic test available for sports performance
Risks of using research for modifying athletes genes
Risks and complications of gene doping
Side effects
Gene silencing
Immune reaction
Integration
Infection of germ cells
Expression
Storage and usage
Long term
Uses of gene therapy
Experimental
90
Potential strategies for detection of gene doping
Detection of viral vectors and monitoring of the host immune response
Methods of gene and proteomic profiling
Can gene doping be detected?
Detection of erythropoietin gene doping
Muscle biopsy
Blood monitoring
Genetic activity tests
Gene profiling
Proteomic profiling
Genetic barcodes
Laboratory techniques for detection of gene doping
Direct methods
Indirect methods
Transgene and nonviral vectors in blood
PCR-based detection of gene transfer vectors
Regulation of gene doping
The ethics of gene doping
The other side of the coin
Bioethical concerns
Definition of enhancement
Genetic enhancement
The thin line between therapy and enhancement
Specific ethical questions
ETHIC ISSUES IN DOPING AND ANTI-DOPING

The aim of sports is victory
Young and immortal
The history of anti-doping ethics
Mid-to-late nineteenth century
Early sports medicine
Enhancing performance
United States Senate committee in 1973
War on doping versus war on drug
Why the war on drugs in sport will never be won
Doping is here to stay
“Everybody take doping drugs”
Arguments for doping and their etical anti-arguments
Human nature
Performance enhancing drugs are not inherently immoral
Regulation could improve safety
The role of rules
Spirit of sport
Argument against doping in sports
Escalating problems
Engineered athletes
Bans can work
Condemned to cheating?
Beta-blockers to musicians
Unfair?
Just for the rich?
Unsafe?
Children
91
Climate of cheating
Prohibition
The problem of strict liability
A health hazard
Test for health, not drugs
Anti-doping agencies suffer from a sort of institutionalized blindness
Alternatives to current anti-doping strategy
A continuing discussion is needed
The reasons for anti-doping rules
The adverse effects of elite competition on health and well-being
A victim of the rules?
The strict liability principle
Arguments for anti-doping
Sports as part of the society
Stakeholder-corporate social responsibility approach to drug control for sport
The unique ethics of sports medicine
Doping as a sign of dehumanization
Making sport possible
Technology introduced but changing the sport
Increased participation and spectatorship
Sponsoring of nutritional supplements and sports drinks
Alternate conceptualizations
Ethical principles
Autonomy
Beneficence
Non-maleficence
Justice
Ethical aspects of “harm” in sports
Harm to the athlete
Harm to other athletes
Harm to society
Ethics of the athletes regarding doping
Athletes’ responsibility to make ethical decisions
Doping as an individual moral fault
Athletes should be involved
Sports Federations’ attitudes
Litigation in sports medicine
Legal and ethical issues in the cardiovascular care of elite athletes
Physician’s role in doping
The challenge of working in sports medicine
Sports doctors and exercise scientists have to take responsibility
Environmental factors
Case reports of the doctor’s fault but athlete’s verdict
Rumanian gymnast
Serbian handball player
Russian basketball player
French basketball player
Spanish basketball player
Regulations
Aims of the regulations
Legislative structures on enhancement
Courtroom medicine
Evidence-based doping?
92
Medically supervised doping
The temptation by human growth hormone
Motherhood goes with gold?
Legitamcy of ban on cannabis and cocaine
The complexity of anti-doping
The case of modafinil
The ethics of doping
Education in moral judgment of participants in team sports
Professionalism and the ethics of the sports physician
Conflicts of interest: the need to minimize
Need to recognize the limits of athlete’s autonomy
The need to maintain informed consent
Confidality in doping
Patient confidality
Fairness in sports
Aspects of hormone abuse in sport
Controversies regarding endogenously produced hormones
Women with hyperandrogenism in elite sports
Women athletes with testes
Critisism of the fairness of the testing procedure
The WADA mission
Sport and the history of ethics in sports medicine
Multiple obligations
Attitudes on anti-doping
Hypotheses on background of doping
Youth and adolescents
Statement by the American Academy of Pediatrics
Environmental factors
Anti-doping programes
The life-cycle model of performance enhancement
A gold medal but also death in 5 years’ time?
Prevention of athletes from harm
Harm reduction for anabolic steroid users
On cheating
Different shades of blood doping
The grey zone of undiscovered doped
Enforcement of anti-doping policy today
Smart drugs (“brain doping”)
Cognitive enhancement among healthy subjects
Non-pharmacological cognitive training
Conceptual issues concerning neuroenhancement
Prevalence
Moral issues concerning neuroenhancement
Swiss university students' attitudes toward pharmacological cognitive
enhancement
University students' attitudes toward pharmacological cognitive enhancement
Legal issues
Performance-enhancing drugs create an uneven playing field
Everybody else is taking them
Performance-enhancing drugs are dangerous
Drug use would be almost impossible to control
Danish ban on gyms not adhering to doping-tests
Cannabis
93
Cannabis and exercise science
Connections between anabolic steroid and heroin use
Gender as more than a binary quantity
On how to make illegal drugs less dangerous
Hidden assumptions and inherent contradictions in anti-doping policy
Setting a good example
A level playing field
Protecting the health of athltese
Preserving the integrity of sport
Policy implications
Problems in research on doping and doping substances
Athletes as guinea pigs?
Visibility
Multiplicity
Consistency
Human guinea pigs in the pharmaceutical industry
Vulnerability
The need for research on enhancements
Pediatric exercise science
Conclusions of an ethic discussion on research on performing-enhancing drugs
Randomized trials
WADA’s statement
Ethical codes
A code of ethics with a foundation in evidence
The complex environment of elite sport
Multiple obligations
Sharing personal information about athletes with others
Risk taking by athletes
Aims for a new code of ethics
Practicalities and negotiating the politics
Scientific cooperation
The formal ethics of research with human subjects
The ethical principles used by review bodies
Further amendments
Sport as an occupation
Medical use of banned drugs
Concerns regarding nutritional supplements and sports drinks
Is it justifed to restrict sponsorship?
An alternative approach to the ethics of supplement and drink company
sponsorship
Australian, Candian and Irish issues
The rights and wrongs of supplement and sports drinks sponsorship in sport
Legislation of drugs aimed for doping
“Naturalness” in sports
An example for the society
Law versus ethics
International doping policy: the WADA and the WADC
Public law versus sport’s law
Guilt, negligence and liability
Privacy
Physician’s responsibility
Sports doctors’ responsibilities
Self-reporting on doping
94
Doping during the Olympics
Statistics published
Hypoxic tents
Pain medication
Injections overall
Injection therapy with local anaestetics
Injection therapy with local corticosteroids
Concluding remarks
The ethics of blood tests
The ethics of not testing for blood doping in athletic competition
Athletes’ safety first
Are we ensuring safety at present?
Safety procedures have already been successfully tested
Screening of athletes for possible diseases
Ethics of preparticipation cardiovascular screening for athletes
Ethical issues raised by epigenetic testing
Social media use in sports and exercise medicine
Publication ethics
VETERINARY
Overview
Horse urine sample preparation methods for metabolomics
Pharmacokinetic and pharmacodynamic approach in the horse
Bacterial hydrolysis of urine without doping substances
Contaminated food for animals
Screening
Anabolic steroids, general
Anabolic hormones used for improvement of meat production
Anabolic steroids in dogs
Anabolic-androgenic steroids naturally present in urine of untreated horses
Elevated testosterone levels due to an XY testicular disorder of sexual
development
Effects of implant strategy on finished body weight of beef cattle
Anabolic steroids in urine
In hair
Differentiation between endogenous steroids and synthetic homologues in
cattle
Homeostatic signature of anabolic steroids in cattle
Testosterone
Testosterone and nandrolone
Testosterone esters and boldenone undecylenate in bovine hair
19-nortestosterone
Nandrolone
Recovery in horses after strenuous physical exercise
19-Norandrostenedione and 19-norandrostenediol
Norethanderolone
1-Testosterone
Stanozolol
In cats
In dogs
Methyltestosterone and mestranolone
Mesterolone
Methandienone (Dianabol)
95
Boldione, boldenone and boldenone esters
Histopathology
Suitability of bovine bile compared to urine
Trebolone
Trenbolone and zeranol residues in cattle muscle and liver
Trenbolone acetate, zeranol and melengestrol acetate
Trebolone and estrogens
In cattle
In steers
In lamb
Dehydroepiandrosterone versus androstenedione
Methandrostenolone
Fluoxymesterone
19-norchlorotestosterone acetate metabolites in cattle urine
17beta-19-nortestosterone in swine
17alpha-ethinylestradiol residue in the hair of cattle
Androsta-1,4,6-triene-3,17-dione (ATD)
Oxyguno
Selective androgen receptor modulators (SARMs)
Steroidal aromatase inhibitors
17beta-estradiol administration to cattle
17beta-estradiol in veal calves
Medroxyprogesterone acetate in pork
Melengestrol acetate (MGA)
Phytosterols
THG
Regucalcin (RGN)
Regucalcin regulation by sex steroid hormones in bovine tissues
Growth hormone
IGF-1
Erythropoietin
Horse
Dog
Darbepoetin
Insulin
Seven peptide hormones
Relaxine
Thyroid hormones
Caffeine
Diphenhydramine
Corticosteroids
Hydrocortisone
Topical glucocorticoids
Betamethasone and dexamethasone
Prednisolone
Triamcinolone
beta2-Agonists
Salbutamol
Clenbuterol and hydrocortisone
Clenbuterol
In meat
Clenbuterol and furosemide
96
Efaproxiral
Filgrastim
Cathinone
Theobromine
Furosemid
Flurbiprofen
Creatine
Branched-chain amino acid and alanine
Somatotropin
Quaternary ammonium drugs
Iron
Bicarbonate
Carnosine
Carnitine
Vitamin C
Tryptophane
Tricyclic antidepressant
Doxepin
Flurbiprofen
Phenylbutazone
Other NSAIDs
Salicylic acid
Bupivacain
Levamisole
3,4-Methylenedioxypyrovalerone (MDPV)
Glycopyrrolate
Dermorphine
Morphine
Alimentary intake of opioid alkaloids by horses
Interleukin-1 receptor antagonist peptide
Biphosphonates
Angiotensin-converting enzyme inhibitors
beta-Hydroxy-beta-methylbutyrate (HMB)
Cobalt
Capsaicin
Acepromazine
Antioxidants
Vitamin E
Alpha-cobratoxin
Piroxicam
Dopamine
Isoxsuprine
Fish oil
GnHR vaccines
Quaternary ammonium drugs
Ethanol
Rhabdomyolysis
Direct-injection differential-gradient LC-LC coupled to hybrid tandem MS/MS
Gas chromatography-mass spectrometry (GC-MS)
AR-LUX
Stable carbon isotope analysis
97
Solid-phase extraction and liquid chromatography-mass spectrometry
Liquid chromatography – Orbitrap mass spectrometry
Proteomics et al
In hair
Identifying individual horses urine by single-nucleotide polymorphisms (SNPs)
TB-500
Primary hepatocytes as a bioassay
Molecularly imprinted polymer applied to the selective isolation of urinary
steroids
Single-nucleotide polymorphism assay
Feeding effect on the 13C/12C isotope ratio of the hormones in bovine urine
Orbitrap
Protein biomarkers
Other apects on forensic medicine
Horses (in general)
Drug metabolism in horses
Report from Iran
Live stock
Intersex conditions
FUTURUM
Upcoming ethical dilemmas
The ABP and confounding factors
Data management: Anti-Doping Administration and Management System
Education
Inadvertent doping
Need for further research
Criminals
Economy and techniques
What if current zero-tolerance anti-doping policy continues?
Social, policy, and public health perspectives on new psychoactive substances
Technology advancement
More complex challenges
Future applications of anabolic steroids
Doping drug candidates
Synthol
Tolperisone
5-hydroxytryptamine (5-HT) agonist
AICAR and peroxisome proliferator-activated receptor delta (PPAR-delta)
Prediction of futures anabolic androgenic steroids
A scientific based statement on the use of performing enhancing drugs
REFERENCES
98
ABBREVIATION
AA anabolic agents
AA amino acid
AA adrenergic agonists
AA ascorbic acid
AA arachidonic acid
AAA aromatic amino acid
AAA American Arbitration Association
AAAAI American Academy of Allergy, Asthma and Immunology
A-AC androsterone acetate
AACE American Association of Clinical Endocrinologists
AAF adverse analytical findings
AAKG L-arginine alpha-ketoglutarate
AAM acrylamide
AAP American Academy of Pediatrics
AAPCC American Association of Poison Control Centers
AAS anabolic androgenic steroids
AASU anabolic steroids users
AAT alanine aminotransferase
AAV adeno-associated virus
AAW African-American women
AB able-bodied
ABAT alanine transaminase
ABB affinity-based biosensors
ABCD Autoridade Brasileira de Controle de Dopagem
ABI autologous blood injection
ABI addiction to body image
aBMD areal bone mineral density
ABP Athlete Biological Passport
ABP arterial blood pressure
ABPS Abnormal Blood Profile Score
ABS anabolic steroids
ABS affinity-based biosensors
ABT autologous blood transfusion
AC analytical column
ACAAI American College of Allergy, Asthma and Immunology
ACC acetyl-CoA carboxylase
ACC anterior cingulate cortex
ACE angiotensin-converting enzyme
ACEA angiotensin-converting enzyme activity
ACEI angiotensin-converting enzyme inhibitor
ACh acetylcholine
ACI anabolic/catabolic index
ACL anterior cruciate ligament
ACP acepromazine
ACRL acute creatine loadings
ACS acute coronary syndrome
ACS autologous conditioned serum
ACSM American College of Sport Medicine
ACSP Australasian College of Sports Physicians
ACTH adrenocorticotropic hormone
ACTN actinin
99
ACTN3 alpha-actinin-3
Act R activin receptors
AD autonomic dysreflexia
AD androstenedione
AD aortic diameter
Ad adrenaline
ADA American Dietetic Association
ADA adenosine deaminase
ADA American Diabetes Association
ADAM Androgen deficiency in Aging Male (questionnaire)
ADAMS Anti‐Doping And Management System
ADD androstenedione, boldione (androst-1,4-diene-3,17-dione)
ADD Danish Anti-Doping Agency
ADD average danish diet
ADH antidiuretic hormone
ADHD attention deficit and hyperactivity disorder
ADHJD attention-deficit hyperactivity disorder
ADI acceptable daily intake
ADIOL androstenediol
A’diol androstenediol
ADION androsta-1,4-dien-3,17-dione
A’dione androstenedione
ADMA-DDAH-NOS ADMA-asymmetrical dimethylarginine
ADME absorption, distribution, metabolism, and elimination
ADO antidoping organisation
ADP adenosine diphosphate
ADR adverse reaction
ADR androsterone
ADRV antidoping rule violation
ADT androgen-deprivation therapy
ADT androsterone
AE aerobic endurance
A/E androsterone to etiocholanolone
AEA anandamide
AEC adenylate enrgy charge
AED androstenedione , 4-androstene-3,17-dione
AED alcohol energy drinks
AEMD atrial electromechanical delay
2-AEPB 2-amino-N-ethyl-1-phenylbutane
AESE acute exhaustive swimming exercise
AET androst-5-ene-3beta,7 beta,17beta-triol
AeT aerobic threshold
AEXS aromatase excess syndrome
AF atrial fibrillation
AF activation function
AFBW adjusted final body weight
AFL Australian Football League
AFM atomic force microscopic images
AFM abdominal fat mass
AG androsterone glucuronide
AG American ginseng
A/G albumin to globulin ratio
Ag silver
100
AGA androgenetic alopecia
AGAT arginine-glycine amindinotransferase
AGHD adult growth hormone deficiency
AGHDA assessment of GH deficiency in adults
AGI aminoglutethimide
AGRP agouti-related protein
AH anterior hypothalamus
AHA automated hematology analyser
AH-AVP anterior hypothalamic-arginine vasopressin
AHF antihemophilic factor
AHR airways hyperresponsiveness
AhR aryl hydrocarbon receptor
aHR adjusted hazard ratio
AHREP acute heavy resistance exercise protocol
AHRET acute heavy resistance exercise test
AI aromatase inhibitor
AIAC acid-insoluble acylcarnitine
AICAR 5-amino-4-imidazolecarboxyamide ribonucleoside
AIDS acquired immunodeficiency syndrome
AIS Australian Institute of Sport
AKG alpha-ketoglutarate
AKI acute kidney injury
AKI acute kidney impairment
AKR aldo-keto reductase
AL albumin
ALA alanine
ALA alpha-linolenic acid
Ala alanin
Ala alpha lipoic acid
ALAT alanine transaminase
ALB albumin
ALLO allopregnanolone
ALP alkaline phosphatase
ALS acid labile subunit
ALT alanine aminotransferase
ALT altitude
AMA Australian Medical Association
AMA arm muscle area
AMDIS automated mass spectral deconvolution and identification system
AMH anti-Müllerian Hormone
AMP amphetamine
AMP adenosine 5′-monophosphate
AMPH amphetamine
AMPK 5'adenosine monophosphate-activated protein kinase
AMS ambient mass spectrometry
aMT6s 6-sulphatoxymelatonin
AN anorexia nervosa
ANCOVA analysis of covariance
ANAD A-nor-5alpha-androstane-2,17-dione
AND Academy of Nutrition and Dietetics
Andro androsterone
ANG angiotensin
ANOVA analysis of variance
101
ANP atrial natriuretic peptide
ANR androgen receptor
ANS autonomic nervous system
AnT anaerobic threshold
AO antioxidants
AOC artificial oxygen carriers
AOC acute oral antioxidant
AOFAS American Orthopedic Foot and Ankle Society
AOGHD adult-onset growth hormon deficiency
AOPP advanced oxidation protein products
AOR adjusted odds ratio
aOR adjusted odds ratios
AORC Association of Official Racing Chemists
AOX antioxidants
AP average power
AP amphetamine
AP anabolic products
APB 6-(2-aminopropyl)benzofuran
APB 2-amino-1-phenylbutane
APCI atmospheric pressure chemical ionization
APD action potential duration
APED appearance- and performance-enhancing drug
APFT Army Physical Fitness Test
APICA N-(1-adamantyl)-1-pentyl-1H-indole-3-carboxamide
APMU athlete passport management unit
APO apomorphine
APO antioxidant status
Apo apolipoprotein
ApoA1 apolipoprotein A1
Apo B apolipoprotein B
ApoE apolipoprotein E
APPI atmospheric pressure photoionization mode
aPTT activated partial thromboplastin time
Aqp Aquaporin
AQP5 aquaporin-5
AQUA Allergy Questionnaire for Athletes
AR androgen receptor
AR adrenergic receptor
5AR 5alpha-reductase
ArAA aromatic amino acids
ARB angiotensin receptor blocker
ARC arcuate nucleus
ARCI Association of Racing Commissioners International
ARCI Addiction Research Centre Inventory
ARDS adult respiratory distress syndrome
ARE androgen responsive element
ARF acute renal failure
ARG arginine
AR-ir androgen-receptor immunoreactivity
ArKO aromatase knockout mice
ART androgen replacement therapy
AS average speed
AS anabolic steroids
102
AS antioxidant supplementation
AS affinity tests
AS Angelica sinensis
ASAC acid-soluble acylcarnitine
ASADA Australian Sports Anti-Doping Authority
ASAP Atmospheric Solids Analysis Probe
ASAT aspartate transaminase
ASC colloid acetyl starch
ASCA Anabolic Steroid Control Act
ASCM American College of Sports Medicine
ASE accelerated solvent extraction
ASIH anabolic steroid-induced hypogonadism
ASP athlete steroidal passport
ASP athlete support personnel
AST aspartate aminotransferase
AST astaxanthin
AT angiotensin
AT anaerobic threshold
ATIII antithrombin III
ATD 1,4,6-androstatriene-3,17-dione
ATHENA Athletes Targeting Healthy Exercise and Nutrition Alternatives
ATF atypical findings
ATG autophagy-related protein
ATP adenosine triphosphate
ATPF atypical passport finding
ATP-PC adenosine triphosphate-phosphocreatine
ATP-PCr adenosine triphosphate-phosphocreatine
ATPS aqueous two-phase systems
ATR attenuated total reflection
AT1-R angiotensin II receptor
ATR-IR attenuated total reflectance-infrared
ATS amphetamine-type psychostimulants
ATS American Thoracic Society
AT-test agility T-test
aTUE abbreviated TUE process
ATUE abbreviated Therapeutic Use Exemption
AU animal units
Au gold
AUC area under the curve
AUDIT Alcohol Use Disorders Identification Test
AUDIT-C Alcohol Use Disorders Identification Test-alcohol consumption
AuNP gold nanoparticle
AUR acute urinary retention
AUC area under the curve
AuNP gold nanoparticle
AUS abdominal ultrasound
AV volume the aorta peak flow
AVF abdominal visceral fat
AVP arginine vasopressin
AVS antioxidant vitamin supplementation
AWB autologous whole blood
AWS alcohol withdrawal symptoms
AWT Anaerobic Wingate Test
103
B1 thiamin
B2 riboflavin
BA beta-alanine
BAC bacterial artificial chromosome
BAI beta-alanine
B-ALA beta-alanine
BALCO Bay Area Laboratory Co-operative
BALP bone alkaline phosphatase
BALT bronchus associated lymphoid tissue
BAmyE Bar adsorptive microextraction
BAP biological antioxidative potential
BAT brown adipose tissue
BB body builders
BBB blood-brain barrier
BC bovine colostrum
BC body composition
BCAA branched-chain amino acids
BCKD branched-chain ketoacid dehydrogenase
BCKDH branched-chain alpha-keto acid dehydrogenase
BCL-6 B-cell lymphoma 6 protein
BCO blood collection officers
BD 1,4-butanediol
1,4-BD 1,4-butanediol
BDD body dysmorphic disorders
BDHI Buss-Durkee Hostility Inventory
BDI Beck Depression Inventory
BDNF brain-derived neurotrophic factor
BE base excess
BEAST Ball-Sport Endurance and Speed Test
BECG benzoylecgonine
BES benzoate
BET betamethasone
17beta-NT 17beta-19-nortestosterone
BF blood flow
%BF percent body fat
B-FGF basic fibroblast growth factor
bFGF basic fibroblast growth factor
BFR blood flow restriction
BFU burst colony forming units
BFU-E burst forming unit‐erythroid
BG beta-glucuronidase
BGE background electrolyte
BGH bioassayable growth hormone
BH bronchial hyperreactivity
BHB beta-hydroxybutyrate
bHb bovine hemoglobin
BHI beverage hydration index
BHK baby hamster kidney
BHP benign prostatic hyperplasia
BHR bronchial hyperresponsiveness
BIA bioelectrical impedance
BIAT Brief Implicit Association Test
BICA bicalutamide
104
BICA bicarbonate
BIL-D direct bilirubin
BIL-T total bilirubin
BioMS bioaffinity liquid chromatography-mass spectrometry
BJ90 maximal 90-s box jump test
BJJ Brazilian Jiu-jitsu
BJR Bezold-Jarisch reflex
Bk-MDMA methylone
BL baseline
BL bioluminescent
BLA basolateral complex of the amygdala
BLa blood lactate
BLC blood lactate concentrations
BM body mass
BMC bone mineral content
BMD bone mineral density
BMI body mass index
BML body mass loss
BMC bone mineral content
BMD bone mineral density
BMP bone morphogenetic protein
BMPEA beta-methylphenethylamine
BMSC bone marrow stem cells
BMX bicycle moto cross
BNP brain natriuretic peptide
BNST bed nucleus of the stria terminalis
BOL boldenone (17-hydroxy-androsta-1,4-diene-3-one)
Bol boldenone
BOLD boldenone
BP bench press
BP blood pressure
BP binding protein
BPA bisphenol A
BPH benign prostatic hyperplasia ¨
BPM lifts on bench maximum
Bpm beats per minute
bPR bovine uterine progestin receptor
BRIC benign recurrent intrahepatic cholestasis
BPT bromopentane
BR bronchial reactivity
BR bed rest
BR beetroot
BrdU 5-bromo-2'-deoxyuridine
BRJ beetroot juice
BRS baroreflex sensitivity
BRUMS Brunel Mood State Inventory
BS best-sprint
BS breeding stallions
BS box squat
BSA body surface area
BSA bovine serum albumin
BSC bovine satellite cell
BSE bovine spongiform encephalopathy
105
BSTFA N,O-bis(trimethylsilyl)trifluoroacetamide
BSTPM posteromedial bed nucleus of the stria terminalis
BT bench throw
BT bioavailable testosterone
BTHC n-butyryl-tri-(n-hexyl)-citrate
BTM betamethasone
BTP Bruce Treadmill Protocol
BUD budesonide
BUF bufotenine
BUN blood urea nitrogen
BV blood volume
BW body weight
BWPLV bioenhanced whey protein
BWS best-worst scaling
bwt body weight
BZ benzodiazepine
BZA bazedoxifene
C cortisol
CA cysteamine
CA catecholamine
CA caffeic acid
CA concentration addition ¨
Ca calcium
CAC consecutive accelerations
CaCl2 calcium chloride
CAD coronary artery disease
CAD collisionally activated dissociation
CAF caffeine
CAF capillaries per fiber
CAFF caffeine
CAH congenital adrenal hyperplasia
CaHMB calcium beta-hydroxy-beta-methylbutyrate
CAL calcium dairy based meal
CAM complementary and alternative medicine
CaMK calmodulin-dependent kinase
cAMP cyclic adenosine monophosphate
CAN acrylonitrile
CAN Center for Alcohol and Narcotic Information
CANTAB Cambridge Neuropsychological Test Automated Battery
CAP capsaicin
CAR central activation ratio
CAR carnitine
CAR carnosine
Carboxy-THC 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid
CARNS carnosine synthase
CAS Court of Arbitration for Sport
CAT catalase
CAT coincidence anticipation timing
CAT Colorado Altitude Training
CATH cathine
Cave time-average concentration
CB cannabinoid
CB caffeinated beverages
106
CBAT calculated bioavailable testosterone
CBC complete blood count
CBD cannabidiol
CBEX chicken breast meat extract
CBG corticosteroid-binding globulin
Cbg cannabigerol
CBM China BioMedicine
CBN cannabinol
CB1R cannabinoid receptors 1
CBT cognitive behavioral therapy
Cbtx cobratoxin
CC caffeinated carbohydrate (gel)
CC16 Clara Cell protein 16
CCalpha decision limit
CCB dihydropyridine calcium channel blockers
CCbeta detection capability
CCDC cerebral creatine deficiency syndromes
CCM curcumin
CCP cornu cervi pantotrichum
cCr cyclocreatine
CCS corticosteroids
CCS collision cross section
CCT cycle capacity test
CD cyclodextrin
CD circular dichroism
C4D capacitively coupled contactless conductivity detection
CDC Centers for Disease Control and Prevention
CDGP constitutional delay of growth and puberty
CDK chronic kidney disease
CDmax maximum amount of conjugated dienes
cDNA complementary DNA
cDNA circulating DNA
CDT carbohydrate deficient transferrin
CE capillary electrophoresis
CE cognitive enhancement
CE conjugated estrogens
CE cycle ergometer
CE-AD capillary electrophoresis-amperometric detection
CEDIA cloned enzyme donor immunoassays
CEE creatine ethyl esther
CE-ECL capillary electrophoresis coupled with electrochemiluminescence
detection
CEL celiprolol
CE-MS capillary electrophoresis-mass spectrometry
CE-MS/MS capillary electrophoresis tandem mass spectrometry
CENTRAL Cochrane Central Register of Controlled Trials
CERA continuous erythropoietin receptor activator
CES carbohydrate-electrolyte solutions
CES-D Center for Epidemiological Studies Depression Scale
CET constant-load exercise test
CET competition exercise test
CE-TOF/MS capillary electrophoresis time-of-flight mass spectrometry
CETP cholesteryl ester transfer protein
107
CEU contrast-enhanced ultrasound
CF cognitive function
CF cardiorespiratory fitness
CF cystic fibrosis
cf core fragment
CFA complete Freund's adjuvant
CFA confirmatory factor analysis
CFTR cystic fibrosis transmembrane conductance regulator
CFIRMS continous-flow isotope ratio mass spectrometry
CFU colony-forming units
CFU-E colony forming unit‐erythroid
CG chromatography
CG chorionic gonadotropin
CG collapsing glomerulopathy
C6G codeine-6-glucuronide
cGH canine growth hormone
cGMP cyclic guanosine monophosphate
CGRP calcitonin-gene-related peptide
CHCA cyano-4-hydroxycinnamic acid
CHD coronary heart disease
CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate
ChAT choline acetyltransferase
CHE cholinesterase activity
CHEM chemiluminescent immunoassay
CHF chronic heart failure
CHF congestive heart failure
CHO Chinese hamster ovary
CHO carbohydrate
CHO-E carbohydrate-electrolyte
CHO-P carbohydrate-protein
CHOPA carbohydrate-protein-antioxidant beverage
CHOS carbohydrate supplementation
CHT oxidative capacity
CI confidence interval
CI chemical ionization
CI calcium ionophore
CI50 concentration causing 50 percent inhibition
CIC ciclesonide
CID collision-induced dissociation
cIEF capillary isoelectric focusing
CIN cinnamon
CINC cytokine-induced neutrophil chemoattractant
CIR carbon isotope ratio
CIR circuit training
CIR circumference
CIT sodium citrate
Cit citrulline
CIR carbon isotope ratio
CK creatine kinase
CK-BB brain creatine kinase
CKD chronic kidney disease
CK-M creatine kinase M-type
CK-MB myocardial creatine kinas
108
CK-MM muscle creatine kinas
CL chemiluminescent
CL confidence limit
CLA conjugated linoleic acid
CLAD chloroandrosterone
CLB clenbuterol
CLE clenbuterol
CLEN clenbuterol
CLT 90-s cycling bout at 110 percent VO2max
CMBCD carboxymethyl-beta-cyclodextrin
CM creatine monohydrate
CM chocolate milk
Cmax maximum concentration
Cmin minimum concentration
CMJ counter-movement jumping
CMJT counter-movement jump test
CML chronic myelogenous leukemia
CMO chief medical officer
CMR carbohydrate mouth rinse
CMS carboxymethyl starch
CMT Charcot-Marie-Toot
CMV cytomegalovirus
CN creatine nitrate
CNB caloric nutritional beverages
CNDP carnosinase
Cne cholestane
CNKI China National Knowledge Infrastructure
CNS central nervous system
CNTF ciliary neurotrophic factor
CO cardiac output
CO carbon monoxide
Co cobalt
COC cocaine
COCET cocaethylene
COD codeine
COF coffee
COL cutoff-line
COL colostrum
COLL collagen
COMT cathecol-O-methyltransferase
CONC concentric
CON control
CONT control
COP colloid osmotic pressure
COPD chronic obstructive pulmonary disease
CoQ coenzyme Q10
CoQ10 co-enzyme Q10
COR cortisol
CORT cortisol
COX cyclooxygenase
COX-2 selective cyclooxygenase-2
COX4 marker cytochrome c oxidase subunit IV
109
CP creatine phosphate
CP carbonylated proteins
CPC colostrum protein concentrate
CPD continuing professional development
CPET cardiopulmonary exercise test
CPK creatine phosphokinase
CPP conditioned place preference
cps counts per second
CPX cardiopulmonary exercise test
CQR Cissus quadrangularis
cQT corrected QT (interval)
CR creatine
CR caloric restriction
Cr creatine
Cr creatinine
CRAT chronic recalcitrant achilles tendinopathies
CrAT carnitine acetyltransferase
CRE creatinine
CRE cyclic AMP response element
CRF corticotrophin releasing factor
CRF-R corticotropin releasing receptor
CRH corticotropin-releasing hormone
CRM certified reference materials
CrM creatine monohydrate
CRM certified reference materials
CRP C-reactive protein
CRT choice reaction time
CRTR creatine transporter
CrS creatine supplementation
CS citrate synthase
CS cage-switching stress
CS creatine supplementation
CS corticosteroid
CS citrate synthase
CSA cross-sectional area
CSF cerebrospinal fluid
CSIA compound-specific isotope analysis
CSR corporate social responsibility
CT computed tomography
CT calcaneal tendon
CT cognitive training
CT concurrent training
CTAB cetyl trimethyl ammonium bromide
cTHC tetrahydrocannabinol-9-carboxylic acid
CTL controls
CTN caffeine-taurine-niacin
CTRL control subjects
CTX C-terminal telopeptide of type-I collagen
Cu copper
Cu/Zn-SOD copper/zinc superoxide dismutase
CV coefficient of variance
CV cardiovascular
CVD cardiovascular disease
110
CVF collagen volumetric fraction
CVP central venous
CVST cerebral venous sinus thrombosis
CVT cerebral venous thrombosis
CW Caucasian women
CWI cold-water immersion
CYP cytochrome P
CZE capillary zone electrophoresis
CZE-UV capillary zone electrophoresis-UV absorbance detection
D deletion
DA dopamine
DA danazol
Da Dalton
dACC dorsal anterior cingulate cortex
DAD diode-array detector
DAG diacylglycerol
DALDA Daily Analysis of Life Demands for Athletes
DAM N,N-dimethylamphetamine
D-AMPH d-amphetamine
DART direct analysis in real time
DAT drug and alcohol testing
DAT dopamine transporter
DBD DNA binding domain
DBD dielectric barrier discharge
DBP diastolic blood pressure
DBS dried blood spots
DC dendritic cells
dcELISA direct competitive enzyme-linked immunoabsorbent assay
DCO doping control officers
DCS doping control stations
DCT direction cue task
DDAH dimethyl-arginine dimethylaminohydrolase
DDAVP desmopressin
ddPCR digital droplet PCR
DDR Deutsche Demokratische Republik
D1DR D1 dopamine receptors
DE differentially expressed
DEA (US) Drug Enforcement Administration
DECA decanoate
DECAF decaffeinated coffee
DFC drug facilitated crimes
DEH dehydration
DEHB di-2-ethyl hexyl phthalate
DEHP di-(2-ethylhexyl)phthalate
DELFIA dissociation enhanced lanthanide fluorescence immunoassay
DEM diethyl maleate
DES diethylstilbestrol
DES dietary energy supplements
DESA Diabetes Exercise and Sports Association
DESI desorption electrospray ionization
DEX dextran
DEX dextrose
DEX dexamethasone
111
DEXA dual-energy x-ray absorptiometry
DFMO difluoromethylornithine
DFSA drug facilitated sexual assault
DFT deep flexor tendon
DFT density functional theory
DG dentate gyrus
DG deoxyglucose
2DG 2-deoxyglucose
dGTE decaffeinated green tea extract
D/H deuterium/hydrogen ratio
DHA docosahexaenoic acid
DHA docosahexaenoic
DHCMT dehydrochloromethyltestosterone
DHDMA dihydroDMA (7alpha,11beta-dimethyl-19-nortestosterone)
DHA 3,4-dihydroxyamphetamine
DHA docosahexanoic acid
DHA-Et docosahexaenoic acid ethyl ester
DHAP dihydroxyacetone phosphate
DHB dihyroxy benzoic acid
DHE dehydration
DHEA 5-androsten-3beta-ol-17-one, dehydroepiandrosterone
DHEAS dehydroepiandrosterone sulfate
DHCMT dehydrochloromethyltestosterone
DHMA 3,4-dihydroxymethamphetamine
DHP 5alpha-pregnane-3,20-dione
2D-HPLC two-dimensional high performance liquid chromatographic
DHMNT dihydroMNT
DHT dihydrotestosterone
DHY dehydrated
DI deletion-insertion
DIAAS digestible indispensable amino acid score
DID Drug Information Database
DILI drug induced liver injury
DIOL androstenediol
DIONE androstenedione
DIPA diisopropylamino-n-alkanes
DIPT dimethyltryptamine
DI-SPME direct immersion solid-phase microextraction
DIY do it yourself
DL decision limits
DLLME dispersive liquid-liquid microextration
DLS dynamic light scattering
DMA 7alpha,11beta-dimethyl-19-nortestosterone
DMAA methylexaneamine (or 1,3 dimethylamylamine)
DMBA dimethylbutylamine
DMBCD heptakis(2,6-di-O-methyl)-beta-cyclodextrin
DME drug metabolizing enzymes
DMG N,N-dimethylglycine
DMH dorsomedial hypothalamus
DMI Doppler myocardial imaging
DMM Drinking Motives Measure
DMSO dimethyl sulphoxide
DMT desoxymethyltestosterone, mandol
112
DMT divalent metal transporter
DMZ dimethazine
DNA deoxyribonucleic acid
D2O deuterium oxide
DOMS delayed onset of muscle soreness
DOPAC 3,4-dihydroxyphenylacetic acid
DOR delta opoid receptor
DOS diversity-oriented synthesis
DoU declaration of use
DP dipyridyl
DP discursive psychology
DP double poling
DPAC dihydroxyphenylacetic acid
DPB diastolic blood pressure
DPG diphosphoglycerate
DPO darbepoetin
DPV differential pulse voltammetry
DPX disposable pipette extraction
DRE dioxin response element
DRI dietary reference intakes
DRIFTS diffuse reflectance infrared Fourier transform spectroscopy
DRN dorsal raphe nucleus
DRS deconvolution reporting software
DS dietary supplements
DS dilute-and-shoot
DSC differential scanning calorimetry
DSD disorders of sexual development
1D SDS-PAGE one-dimensional sodium dodecyl sulfate/polyacrylamide gel
electrophoresis
DSHEA Drugs Supplement Health and Education Act
DSM Diagnostic and Statistical Manual of Mental Disorders
DSM-IV Diagnostic and Statistical Manual of Mental Disorders, Fourth
edition
dSPE dispersive solid phase extraction
DTR detraining
DUB dysfunctional uterine bleeding
DUB doping use belief
DUID driving under the influence of drugs
DUS dried urine spot
DUT dutasteride
DVT deep vein thrombosis
DWTP drinking water treatment plant
DYN-MRM dynamic multiple reaction monitoring
DXA dual energy X-ray absorptiometry
DXM dextromethorphan
DXM dexamethasone
DZOase diazoxon
E1 estrone
E2 17beta-estradiol
EA energy availability
EAA essential amino acid
EAA European Academy of Andrology
EAACI European Academy of Allergy and Clinical Immunology
113
EAAS endogenous anabolic androgenic steroids
EABP ethylamino-1-phenylbutane
EAC exercise-associated collapse
EAD 5alpha-estrane-3beta,17alpha-diol
EADR epi-androsterone
EAH exercise-associated hyponatremia
EAHE exercise-associated hyponatraemic encephalopathy
EAMC exercise-associated muscle cramps
EAPH exercise-associated postural hypotension
EAS erythropoiesis-stimulating agents
EASIA enzyme amplified sensitivity immunoassay
eAST erythrocyte aspartate aminotransferase activity
EAU European Association of Urology
EB energy beverages
EB estradiol benzoate
EBGM empirical Bayes geometric mean
EBW effective body water
EC epicatechin
EC European Community
EC European Commission
EC external capsule
EC eccentric contractions
eCB endocannabinoid
ECC eccentric
Ecdy phytoectysteroid ecdysterone
ECF extracellular fluid
ECG electrocardiogram
ECG epicatechin gallate
ECGC epigallocatechin gallate
ECH echinacea
ECL electrochemiluminescence
ECM extracellular matrix
ECME ecgonine methyester
ECNI-MS electron capture negative ionization mass spectrometry
ECS endogenous cannabinoid system
ECT exercise challenge tets
ECW extracellular water
ED endocrine disruptors
ED emergency department
ED energy drinks
ED eating disorder
ED energy deficit
ED erectile dysfunction
ED effort discounting
EDC ethyl-3-(dimethylaminopropyl)carbodiimide hydrochloride
EDC endocrine disrupting compounds
EDC endocrine disrupting chemicals
EDC endocrine chemical
EDDP 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine
EDL extensor digitorum longus
EDT electron-transfer dissociation
EDTA ethylenediamine tetraacetic acid
EDMA ethylene glycol dimethacrylate
114
EDNO endothelium-derived nitric oxide
EE energy expenditure
EE eccentric exercise
EE2 alpha-ethynyl estradiol
EEA essential amino acid
EED 5(10)-estrene-3beta,17alpha-diol
EEG electroencephalographic
eEPO endogenous erythropoietin
EES ethinylestradiol
EESI extractive electrospray ionization
EF ephedrine
EF enrichment factor
EF ejection fraction
EF elbow flexors
EFL expiratory flow limitation
EFSA European Food Safety Agency
EG epitestosterone glucuronide
EG etiocholanolone glucuronide
EG epitestosterone glucuronide
EGC epigallocatechin
EGCG epigallocatechin-3-gallate
EGF epidermal growth factor
EGFP enhanced green fluorescence protein
eGFR estimated glomerular filtration rate
eGH equine growth hormone
EGP endogenous glucose production
EGR erythrocyte glutathione reductase
EHSLC European Horserace Scientific Liaison Committee
EI electron ionisation
EI electron impact
EI energy ingestion
EIA exercise-induced asthma
EIA enzyme-immunoassay
EIAH exercise induced arterial hypoxaemia
EIB exercise induced bronchoconstriction
EID exercise-induced dehydration
eIF eukaryotic initiation factor
eIEFE eukaryotic initiation factor 4E
eIF4E-BP1 eukaryotic initiation factor 4E-binding protein 1
ElJ Eurycoma longifolia Jack
EIMD exercise-induced muscle damage
EI-MS electron ionization mass spectrometry
EIPH exercise-induced pulmonary hemorrhage
EIS electrochemical impedance spectra
ELISA enzyme-linked immunosorbent assay
Elt euglobulin-lysis-time
EMA European Medicines Agency
EMA erythropoietin-mimetic agents
EMAS European Male Aging Study
EMCDDA European Monitoring Centre for Drugs and Drug Addiction
EME electromembrane extraction
EMD electromechanical delay
EMG electromyogram
115
EMG education model guidelines
EMg erythrocyte Mg2+
EMGFT electromyographic fatigue threshold
EMIT enzyme multiplied immuno technique
EMP erythropoietin mimetic peptides
EMS electromyostimulation
EMS eosinophilia-myalgia syndrome
eNO exhaled nitric oxide
eNOS endothelial nitric acid synthase
EOGHD early-onset growth hormon deficiency
EP ephedrine
EP erythroid progenitor
EP endurance performance
EPA eicosapentaenoic acid
EPA-Et eicosapentaenoic acid ethyl ester
4EPB 4E-binding protein 1
EPC effective concentration
EPH ephedrine
EPHE ephedrine
EPI epinephrine
EPI enhanced product ion
EPI epididymal adipose tissue
epiDHT 5alpha-androstane-17alpha-ol-3-one
EpiT epitestosterone
epiTG epitestosterone glucuronide
EPM elevated plus maze
EPO erythropoietin
Epo erythropoietin
EPOC excess postexercise oxygen consumption
EPOR erythropoietin receptor
EpoR erythropoietin receptor
EPPGE edge plane pyrolytic graphite electrode
EPR electron paramagnetic resonance
EQAS External Quality Assessment Scheme
ER estrogen receptors
ERC endogenous reference compounds
ERD exercise-related arterial oxygen desaturation
ERE estrogen response elements
ERK extracellular regulated protein kinases
ERP event-related potential
ERS European Respiratory Society
ERS exercise-induced rhadomyolysis
ES estradiol
ES effect size
ES electrospray ionization
ES etiocholanolone sulfate
ES energy shots
ES electrical stimulation
ES Eleutherococcus senticosus
ESA erythropoiesis stimulating agents
ESA elite student athlete
ESI electrospray ionization
ESI-MS electrospray ionisation-mass spectrometry
116
ESI-MS/MS electrospray tandem mass spectrometry
ESI-TOFMS electrospray ionization time-of-flight mass spectrometry
ESN estrone
ESPAD European School Survey Project on Alcohol and Other Drugs
ESR estrogen receptor
ESR electron spin resonance
ESRD end-stage renal disease
EST estriol
eST equine somatotropin
ET epitestosterone
ET ethynyl testosterone
ET exhaustive test
ET exercise training
ET exercise training
ET essential tremor
ETCT estrogen-testosterone combination therapy
ETG epitestosterone glucuronide
ETG etiocholanolone glucuronide
EtG ethylglucuronide
ETH ethyl-alpha-ethyl-phenethylamine
ETIC eticlopride
ETIO etiocholanolone
Etio etiocholanolone
ETK erythrocyte transketolase
EtOH ethanol
ETS epitestosterone
EU European Union
EUH euhydration
EuMoCEDA European Monitoring Center for Emerging Doping Agents
EV ecologically valid
EVH eucapnic voluntary hyperpnoea
EVOK evoked strength
EWB equine whole blood
EWS Early Warning System
EXP experimental
FA ferulic acid
FA fatty acids
FA fractional anisotropy
FA free acid
FAAH fatty acid amid hydrolase
FABP fatty acid-binding protein
FAI free androgen index
FAK focal adhesion kinase
FAM fat metabolism
FAO fatty acid oxidation
FAO/WHO Food & Agriculture Organization and World Health Organization
FARS Fatality Analysis Reporting System
FAS felt arousal scale
FASI field amplified sample injection
FASM Faculty of Sport and Exercise Medicine
FASS field-amplified sample stacking
F-AuNP functional gold nanoparticle
FBF forearm blood flow
117
FBS fragment-based screening
FCE false consensus effect
FCMIA fluorescence covalent microbead immunosorbent assay
fCSA fibre cross-sectional area
FDA (US) Food and Drug Administration
FDG fluorodeoxyglucose
FDI first dorsal interosseus
FEARS United States Food and Drug Administration Adverse Event
Reporting System
FED functional energy drinks
FEI Fédération Equestre Internationale
FeNO fraction of exhaled nitric oxide
FER ferritin
FEV fast eccentric velocity
FEV1 forced expiratory volume in 1 second
FEV1/FVC forced expiratory volume in 1 s to forced vital capacity ratio
FFA free fatty acid
FFDCA Federal Food, Drug, and Cosmetic Act
FFM fat-free mass
FFQ Food Frequency Questionnaire
FFST fat-free soft tissue
FFW fat-free weight
FGF fibroblastic growth factor
FHA functional hypothalamic amenorrhea
FHL flexor hallucis longus
FI fatigue index
FI flow injection
FIFA Fédération Internationale de Football Association
FIMS Fédération International de Médecine du Sport
FIN finasteride
FINA Fédération Internationale de Natation
FIO2 inspired fraction of oxygen
FIS International Ski Federation
FISH fluorescence in situ hybridization
FITC fluorescein isothiocyanate
FITR Fourier transform infrared
FLST follistatin
FLU flutamide
FLX fluoxetine
FM fat mass
F-MARC FIFA Medical Assessment and Research Centre
FMC fluoromethcathinone
FMD flow-mediated dilation
FNH focal nodular hyperplasia
FO fish oil
FOLLI follistatin
FOS fructo-oligosaccharides
FOT forced oscillation technique
FOXO forkhead homeobox type O
FPG fasting plasma glucose
FPP fermented papaya preparation
FP follicular phase
FR fructose
118
FR fixed-ratio
FRAST free-running asthma screening test
FRAP ferric-reducing ability of plasma
FRI fatigue resistance index
FRSA free radical scavenger activity
FS feeling scale
FS first-sprint
FSGS focal segmental glomerulosclerosis
FSH follicle-stimulating hormone
FSR fractional synthesis rate
FST forced swimming test
FT free testosterone
FT Fourier transform (orbitrap)
FT3 triiodothyronine
fTC free testosterone to cortisol ratio
FTE full time equivalents
FTICR Fourier transform ion cyclotron resonance
FT-ICR/MS Fourier transform ion cyclotron resonance mass spectrometry
FTIR Fourier transform infrared
FTM Fahn-Tolosa-Marin (tremor rating scale)
F20TPP meso-tetrakis(pentafluorophenyl) porphyrin
f-TRP free-tryptophan
fTRYP free tryptophan
FV fruits and vegetables
FVC forced vital capacity
FVC forearm vascular conductance
FVIII factor VIII-activity
FW fat weight
FW flavored water
FWHM full-width at half maximum
GA gastrocnemius
GAA guanidinoacetic acid
GABA gamma-aminobutyric acid
GABAA gamma-aminobutyric acid subtype A
GABAAR gamma-aminobutyric acid receptor
GABAB gamma-aminobutyric acid subtype B
GAD glutamic acid decarboxylase
GAKIC glycine-arginine-alpha-ketoisocaproic acid
GAL galactose
GAMT guanidino-acetate methyltransferase
Gas musculus gastrocnemius
GASP-1 growth and differentiation factor-associated serum protein-1
GAST gastrocnemius
GBL gamma-butyrolactone
GBR gamma-aminobutyric acid (GABA)/benzodiazepine receptor
complexes
GBS global positioning system
GC glucocorticoid
GC gas chromatography
GCC gas chromatography combustion
GC/C/IRMS chromatography/combustion/isotope ratio mass spectrometry
GCD gluten-containing diet
GC/EI/MS/MS gas chromatography tandem mass spectrometry
119
GC-ES gas chromatography with an electron ionization source
GC/FID gas chromatography-flame ionization detector
GC/GC/TOF/MS two-dimensional gas chromatography coupled to time-of-flight
mass spectrometry
GC/HRMS gas chromatography-high-resolution mass spectrometry
GC/GC two-dimensional gas chromatography
GC/GC/TOFMS two-dimensional gas chromatography with time-of-flight mass
spectrometry
GC/IT/MS gas chromatography-ion trap-mass spectrometry
GC/MS gas chromatography/mass spectrometry
GC/MSD gas chromatograph-mass spectrometer
GC/MS/MS gas chromatography-tandem mass spectrometry
GC-MS/NPD gas chromatography-mass spectrometry and nitrogen
phosphorus-specific detection
GC-NCI-MS/MS gas chromatography-negative chemical ionization-tandem mass
spectrometry
GC-NPD gas chromatography-nitrogen-phosphorus detection
GCoaTOFMS gas chromatography/electron ionization orthogonal acceleration
time-of-flight mass spectrometry
GcR glucocorticoid receptors
GCS glutamylcysteine synthetase
GCS Glasgow Coma Scale
G-CSF granulocyte colony-stimulating factor
GC/TOF/MS gas chromatography time-of-flight mass spectrometry
GD gastrointestinal distress
GDH growth-hormone deficiency
GDR glucose disposal rate
GDR German Democratic Republic
GET gas exchange threshold
GF growth factor
GFAP glial fibrillary acidic protein
GFD gluten-free diet
GFP green fluorescent protein
GFP general factor of personality
GFPQ General Factor of Personality Questionnaire
GFR glomerular filtration rate
GGE gradient gel electrophoresis
GGT gamma-glutamyl-transferase
GH growth hormone
GHB gamma-hydroxybutyric acid
GHBP growth hormone binding protein
GHD growth hormon deficiency
GHif immunofunctional growth hormone
GHir immunoreactive growth hormone
GHR growth hormone receptor
GHRH growth hormone-releasing hormone
GHRh growth hormone releasing factor/somatoliberin
GHRP growth hormone releasing peptides
GHS growth hormone secretagogue
GHV gamma-hydroxyvalerate
GI gastrointestinal
GI glycemic index
GIH glycerol-induced hyperhydration
120
GINA Global Initiative for Asthma
GIP glucose-dependent insulinotropic polypeptide
GIR glucose infusion rate
GL glycemic load
GLA gamma-linolenic acid
GLC gas-liquid chromatography
Gln glutamine
GLP glucagon-like peptide
GLU glucose
Glu glucose
Gly glycerol
GLUC glucose-maltodextrin
GLUT glucose transporter
GLUT4 glucose transporter 4
GMC General Medical Council
GMFI geometric mean fluorescence intensity
GMP guanosine monophosphate
GnIH gonadotropin inhibitory hormone
GNP gold nanoparticle
GnRH gonadotrophin-releasing hormone
GnRH-a gonadotropin-releasing hormone agonist
GOS galacto-oligosaccharides
GP General Practitioner
GP glucose polymer
GP growth promoters
GPA growth-promoting agents
GPA grade point average
GPCR G protein coupled receptors
GPER G protein-coupled estrogen receptor
GPLC glycine-propionyl-L-carnitine
GPR30 G protein-coupled receptor-30
GPS global positioning devices
GPx glutathione peroxidase
GR glutathione reductase
GR glucocorticoid receptor
GPS global positioning systems
GPx glutathione peroxidase
GR glutathione reductase
GR glucocorticoid receptor
GR graphene
GR glycemic response
GRS genomic risk score
GS glycogen synthase
GS gas spectroscopy
GSH/GSSSG reduced to oxidized glutathione ratio
GS/GS/TOFMS gas chromatography coupled two time-of-flight mass spectrometry
GSH glutathione
GSH-Px glutathione peroxidase
GSHt total glutathione
GS-MS gas chromatography-mass spectrometry
GSSG glutathione disulphide
GST glutathione-s-transferase
GT glutamyl-transferase
121
GT green tea
GTC green tea catechins
GTE green tea extract
GTN gastrocnemius
GTN nitroglycerin
GVL gamma-valerolactone
GXT graded exercise tests
[H+] hydrogen ion concentration
HA hepatic adenomas
HAA hydroxyanthranilic acid
HABA 2-(4-hydroxyphenylazo)benzoic acid
HAD hydroxyacyl-CoA dehydrogenase
HAM-D Hamilton Rating Scale for Depression
hAR human androgen receptor
HAS human serum albumin
Hb hemoglobin
[Hb] haemoglobin concentration
HBA hydroxybutyrate
HBC hyperimmune bovine colostrums
HBc hepatitis B core antigen
HbCO carboxyhemoglobin
%HbCO percent carboxyhemoglobin
HBM beta-hydroxy-beta-methylbutyrate
Hbmass total haemoglobin mass
HBC hepatitis virus C
HBD hepcidin-binding
HBM-FA beta-hydroxy-beta-methylbutyrate free acid
HBOC hemoglobin-based oxygen carriers
HbP hemoglobin-loaded nanoparticle
HbSaO2 arterial oxyhemoglobin saturation
HbP hemoglobin particles
HBV hepatitis B virus
HbV hemoglobin-vesicles
HC hormonal contraceptives
HC high carbohydrate
HCA hydroxycitric acid
HCA hierarchical cluster analysis
HCA hepatocellular adenoma
HCAR homocarnosine
HCC hepatocellular carcinoma
HCD histidine containing dipeptides
HCD higher collision energy dissociation
HCD high-carbohydrate diet
HCG human chorionic gonadotrophin
hCG human chorionic gonadotropin
H-CHO high-carbohydrate diet
HCM high carbohydrate meal
HC-MS/MS headspace-gas chromatography-tandem mass spectrometry
HCP hematopoietic cell phosphatase
HCT hematocrit
HCTZ hydrochlorothiazide
HCV hepatitis C virus
HCVR hypercapnic ventilatory responses
122
Hct haematocrit
Hcy homocysteine
HDC histidine decarboxylase
HDC high-density cholesterol
HDHQ Hostility and Direction of Hostility Questionnaire
HDL high-density-lioprotein
HDL-C high-density lipoprotein cholesterol
HDP hydroxypropyl-distarch phosphate
HDT head down tilt
HE haematoxylin-eosin
HE hypertensive encephalopathy
HEA hereditary angioedema
HED heavy episodic drinking
HEK human embryonic kidney
hEPO human erythropoietin
hEPOR human erythropoietin receptor
HEPS (hydroxyethyl)promazine
HER heroin
hERG human ether-a-go-go-related gene
HES hydroxyethyl starches
HF heart failure
HFBA heptafluorobutyric anhydride
HFD high-fat diet
HFD-CHO high-fat diet plus carbohydrate
HF-LC high-fat/low-carbohydrate diet
HF-LPME hollow fiber liquid phase microextraction
HFM high fat meal
HFMP high-fat/moderate protein
HG handgrip strength
HGB hemoglobin
HGF hepatocyte growth factor
HGF human gingival fibroblasts
hGH human growth hormone
hGHRH human growth hormone releasing hormone
HGI high glycaemic index
HGP hormone growth promotants
HH hypobaric hypoxia
HHRE hypertrophic heavy resistance exercise
HHQ hydroxyhydroquinone
5-HIAA 5-hydroxyindoleacetic acid
HIE high-intensity exercise
HIE hypoxic-ischaemic encephalopathy
HIF hypoxia-inducible factor
HIEGEAA hyperinsulinaemic-euglycaemic-euaminoacidaemic
HiHiLo live high-base train high-interval train low
HIIRT high-intensity interval and resistance training
HIIT high-intensity interval training
HILIC hydrophilic interaction chromatography
HILIC/MS/MS hydrophilic interaction chromatography-tandem mass
spectrometry
HILIC-HRMS hydrophilic interaction liquid chromatography-high
resolution/high accuracy mass spectrometry
HILIC/TOFMS hydrophilic interaction liquid chromatography-time-of-flight mass
123
spectrometry
Hi-Lo living high-training low
HIPP hippocampus
HIR hydrogen isotope ratios
HIRT high-intensity resistance training
HIS high-intensity intermittent sprints
HIS hydroxyethyl starch
HIT high-intensity training
HIV human immunodeficiency virus
HK hexokinase
HK hydroxykynurenine
HK hypokinesia
HLB hydrophilic lipophilic balanced (particles)
HLM human liver microsomes
HMB beta-hydroxy-beta-methylbutyrate
HMB/ATP beta-hydroxy-beta-methylbutyrate plus adenosine triphosphate
HMBC heteronuclear multiple bond connectivity
HMB-Ca calcium salt of beta-hydroxy-beta-methylbutyrate
HMBCr beta-hydroxy-beta-methylbutyrate plus creatine monohydrate
HMB-FA free acid of beta-hydroxybeta-methylbutyrate
HMF hydroxymethylfurfural
HMG human menopausal gonadotrophin
hMG human menopausal gonadotropin
HMG-CoA hydroxy-methylglutaryl-CoA
HMMA 4-hydroxy-3-methoxymethamphetamine
HMW high-molecular-weight
HN heated nebulizer
HO heme oxygenase
H2O2 hydrogen peroxide
HOI highest observed intake
HOMA homeostasis model assessment
HOMA-IR homeostatic metabolic assessment for insulin resistance
HORMA Hormonal Regulators of Muscle and Metabolism in Aging
HP heat production
HP hydroxyproline
HP hydroxylysylpyridinoline
HP high protein
Hp haptoglobin
HPA hypothalamic-pituitary-adrenal
HPAA hypothalamic-pituitary-adrenal axis
HPF human oral periosteal fibroblasts
HPG hypothalamic-pituitary-gonadal (axis)
HPLC low-carbohydrate/high-protein
HPLC high performance liquid chromatography
HPLC-DAD high resolution liquid chromatography-diode array detector
HPLC/ESI-MS/MS high-performance liquid chromatography/electrospray ionization
tandem mass spectrometry
HPLC/(HR)MS high-performance liquid chromatography-high resolution mass
spectrometry
HPLC-MS high-performance liquid chromatography/mass spectrometry
HPLC/MS/MS high-performance liquid chromatography tandem mass spectrometry
HPLC/UV high performance liquid chromatography and ultraviolet detection
HPLF high-protein low-fat
124
HPMC (hydroxypropyl)methyl cellulose
HPP hexyloxyphenylpropionate
HPS hydroxypropyl starch
HPT hypothalamic-pituitary-testicular
HPT haptoglobin
HRP horse radish peroxidase
HPLC high-performance liquid chromatography
HR hazard ratio
HR high-resolution
HR heart rate
HRAM high resolution and accurate mass
HRAMS high resolution accurate mass screening
HRE hypertrophy-type resistance exercise
HRE heavy resistance exercise
HRE hypoxia response elements
HRmax maximal heart rate
%HRmax percentage of maximal heart rate
HRMS high resolution mass spectrometry
HRP horseradish peroxidase
HRR heart rate recovery
%HRR percentage of heart rate reserve
HRR60s heart rate recorded 60-s after
HRT hormone replacement therapy
HRT half relaxation time
HRV heart rate variability
hs-CRP high-sensitivity C-reactive protein
HSD hydroxysteroid dehydrogenase
HSDD hypoactive sexual desire syndrome
HSF heat shock factor
hSHBG human sex-hormone binding globulin
hSKMC primary human skeletal muscle cell
HSL harmonised screening limits
HSL hormone sensitive lipase
HSP heat-shock protein
HSV herpes simplex virus
HT Hashimoto's thyroiditis
5-HT 5-hydroxytryptamine
5-HT serotonin
5-HT-ir serotonin immunoreactive
Hto hematocrit
5-HTT serotonin transporter
HTLV human T-lymphotropic virus
HTS hybrid training system
huEPO human endogenous etythropoietin
HUGO Human Genome Project
HuREPO human recombinant erytropoietin
HUVEC human umbilical vein endothelial cell
HVA homovanillic acid
HVLT Hopkins Verbal Learning Test
HVR hyperoxic ventilatory responses
HVT high volume training
125
HWP hydrolyzed wheat protein
HYD hydrated
HYD hydrocortisone
HYP hydroxyproline
HZ heterozygous rats
I insertion
IA immunoassay
IAA trans-3-indoleacrylic acid
IAAF International Association of Athletic Federations
IAC immunoaffinity column
IAC immunoaffinity chromatography
IADA International Anti-Doping Arrangement
IAHGD Idiopathic adult growth hormone deficiency
5-IAI 5-iodo-2-aminoindane
IAP immunoaffinity micro well plate
IAT Implicit Associations Test
IAT individual anaerobic threshold
IAU International Association of Ultrarunners
IAUC incremental area under the curve
IBA inhaled beta2-agonists
IBD inflammatory bowel disease
IBM inclusion body myositis
IC in-competition
IC inspiratory capacity
iCHO isocaloric carbohydrate drink
ICL immunochemiluminometric
ICP inductively-coupled plasma
ICP-MS inductively coupled plasma-mass spectrometry
ICQC International Carbohydrate Quality Consortium
ICS inhaled corticosteroids
ICTP C-terminal telopeptide of type I collagen
ICTP type I collagen cross-linked carboxy-terminal telopeptide
ICV intracerebroventricular
icv intracerebroventricular
ICW intracellular water
ID illicit drug
ID iron deficiency
ID index of difficulty
I/D insertion/deletion
IDA iron deficiency anaemia
IDA information-dependent acquisition
IDA independent data acquisition
ID-LC-MS/MS isotope-dilution liquid chromatography-tandem mass spectrometry
IDNA iron-deficient but non-anaemic
IDP indapamide
IEE intense and exhaustive exercise
IEF isoelectric focusing
IEF-PAGE isoelectric focusing in polyacrylamide slab gels
IEMG integrated electromyogram
IET intermittent endurance test
IF interfibrillar
IF immunfunction
IF international federation
126
iFA isoferulic acid
IFBB International Federation of Bodybuilding and Fitness
IFG impaired fasting glucose
IFHA International Federation of Horseracing Authorities
IFN interferon
IFSP International Federation of Sports Physiotherapy
Ig immunoglobulins
IgA immunoglobulin A
IgAN immunoglobulin A nephropathy
IGF insulin-like growth factor
IGFBP insulin-like growth factor-1 binding protein
IGF1-R IGF-I receptor
IHD ischemic heart disease
IHE intermittent hypoxic exposure
IHI intermittent high-intensity
IHIT intermittent hypoxic exposure with intermittent hypoxic training
IHT intermittent hypoxic training
IIEF International Index of Erectile Dysfunction
IJSM International Journal of Sports Medicine
IkappaB inhibitor-kappaB
IL ionic liquid
IL interleukin
IL-6 interleukin-6
IL-ISTD isotope-labeled internal standard
IL-1ra interleukin-1 receptor antagonist
ILT incremental load test
IM intramuscular
IM immobilization
IMBE immuno-magnetic beads-based extraction
IMCL intramyocellular lipid
iMg ionic Mg2+
IMHH idiopathic male hypogonadotropic hypogonadism
IMP inosine monophosphate
IMS-MS ion mobility spectrometry-mass spectrometry
IMT inspiratory muscle training
IMT intima-media thickness
IMTP isometric midthigh pulls
IN intranasal
IND indomethacin
INF interferon
iNOS inducible nitric oxide synthase
INS insulin
Ins insulin
IOC International Olympic Committee
IOC-MC International Olympic Committee-Medical Commission
IOM Institute of Medicine
IOMT illicit drugs other than marijuana
i.p. intraperitoneal
IPA Interpretative Phenomenological Analysis
IPC International Paralympic Committee
IPC irrelevant plasma concentration
IPED image and performance enhancement drugs
IPF idiopathic pulmonary fibrosis
127
iPTH intact parathyroid hormone
IQR interquartile range
IR immunoreactivity
I/R ischemia/reperfusion
IRF immature reticulocyte fraction
IRIDA iron-refractory iron-deficiency anemia
IRMA immunoradiometric assay
IRMPD infrared multiple photon dissociation
IRMS isotope ratio mass spectrometry
IRS intermittent repeated sprint
IS internal standards
ISE intermittent sprinting exercise
ISA International Society of Andrology
ISAAC International Study of Asthma and Allergies in Childhood
ISCID in-source collision induced dissociation
ISF isoflavone
ISL International Standard for Laboratories
ISO International Standards Organization
ISO isoflavones
ISO isomaltulose
ISO isokinetic strength
ISO isoproterenol
isoBOC isobutyloxycarbonyl
ISP infraspinatus
ISP-MS inductively coupled plasma mass spectrometry
iSPT intermittent soccer performance test
ISSAM International Society for the Study of the Aging Male
ISSN International Society of Sports Nutrition
IST intermittent sprint test
IST International Standard on Testing
ISTD internal standard
ISTI International Standard on Testing and Investigations
ISU International Skating Union
ISWT incremental shuttle walk test
IT intratesticular
IT intensified training
ITC internal threshold control
IT-MS ion trap mass spectrometry
ITPP inositol trispyrophosphate
ITS swimming intervals
ITT insulin tolerance test
ITT intratesticular testosterone
IUC irrelevant urine concentration
IV intravenous
iv intravenous
IVS interventricular septum
IVSA intravenous self-administration
IVUS intravascular ultrasound
IWS water-immersion stress
JAK janus kinases
JCAAI Joint Council of Allergy, Asthma and Immunology
JECFA Joint FAO/WHO Expert Committee on Food Additives
JL jet lag
128
KA kynurenic acid
KAAA keto analogues and amino acids
11k-AC 11-ketoetiocholanolone acetate
KD knowledge of doping
KET ketamine
KG ketoglutarate
KG ketoglutaric acid
KIA knowledge, incidence and attitudes
KIC ketoisocaproate
KIE kinetic isotope effects
KJRM knee range of motion
k-NN k-Nearest Neighbours
KOOS knee injury and osteoarthritis outcome score
KP ketoprofen
KROM knee range of motion
KSN knowledge of sports nutrition
KST kamishimotsuto
Kyn kynurenine
LA (musculus) levator ani
LA lactate acid
LA left atrium (diameter)
LA lactalbumin
LA linoleic acid
[LA] lactate concentration
[La] concentration of lactate
LAB long-acting beta2-agonist
LABA long-acting beta-2-agonists
LABC levator ani/bulbocavernosus
Lac lactate
LAD left anterior descending
LAH latero-anterior subdivision of the hypothalamus
LAT L transporter
LBD ligand binding domain
LBM lean body mass
LC liquid chromatography
LC L-carnitine
LC slow carbohydrate
LC locus coeruleus
LCA latent class analysis
LC-Ag+-CIS liquid chromatography with a silver ion coordination ion spray
source
LC/APCI/MS/MS liquid chromatography-tandem mass spectrometry using
atmospheric pressure chemical ionization
LCB low calorie beverages
LC-DAD liquid chromatography coupled with a diode-array detector
LC/ESI liquid chromatography-electrospray ionisation
LC/ESI/HRMS liquid chromatography/electrospray ionization high resolution/high
accuracy tandem mass spectrometry
LC/ESI/MS liquid chromatography-electrospray ionization-mass spectrometry
LC/ESI/MS/MS liquid chromatography electrospray ionization tandem mass
spectrometry
LC liquid chromatography
L-CHO low-carbohydrate diet
129
LC-HRMS liquid chromatography high resolution mass spectrometry
LCL lowest calibration level
LCLT L-carnitine L-tartrate
LC/MS liquid chromatography/mass spectrometry
LC/MS/MS liquid chromatography-tandem mass spectrometry
LCn-3PUF A long-chain n-3 polyunsaturated fatty acid
LCoaTOFMS liquid chromatography/electrospray ionization orthogonal
acceleration time-of-flight mass spectrometry
LCPUFA long chain polyunsaturated fatty
LC-QqQ liquid chromatography tandem mass spectrometry
LC-QqQ-MS liquid chromatography coupled to triple-quadrupole mass
spectrometry
LC-Q-TOF-MS liquid chromatography/ electrospray ionization quadrupole time-of-
flight mass tandem mass spectrometry
LCT long-chain triglyceride
LC/TOF/MS liquid chromatography/ time-of-flight MS
LDA linear discriminant analysis
LD detection limit
LD longissimus dorsi
LD low-dose
LD Leydig cells
L/D light/dark
LDD least detectable doses
LDH lactate dehydrogenase
LDL low-density lipoprotein
LDL lipoprotein cholesterol levels
LDL-C low-density lipoprotein cholesterol
LDS lipodermatosclerosis
LE leg extension
LEAA leucine-enriched essential amino acids
LEFS Lower Extremity Functional Scale
LEU leucine
LF Langmuir-Freundlich
LF-HC low-fat/high-carbohydrate diet
LFOR low functional ovarian reserve
LG lactoglobulin
LGI low glycaemic index
LH luteinizing hormone
LH liver hemangiomas
LH lipid hydroperoxide
LHb lateral habenula
LH-RH luteinizing hormone-releasing hormone
LHrh recombinant luteinizing hormone
LHTH live high-train high
LHTL live high-train low
LIF leukemia inhibitory factor
LIF laser-induced fluorescence detection
LIP lipoic acid
LIPA low intensity physical activity
LIPOXmax maximal lipid oxidation
LIQ liquid (source)
LIST Loughborough Intermittent Shuttle Test
LIT/MS linear ion trap mass spectrometry
130
LLE liquid-liquid extraction
LLE lipophilic ligand efficiency
LLLME liquid-liquid-liquid microextraction
LLOD lower limits of detection
LLOQ lower limit of quantification
LLTH live low – train high
LM lean mass
LMA locomotor activity
LMA Longissimus muscle areas
LMCR light meal combat ration
LMS lactate-minimum speed
LMT lactate-minimum test
LMW low-molecular-weight
LMWLH low molecular weight luteinizing hormone
LNAA large neutral amino acid
L-NAME N-nitro-L-arginine methyl ester hydrochloride
L-NMMA N-monomethyl-L-arginine
vLOC limit of confirmation
L-NNA nitro-L-arginine
LOD limit of detection
LOOH lipid hydroperoxides
LOQ limit of quantification
LOV lacto-ovo-vegetarian
LP leg press
LP luteal phase
LP lactate paradox
LP lysylpyridinoline
Lp lipoprotein
Lpa lipoproteina
LPD luteal phase deficient
LPDT Leidenfrost phenomenon assisted thermal desorption
LPL lipoprotein lipase
LPM leg press maximum
LPME liquid-phase microextraction
LPO lipid hydroperoxide
LPO lipoperoxides
L-PRF leukocyte- and platelet-rich fibrin
L-PRP leucocyte and platelet-rich plasma
LPS lipopolysaccharide
LPTD Leidenfrost phenomenon assisted thermal desorption
LPUS lower-body power and upper-body strength
LRA leukotriene receptor antagonists
LRS long run session
LS lateral septum
LSD diethilamid lisergic acid
LSP localised surface plasmons
LSPT Loughborough Soccer Passing Test
LT lactate threshold
LTA latent trait analysis
LTCS long-term cigarette smoking
LTM long-term metabolites
LTP long-term potentiation
LUTS lower urinary tract
131
LV left ventricular
LVAD left ventricular assist device
LVEDD left ventricle end-diastolic diameter
LVET left ventricular ejection times
LVLD left ventricular lumen diameter
LVH left ventricular hypertrophy
LVI large-volume injection
LVM left ventriculus mass
LVMI left ventriculus mass index
LVT light visual task
MA methamphetamine
MA megestrol acetate
MA masters athletes
MAA methacrylic acid
MAACL-R Multiple Affect Adjective Checklist Revised
MAB metastable atom bombardment
mAbs monoclonal antibodies
MAC medications advisory committee
MAC methamphetamine-associated congestive heart failure
%Macro percent macrocytes
MAIIA membrane-assisted isoform immunoassay
MALDI matrix-assisted laser desorption/ionisation
MALDI-MS matrix-assisted laser desorption/ionization-mass spectrometry
MALDI-MSI matrix-assisted laser desorption/ionization-mass spectrometry
imaging
MALDI/TOF/MS matrix-assisted laser desorption/ionisation time-of-flight mass
spectrometry
MALT maltodextrin
MAMP methamphetamine
MAMS acid/alcohol-modified cornstarches
mAMU milli albumin mobility units
MAO monoamine oxidases
MAOD maximal accumulated oxygen deficit
MAODALT maximal accumulated oxygen deficit at a single supramaximal
effort
MAP mean arterial pressure
MAPK mitogen activated protein kinase
MAPK/ERK mitogen-activated protein kinase/extracellular signal-regulated
protein kinase
MARCO macrophage receptor with collagenous structure
MARE maximal anaerobic running exercise session
MAV maximal aerobic velocity
Mb myoglobin
MBA methane boronic acid
MBAS Male Body Attitudes Scale
MBDB N-methyl-benzodioxolylbutanamine
MBL mannan-binding lectin
MBP muscle protein breakdown
MC micellar casein
MC Medical Committee
MCA middle cerebral artery
MCAD medium-chain acyl-CoA dehydrogenase
MCFA medium chain fatty acids
132
MCH methacholine
MCH mean corpuscular hemoglobin
MCHC mean corpuscular hemoglobin concentration
MCP menstrual cycle phase
MCP monocyte chemotactic protein
MCR metabolic clearance rate
M-CSF macrophage colony-stimulating factor-1
MCT medium-chain triacylglycerol
MCT monocarboxylate transporters
MCV maximum voluntary contraction
MCV mean corpuscular volume
MD maltodextrin
MD mood
MD mean differences
MD muscular dystrophies
MD modafinil
MD molecular docking
Md methandienone
Md maltodextrin
MDA malondialdehyde
MDA methylenedioxyamphetamine
MDA 3,4-methylenedioxyamphetamine
MDAI 5,6-methylenedioxy-2-aminoindane
MDEA 3,4-ethylendioxyethylamphetamine
MDI metered dose inhaler
MDMA 3,4-methylenedioxymethamphetamine, “ecstasy”
MDP methylephedrine
MDPA methylenedioxypropylamphetamine
MDPF median signal's power spectrum
MDPV 3,4-methylenedioxypyrovalerone
MDRD modification of diet in renal disease
MDRT multistage discontinuous incremental running test
ME methylephedrine
ME metenolone enanthate
ME muscular endurance
MeA medial amygdala
MEAP met-enkephalin-Arg-Phe
MEC methylethcathinone
MECC micellar electrokinetic capillary chromatography
MED medroxyprogesterone
MF-REP myofibroblast-transformed erythropoietin-producing cells
MEHHP mono-(2-ethyl-5-hydroxyhexyl)phthalate
MEHP mono(2-ethylhexyl)phthalate
MEKC-UV micellar electrokinetic chromatography-UV absorbance detection
mass spectrometry
MENT 7alpha-methyl-19-nortestosterone
MEOHP mono-(2-ethyl-5-oxohexyl)phthalate
MeO-PCP methoxyphencyclidine
MEP motor evoked potential
MeP posteromedial amygdala
MEPH methylephedrine
MEPP mono-(2-ethyl-5-carboxypentyl)phthalate
MEPS microextraction by packed-sorbents
133
MePV medial amygdala
MES mesenteric adipose tissue
MET methadone
MeT methyltestosterone
METH methamphetamine
MetS metabolic syndrome
MeT methyltestosterone
metHB methemoglobin
MFGM milk fat globule membrane
mfMRI muscle functional magnetic resonance imaging
3MG 3-O-methyl-d-glucose
MGA melengestrol acetate
MGF mechano growth factor
M3G morphine-3-glucuronide
M6G morphine-6-glucuronide
Mg myoglobin
MGM milk fat globule membrane
MGF mechano growth factor
MGP matrix Gla protein
MH methylhistidine
3-MH 3-methyl-histidine
MHA methylhexaneamine
MHC myosin heavy chain
MHPG 4-hydroxy-3-methoxyphenylglycol
MHPG-S 3-methoxy-4-hydroxyphenyl glycol sulfate
MHRA Medicines and Healthcare products Regulatory Agency (UK)
MHS maximal handgrip strength
MI myocardial infarction
MIF maximal isometric force
MINORS methodological index for nonrandomized studies
MIP maximum inspiratory pressure
MIP molecular imprinted polymer
MIP macrophage inflammatory protein
MIPF molecularly imprinted polymer filaments
MIPS multi-ingredient performance supplements
miRNA microRNA
MISPE molecularly imprinted solid-phase extraction
MIVC maximal isometric voluntary contractions
MLM medium-, long-, and medium-chain fatty acid
MLOR maximal lipid oxidation rate
MLP mid- to late-pubertal
MLR multiple linear regression
MLSS maximal lactate steady state
MM muscle mass
MMA mixed martial arts
4-MMC mephedrone
MMG mechanomyographic
MMHP mono-(2-carboxymethylhexyl)phthalate
MMP matrix metallopeptidases
MMSE mini-mental state examination
MMT manual muscle testing
MMTV mouse mammary tumour virus
MNC micronucleated cells
134
MnR median raphe
MN metanephrine
MNGO magnetic nanographene oxide
Mn-SOD manganese superoxide dismutase
MNT 11beta-methyl-19-nortestosterone
MO maize oil
Mor/cod morphine/codeine
MP methylprednisolone
MP medroxyprogesterone
MP mean power
MPA medroxyprogesterone acetate
MPB muscle protein breakdown
MPB male-pattern baldness
MPC milk protein concentrate
mPFC medial prefrontal cortex
MPE methylpseudoephedrine
MPFS mean power frequency spectrum
MPH methylphenidate
MPH milk protein hydrolysates
MPHL male pattern hair loss
MPI myocardial perfusion imaging
MPI myocardial performance index
MPL maximum permitted levels
MPMF moderate-protein moderate-fat
MPO mean power output
MPO myeloperoxidase
mPOA medial preoptic area
MPOM medial preoptic area
MPPI microplasma photoionization
MPS myofibrillar protein synthesis
MPS muscle protein synthesis
MPS misuse of prescription stimulant
mPSc mid-pachytene spermatocytes
MPV mean platelet volume
MR mineralocorticoid receptor
MR mouth rinse
MRA magnetic resonance angiography
MRF myogenic regulatory factor
MRI magnetic resonance imaging
MRL maximum residue limits
MRM multiple reaction monitoring
mRNA messenger RNA
MRP meal replacement powders
MRPB muscle power production during repetitive high-power-output
exercise bouts
MRPL minimum required performance limits
MRS magnetic resonance spectroscopy
MRUCR mean urinary creatine
MRSA methicillin-resistant Staphylococcus aureus
MS mass spectrometry
MS muscle soreness
MS muscular strength
MSCA multilevel simultaneous data-analysis
135
MSG monosodium glutamate
MSH melanocyte-stimulating hormones
MSIA mass spectrometry immunoassay
MSL mestanolone
MSM methylsulphonylmethane
MS/MS tandem mass spectrometry
MSNA muscle sympathetic nerve activity
MSTFA N-methyl-N-(trimethylsilyl)trifluoroacetamide
MSIA mass spectrometric immunoassay
MSIA-HRAM mass spectrometric immunoassay with high resolution and
accurate mass
MSI-HR/MS mass spectrometric immunoassay with high resolution and
accurate mass detection
MSLT multiple sleep latency test
MS-MS tandem mass spectrometric
MSNA muscle sympathetic nerve activity
mSON medial supraoptic nucleus
MSPD matrix solid-phase dispersion
MSPE magnetic solid phase extraction
MST maximal speed training
MSTFA N-methyl-N-trimethylsilyltrifluoroacetamide
MSTN myostatin
MT methyltestosterone
M1T methyl-1-testosterone
3MT 3-methoxytyramine
mTb mean body temperature
MTB mountain bike
MTBE methyl tert-butyl ether
MT methyltestosterone
MT muscle thickness
MTC multiple transportable carbohydrates
mtDNA mitochondrial DNA
MTFA N-methyltrifluoroacetamide
mTOR mammalian target of rapamycin
mTORC mammalian target of rapamycin complex
mtPTP mitochondrial permeability transition pore
MTS methyltestosterone
mtTFA mitochondrial transcription factor A
MU methylnortestosterone undecanoate
MUFA monounsaturated fatty acids
MuRF1 muscle RING finger 1 protein
MVC maximal voluntary contractions
MVIC maximal voluntary isometric contractions
MyHC myosin heavy chain
MYOPRO myostatin propeptide
MV muscle volume
MVC maximal voluntary contraction
MVICT maximal voluntary isometric contraction testing
MW molecular weight
MWCNT multi-walled carbon nanotubes
MVIC maximum voluntary isometric contractions
MW molecular weight
136
6MWD 6-min walk distance
MWM Morris Water Maze
6MWT six-minute walk test
MX methylxanthine
MyHC myosin heavy chain
myoFSR myofibrillar fractional synthesis rate
m/z mass-to-charge ratio
N-3 omega-3 polyunsaturated fatty acids
NA nandrolone
NA noradrenaline
NA norandrosterone
NA nicotinic acid
NA nucleus accumbens
19-NA 19-norandrosterone
NAC N-acetylcysteine
NAc nucleus accumbens
Na₃C₆H₅O₇ sodium citrate
nAChR neural nicotinic acetylcholine receptor
NAd noradrenaline
NADH-TR nicotinamide adenine dinucleotide-tetrazolium reductase
NADO national anti-doping organisations
NAED 19-Norandrostenedione
NaHCO₃ sodium bicarbonate
NAN nandrolone
NANA N-acetylneuraminic acid
nanoLC-MS/MS nano liquid chromatography-tandem mass spectrometry
NARI noradrenergic reuptake inhibitor
NAS norandrosterone sulfate
NB net balance
NC nucleus circularis
NCAA National Collegiate Athletic Association
NCOLA noncola soft drink intake
NCE normochromatic erytrocyte
NCITA 4-chloro-19-nortestosterone acetate
ND nandrolone decanoate
NDP N-diethyl derivative
NE norepinephrine
NE noretiocholanolone
NE norephedrine
NE neuroenhancement
19-NE 19-noretiocholanolone
NEAA nonessential amino acids
NEC nonesterified carnitine
NECA 5'-N-ethylcarboxamidoadenosine
NEFA non-esterified fatty acids
NEJM New England Journal of Medicine
Neo5Gc N-glycolyl-neuraminic acid
NESP novel erythropoiesis stimulating protein
NET noradrenaline transporter
NEV non-ecologically valid
NF nuclear factor
NFL National Football League
NFLX norfluoxetine
137
NGB national governingbBodies
NGF nerve growth factor
NF nuclear factor
NFALD non-alcoholic fatty liver disease
NFkappaB nuclear factor kappaB
NFL National Football League
NG nandrolone glucuronide
NH normobaric hypoxia
NHANES National Health and Nutrition Examination Survey
NHL National Hockey League
NHTSA National Highway Traffic Safety Administration
NIC nicotine
NIIINP N-terminal propeptide of type III procollagen
NIR near infrared
NK natural killer
NMDA N-methyl-D-aspartate
NMDAr N-methyl-d-aspartate receptor
NMN normetanephrine
NMR nuclear magnetic resonance
NMT non-motorized treadmill
NND new nordic diet
NN-DMPPA N,N-dimethyl-2-phenylpropan-1-amine
NNH numbers needed to treat harm
nNOS neuronal nitric oxide synthase
NNT numbers needed to treat
NO nitric oxide
NO2− nitrite
NO3- nitrate
NOC National Olympic Committee
NOC normal menstrual cycles
NOLD nonoxidative leucine disposal
NOR norandrostenedione
NOR novel object recognition
NOR norepinephrine
NOREPH norephedrine
NOS nitric oxide synthase
NOS2 nitric oxide synthase
NOX NADPH oxidase
NOx nitrite + nitrate
NP nanoparticles
NP normal protein
NP nocturnal polyuria
NPB net protein balance
NPC neural progenitor cells
NPCE non-pharmacological cognitive enhancement
NPD nitrogen-phosphorus detector
NPE norpseudoephedrine
NPN nonprotein nitrogen
NPS novel psychoactive substances
NPS nonmedical use of prescription stimulants
NQI nutritional quality index
NQO nicotinamide adenine dinucleotide phosphate:quinone
oxidoreductase
138
NQO NAD(P)H dehydrogenase, Quinone
NR nuclear receptor
NR nonresponder
NRF nuclear respiratory factor
NRF NF-E2-Related Factor
NS nutritional supplement
NSAID nonsteroidal anti-inflammatory drug
NT nandrolone
NT nortestosterone
NTBI nontransferrin-bound iron
NTD amino-terminal domain
NTD amino-terminal trans-activation domain
NTF neuromuscular transmission failure
NW normal-weight
O2 oxygen
O2- superoxide anion radical
1
O2 singlet oxygen
OA osteoarthritis
OATP organic anion transporting polypeptides
OBLA onset of blood lactate accumulation
OC oral contraceptive
OC out of competition
OC organochlorine (pesticide)
OCD obsessive-compulsive disorder
3-OCMO-G gestrinone 3-carboxy-methyloxime
oCOR optimized carbon monoxide-rebreathing
OCP oral contraceptive pills
OCS oral contraceptive steroids
ODT oral disintegrating tablet
OCTN2 organic cation transporter 2
Odc ornithine decarboxylase
ODT Olympic-distance triathlon
OF oral fluid
OFF-Hr Score erythropoietic stimulation index
OFT open-field tests
OGTT oral glucose tolerance tests
OHA hydroxyandrosterone
OH-And hydroxyandrosterone
OH-Etio hydroxyetiocholanolone
4-OHA 4-hydroxy-androst-4-ene-17-dione
OhdG hydroxy-2-deoxyguanosine
OHE hydroxyetiocholanolone
16-OHE1 16alpha-hydroxyestrone
2-OHE1 2-hydroxyestrone
OHP hydroxyprogesterone
OH-P4 hydroxy-progesterone
OH-THC hydroxy-tetrahydrocannabinol
35-OHvitD3 11-deoxycortisol and 25-hydroxy-vitamin D3
OKG ornithine-alpha-ketoglutarate
OOC out-of-competition
ONS oral nutritional supplement
OPB oxyphenbutazone
139
OPIAD opioid-induced androgen deficiency
OPLS-DA orthogonal projection of latent structure discriminant analysis
OPN osteopontin
OR odds ratio
ORAC oxygen radical absorptive capacity
OROS-MPH osmotic-release oral system methylphenidate
Orn ornithine
ORS oral rehydration solution
ORX orchiectomized
OSL observed safe level
OSM osmolality
OST oral smokeless tobacco
OST osteocalcin
OT overtraining
OT oxytocin
OTC over-the-counter
OTSOC One-Touch Stockings of Cambridge
OVX ovariectomy
OW overweight
6-OXO androst-4-ene-3,6,17-trione
OXY oxyglobin
P4 progesterone
P5 pregnenolone
PA propionic acid
PAB pro-oxidant-antioxidant balance
PAD peripheral arterial disease
PADAM partial androgen deficiency of the aging male
PAG periaqueductal gray
PAGE polyacrylamide gel electrophoresis
PAH polycyclic aromatic hydrocarbons
PAH pulmonary arterial hypertension
PAI plasminogen activator inhibitor
PAP pulmonary arterial pressure
PAQ-C Physical Activity Questionnaire for Older Children
PARAFAC parallel factor analysis
PARP peroxisome proliferator-activated receptor
PASP Point Subtraction Aggression Paradigm
PAT pharmacological androgen therapy
p-ATP p-aminobenzenethiol
PAV peak angular velocity
Pax7 paired box gene7
PB protein breakdown
PB phenylbutazone
PBA pure beta-alanine
PBF percentage body fat
PBL peripheral blood lymphocytes
PBMC peripheral blood mononuclear cell
PC protein carbonyls
PC plasma caffeine
PC phosphatidylcholine
PCA principal component analysis
PCa prostate cancer
PCB polychlorinated biphenyls
140
PCB placebo
PCC protein carbonyl content
PcCr phospho-cyclocreatine
PCDD/PCDF polychlorinated dibenzo-p-dioxins/polychlorinated dibenzofurans
PCE polychromatic erytrocytes
PCE pharmacological cognitive enhancement
PCG peroxisome proliferator-activated receptor gamma co-activator
PCI positive chemical ionization
PCK phosphoenolpyruvate carboxykinase
PCO proanthocyanidin
PCOS polycystic ovary syndrome
PCR polymerase chain reaction
PCr phosphocreatine
PCV packed cell volume
PD pharmacodynamic
PD pregnandiol
PD probability discounting
PDA photodiode array
PDCAAS protein digestibility-corrected amino acid score
PDE phosphodiesterase
PDEGF platelet-derived epidermal growth factor
PDGF platelet derived growth factor
PDI protein disulphide isomerase
PDMS polydimethylsiloxane
Pdiol 5beta-pregnane-3alpha,20alpha-diol
PDQ-R Personality Disorder Questionnaire
PDTC pyrolidine dithiocarbamate
PDUS power-doppler ultrasonography
PE pseudoephedrine
PE performance enhancement
PE phytoecdysteroid
PE plasma expander
PE pulmonary embolism
PE phosphatidylethanolamine
PEA phenylethanamine
PEA phenethylamine
PEAS Performance Enhancement Attitude Scale
PED performance enhancing drug
PEDro Physiotherapy Evidence Database
PEF peak expiratory flow
PEG polyethylene glycol
PEG-EPO methoxy polyethylene glycol-epoetin beta
PEG-HAS polyethylene glycol conjugated with human serum albumin
PEH post-exercise hypotension
PE-IAT performance-enhancement related Implicit Associations Test
PEP performance enhancing polymorphisms
PEP performance enhancing products
PEP prepubertal
PEP pre- to early pubertal
Pep Pepsin
PEPCK phosphoenolpyruvate carboxykinase
PEPCK-C phosphoenolpyruvate carboxykinase
PEPH pseudoephedrine
141
PES performance enhancing substances
PES pooled effect size
PESA performance enhancement and sleep avoidance
PET positron-emission tomography
PetO2 O2 partial pressure
PF peak force
PF performance test
PF physical functioning
PF plantar fasciopathy
PF4 platelet factor 4
PFA perfluorocarbon
PFA Professional Footballers Association (of Great Britain)
PFC perfluorocarbons
PFC prefrontal cortex
PFC prefrontal cortical
PFC perfluorocarbon
PFD perfluorodecalin
PFMD perfluoro(methyldecalin)
PFCOC perfluorocarbon-based O2 carriers
PFPA pentafluoropropionic anhydride
PFT pulmonary function tests
PG progesterone
PGC peroxisome proliferator-activated receptor-gamma coactivator
PGE2 prostaglandin E2
PGF prostaglandin-F
pGH plasma growth hormone
pGHBP growth hormone binding protein
PGR progesterone receptor gene
PgR progesterone receptor
phGH pituitary human growth hormone
pHi intramuscular pH
PHR peak heart
PHY phytoestrogens
PHZ phenylhydrazine
PI phosphatidylinositol
Pi inorganic phosphate
PICI positive ion chemical ionization
PICP procollagen type I carboxy-terminal propeptide
PIED performance- and image-enhancing drugs/substances
PIF proteolysis-inducing factor
PIIINP N-terminal propeptide of type III procollagen
PI3K phosphatidylinositol 3-kinase
PINP N-terminal propeptide of type I procollagen
PIO2 inspired partial pressure of oxygen
PIP potentially inappropriate prescriptions
PIT pituitary
PK pharmacokinetic
pKa equilibrium constant
PKC protein kinase C
PKC proteolysis-inducing factor-induced activation of protein kinase C
PK/PD pharmacokinetic/pharmacodynamic
PKR protein kinase R
PL placebo
142
PL prohibited list
PLA placebo
PLAC placebo
PLC phospholipase C
PLCL L-lactide and epsilon-caprolactone
PLP pyridoxal 5'-phosphate
PLS partial least squares
pLSc preleptotine spermatocytes
PLS-DA partial-least squares-discriminant analysis
PM particulate matter
PMA methoxyamphetamine
PMA myristate-acetate
PMD phonomechanical delay
PMMA para-methoxy-N-methylamphetamine
PMMA polymethyl metacrylate
PMI percentage of migrated isoforms
PMM pectoralis major muscle
PMN polymorphoneutrophil
PMNL human polymorphonuclear leucocytes
plIGF plasma insulin-like growth factor
P-III-NP N-terminal pro-peptide of type III collagen, type 3 procollagen
Pn pontine nucleus
PNB purine nucleotide biosynthesis
PND postnatal day
PNS parasympathetic
PO plasma osmolality
PO power output
PO per oral
PO/AH preoptic area and anterior hypothalamus
POase paraoxonase activity toward paraoxon
POC polycosanols
POCIS polar organic chemical integrative samplers
POE polyoxyethylene
POE-PLP-Hb polyoxyethylene-conjugated pyridoxalated hemoglobin
POI primary ovarian insufficiency
polyQ polyglutamine
Poly-XLHb polymerized cross-linked hemoglobine
POMC proopiomelanocortin
POMS Profiles of Mood States
POP persistent organic pollutants
PP peak power
PP protein precipitation
P-III-P type 3 pro-collagen
PPA physical activity per week
PPA phenylpropanolamine
PPAR peroxisome proliferator-activated receptors
PPARD peroxisome proliferator-activated receptor delta
PPC Partnership for Clean Competition
PPCP pharmaceuticals and personal care products
PPI peak power improvement
PPI prepulse inhibition (of the startle response)
PPIB cyclophilin b
143
PPO peak power output
P-PRF pure platelet-rich fibrin
P-PRP pure platelet-rich plasma
PPT pressure pain threshold
PPT postprandial triglyceridaemia
PPY peptide YY
pQCT peripheral quantitative computed tomography
PR progressive ratio
PR progesterone receptor
PR progestin receptors
PRC peroxisome proliferator-activated receptor gamma co-activator
PRDX peroxiredoxin
PREG pregnenolone
PRF platelet-rich fibrin
PRFM platelet rich fibrin matrix
PRGF plasma rich in growth factors
P-PRP pure platele.trich plasma
PRC packed red cells
PRCA pure red cell aplasia
PRED prednisone
PRED prednisolone
PRES posterior reversible encephalopathy syndrome
PRFD peak rate of force development
PRICE protection, rest, ice, compression, elevation
PRISMA preferred reporting items for systematic reviews and meta-
analyses
PRL prolactin
PRO protein
PRO propranolol hydrochlorid
PRO probiotic
PROG progesterone
PROP-1 prophet of pit-1
PROT protein
PRP platelet-rich plasma
PRPI platelet-rich plasma injection
PRPT platelet-rich plasma therapy
PRT periodized resistance training
PRX pre-exercise sports drinks
PS phosphatidylserine
PS protein synthesis
PSA prostatic specific antigen
PSA pseudoephedrine
PSAE participation in sports, athletics or exercising
PSE pseudoephedrine
PSG polysomnography
PSH plasma thiols
p70(S6K) ribosomal protein S6 kinase
PSS poly(sodium 4-styrenesulfonate)
PSS predator-scent stress
PT performance test
PT peak torque
PT thromboplastin time
144
PTF peak twitch force
PTFE polytetrafluoroethylene
PTH parathyroid hormone
PTM post-translational modifications
PTOA posttraumatic osteoarthritis
PTS propionate
PTSD post-traumatic stress disorder
PTU propylthiouracil
PUFA polyunsaturated fatty-acid
PV plasma volume
P-V pressure-volume
PVC polyvinyl chloride
PVC premature ventricular contractions
PVD peripheral vascular disease
PVE plasma volume expanders
PVN paraventricular nucleus of the hypothalamus
PVP polyvinylpyrrolidone
PVT power output at ventilatory threshold
PVT paraventricular thalamus
PWC physical working capacity
PWCFT physical working capacity at fatigue threshold
PWCFT physical working capacity at fatigue threshold
PWEDWT posterior wall end diastolic wall thickness
PWS pre-workout supplements
PYK2 proline-rich tyrosine kinase 2
Py-MAB-TOF-MS pyrolysis metastable atom bombardment time-of-flight mass
spectrometry
PYY peptide YY
Q10 coenzyme Q10
QAD quaternary ammonium drugs
QC quality control
QD quantum dot
QCM quartz crystal microbalance
QED-MS/MS quantitation-enhanced data-dependent MS/MS
QF quadriceps femoris
qNMR quantitative nuclear magnetic resonance
QOL quality of life
qPCR quantitative polymerase chain reaction
qRT quantitative real-time
qRT-PCR quantitative reverse transcriptase polymerase chain reaction
QSAR quantitative structure-activity relationships
QSRR quantitative structure-retention relationship
QqQ triple quadrupole
QqToF quadrupoleTime-of-Flight
QTOFMMS/MS dual-sprayer quadrupole time-of-flight mass spectrometry
QSAR quantitative structure-activity relationship
QSRR quantitative structure-retention relationship
QTc corrected QT interval
QTOF quadrupole-time of flight
QToF quadrupole time-of-flight
QTOFMS quadrupole time-of-flight mass spectrometry
QTR quarters
QTRAP quadrupole linear ion trap
145
Ra rates of appearance
RAAM reagent array analysis method
RAAP antiandrogenic potency relative to bicalutamide
rAAV recombinant adeno-associated viral vector
RAB risk assessment behaviors
RAC ractopamine
RANG relaxed arm angle
RANKL receptor activator nuclear factor-kappaB ligand
rANOVA repeated measures analysis of variance
RANTES regulated on activation, normal T cell expressed and secreted
R-AO restricted-antioxidant
R-AO restricted-antioxidant
RAS renin-angiotensin system
RAST running anaerobic sprint test
RAT reactive agility test
Rb retinoblastoma
RBC red blood cell
RBC# absolute red blood cell
RBCM red blood cell mass
RBCV red blood cell volume
RBL residual bilinearization
RBML rapid body mass loss
RCAN regulator of calcineurin
RCI Racing Commissioners International
RCP respiratory compensation point
RCT respiratory compensation threshold
RCT randomised controlled trials
RCV red cell volume
RD recreational drugs
Rd rates of disappearance
RDA recommended dietary allowances
RDS relative standard deviation
RDW red blood cell distribution width
RE resistance exercise
RE running economy
REE resting energy expenditure
reGH recombinant equine growth hormone
REI relative energy intake
REM rapid eye movement
REP erythropoietin-producing cells
rEPO recombinant erythropoietin
RER respiratory exchange ratio
RES resistance exercise scheme
RES resveratrol
RET resistance exercise training
RET retroperitonial adipose tissue
Ret reticulocytes
Ret % reticulocytes percentage
RET# absolute reticulocyte
RetHct reticulocyte hematocrit
RETN resistin
RF rectus femoris
RFD rate of force development
146
rFSH recombinant FSH
rFst recombinant follistatin
RGA reporter gene assays
rGH recombinant growth hormone
RGN regucalcin
rhAR recombinant human androgen receptor
rhCG recombinant human chorizon gonadotropin
rHCG recombinant HCG
rhEPO recombinant human erythropoietin
rhFSH recombinant human follicle-stimulating hormone
rhG-CSF recombinant human granulocyte colony-stimulating factor
rhGH recombinant human growth hormone
RHI reactive hyperemia index
rhIGF recombinant human insulin like growth factor
rhIGFBP recombinant human insulin-like growth factor-I binding protein
RHS rebound hypersomnolence
rhSHBG recombinant human sex hormone-binding globulin
rHuEPO recombinant human erythropoietin
RIA radioimmunoassay
RIM racing intact males
rLH recombinant LH
RLX relaxin
RM remobilization
RM repetition maximum
1-RM one repetition-maximum
10-RM ten repetition maximum
Rmax maximum rate of oxidation
1RMecc one repetition maximum eccentric
RMR resting metabolic rate
RMS root mean square
RMSEP root mean squares error of prediction
RMSSD root mean square of successive differences
RMST reactive motor skills test
rMV relative mobility values
RNA ribonucleic acid
RNAi RNA interference
RNI recommended nutrient intake
ROM range of movement
ROM reactive oxygen metabolites
RONS reactive oxygen and nitrogen species
ROOM ranges of motions
ROS reactive oxygen species
ROS/RNS reactive oxygen/nitrogen species
RP relative potencies
RP reversed-phase
RPE ratings of perceived exertion
RPE-C ratings of perceived exertion for the chest
RPE-L ratings of perceived exertion for the leg
RPE-O ratings of perceived exertion for the overall body
RPF ratings of perceived fatigue
RPF renal plasma flow
RP-HPLC reversed phase high performance liquid chromatographic
RPLC reversed phase liquid chromatography
147
RP-LC-HRMS reversed-phase interaction chromatography systems
RPP rate pressure product
RQ respiratory quotient
RR respiration rate
RR Rhodiola rosea
RR relative risk
RR risk ratios
rRE resistance exercise
RRT randomized response technique
RRT relative retention time
RS restraint stress
RSA roentgen stereophotogrammetric analysis
RSA repeated sprint ability
RSb best repeated sprint
RSD relative standard deviation
rsFC resting-state functional connectivity
RSH repeated sprint training in hypoxia
RSI reactive strength index
RSI reactive strength index
RSm mean repeated sprint
RST repeated-sprint tests
RT resistance training
RT reaction time
RT reverse-transcriptase
RT rectal temperature
RTD ready to drink supplements
RTD rate of torque development
RTF repetitions to fatigue
RTFF run time to fatigue
RTIME readiness to invest mental effort
RTIPE readiness to invest physical effort
RTLM right thigh non-osseous fat-free mass
RT-PCR real-time polymerase chain reaction
RU response unit
RUCAM Roussel Uclaf Causality Assessment Method
RV right ventricular hypertrophy
RVD right ventricular diameter
RVH right ventricular hypertrophy
RVP rapid visual ipnformation processing
RVSD right ventricular systolic dysfunction
RVSP right ventricular systolic pressure
RW raw wastewater
RWL rapid weight loss
RXR retinoic X receptor
SA synthetic androgen
SA semen analysis
sAA salivary alpha amylase
SABA short-acting beta2-adrenergic receptor agonist
SACS supplemented ambulatory control subjects
SAFE Safe and Fair Events
SAGE Serial Analysis of Gene Expression
SAL salbutamol
SAL saline
148
SaO2 oxygen saturation
SAR sodium N-lauroylsarcosinate
SAR sarcosyl
SARM selective androgen receptor modulator
SAR-PAGE sarcosyl polyacrylamide gel electrophoresis
SB sodium bicarbonate
SB sibutramine
SBC sodium bicarbonate
SBJ standing broad jump
SBMA spinal and bulbar muscular atrophy
SBP systolic blood pressure
SBPV systolic blood pressure variability
SC subcutaneous
SC synthetic cannabinoids
SC satellite cells
SC Sertoli cells
s.c. subcutaneous
SCA cross-sectional area
SCAD severe combined auto-immuno deficiency
SCB synthetic cannabinoids
SCD sudden cardiac death
scFv single-chain antibody fragment of variable region
SCG sodium cromoglycate
SCI spinal cord injury
SCID severe combined immunodeficiency
SCL skin conductance level
SCL-90 Symptoms Check List-90
SCM combat maneuvers
SCRTT choice serial reaction time task
SD socially desirable
SD sports drink
SD sexual dysfunction
SD sleep deprivation
Sd spermatids
SDA Sports Dietitians Australia
SDMA symmetrical dimethylarginine
SDME single-drop microextraction
SDS standard deviation score
SDS sodium dodecyl sulfate
SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis
SDS/SAR-PAGE sodium dodecyl sulfate/sarcosyl polyacrylamide gel
electrophoresis
SDT signal detection task
SDT self-determination theory
SE strength endurance
Se selenium
SEC size exclusion chromatography
SEC-HPLC size-exclusion high performance liquid chromatography
SED sports and energy drink
SEF sensitivity enhancement factors
SELDI surface enhanced laser desorption/ionization
SELDI/TOF surface-enhanced laser desorption/ionization time-of-flight
SEM structural equation modeling
149
SEM scanning electron microscopy
SEM sport and exercise medicine
SEP synthetic erythropoiesis stimulating protein
sEpo serum erythropoietin
SePP selenoprotein P
SERM selective estrogen receptor modulator
SERS surface-enhanced Raman scattering
SERT serotonin transporter
SES socioeconomic status
SES strength exercise session
SET standard exercise test
SEV slow eccentric velocity
SF safety factor
SF serum ferritin
SFA saturated fatty acids
sFe serum iron
sFer serum ferritin
SF-Hb stroma free hemoglobin
SFR structure-fragmentation relationship
SFT superficial flexor tendon
SG specific gravity
SGLT sodium dependent transporter
SGRQ St. George's Respiratory Questionnaire
SH sulphydryl
-SH thiol
SHBG sex hormone-binding globulin
SHKS supplemented hypokinetic subjects
SHR spontaneously hypertensive rats
SI System of Units
SI socially isolated
SI serum iron
SI suicidal ideation
SIADH syndrome of inappropriate antidiuretic hormone secretion
SID stable isotope dilution
SID-LC/ESI/SRM/MS stable isotope dilution liquid chromatography electrospray
ionization selected reaction monitoring mass spectrometry
SIgA salivary immunoglobulin A
sIGF-1 saliva insulin-like growth factor-1
SIH sodium-induced hyperhydration
SIM selected ion monitoring
sIns submaximal insulin
SIR sirtuin
siRNA small interfering ribonucleic acid
SIRT sirtuin
Sirt silent information regulator transcript
SIRT silent information regulator transcript
SIS steroid isotopic standards
SJ static jumping
SJ squat jump
SJFT Special Judo Fitness Tests
SL sheath liquid
SL sea level
SLCO solute carrier organic anion transporter
150
SLE sorbent-supported liquid extraction
SLE solid-liquid extraction
SLF superior longitudinal fasciculus
SLGT sodium-dependent glucose transporter
SLM supported liquid membrane
SLR systematic literature review
SLT smokeless tobacco
SM sphingomyelin
SM skim milk
SM secondary metabolites
SM skeletal muscle
SMD standardized mean differences
SME submaximal heavy resistance exercise
SMR standardized mortality ratio
SMP soluble milk protein
SMT sulfatoxymelatonin
S/N signal to noise ratio
SNAT system A transporter
SNB spinal nucleus of the bulbocavernosus
sNGF salivary nerve growth factor
SNP single-nucleotide polymorphism
SNS sympathetic nervous system
SOD superoxide dismutase
SOL soleus
SORT strength of recommendation taxonomy
SOT sotalol
SP sodium phosphate
SP substance P
SPD sports drinks
SPE solid phase extraction
SPE-ELISA sensitive solid-phase extraction-enzyme-linked immunosorbent
assay
SPE-XCW exchange solid phase extraction
SPF sun protection factor
SPG Shirasu porous glass
spiPCR primer-internal, intron-spanning PCR
SPM suspended particulate matter
SPME solid-phase microextraction
SpO2 oxygen saturations
SPR surface plasmon resonance
SPRi surface plasmon resonance imaging
SPS speed-precision-success
SPT sucrose preference test
SQ standardized questionnaire
SQ squat
SR sarcoplasmic reticulum
SRBA slow-release beta-alanine
SRF strength of religious faith
SRH self-rated health
SRI strain rate imaging
SRM selected reaction monitoring
SRS short run session
SRY sex-determining region of Y
151
SS steady status
SS somatostatin
SS subsarcolemmal
SSA succinic semialdehyde
SSB sugar-sweetened beverages
SSC spermatogonial stem cells
SSE steadystate endurance exercise
SSF subcutaneous fat
SSH sex steroid hormones
SSI sonic spray ionization
SSIW somatostatine infusion withdrawal
SSO sedentary sham-operated
SST serial subtraction test
SSTF (Endocrine) Society’s Scientific Statement Task Force
SSTI skin and soft tissue infection
ST smokeless tobacco
ST stanozolol
ST somatotropin
STA systematic toxicological analysis
STAC sirtuin 1 activating compounds
STAGE Swedish Twin Adults: Genes and Environment
STAN stanozolol
StAR steroidogenic acute regulatory protein
STAT signal transducers and activators of transcription
STB standardbred
STBP stanozolol-binding protein
STD seminiferous tubuler diameter
STEMI ST-elevation myocardial infarction
sTfR soluble transferrin receptor
STGC simulated team game circuit
sTnI skeletal troponin I
STP sewage treatment plants
sTP saliva total protein
STR short tandem repeat
STR striatum
s-TRAP serum antioxidant potential
STS strength training session
STZ stanozolol
SUD substance use disorder
SULT sulphate transferase
SUM substance use and misuse
SUM sum of the adipose tissues
SUN serum urea-N
SUNSR standardized rate of urea nitrogen synthesis
SUP supplement
SV stroke volume
SVM support vector machine
SPE solid-phase extraction
SPR surface plasmon resonance
SVR systemic vascular resistance
SWNT single-wall carbon nanotubes
SWT swim-trained
Sz stanozolol
152
T testosterone
T3 triiodothyronine
T4 thyroxine
TA triamcinolone acetonide
TA tibialis anterior
TAA total antioxidant activity
TAA total amino acids
TAC total antioxidant capacity
TAC total alkaloids content
TACO transfusion associated circulatory overload
tACS transcranial alternating current
TAFLD toxicant-associated fatty liver disease
TAG triglycerides
TAG triacylglycerol
TAM tamoxifen
Tam tamoxifen
TAP tapering period
TAS total antioxidant status
TASH toxicant-associated steatohepatitis
TAT tyrosine aminotransferase
TAU taurine
TB thoroughbred
TB trenbolone
TBA thiobarbituric acid
TBA trenbolone acetate
TbA trenbolone acetate
TBARS thiobarbituric-acid-reactive substances
TBG thyroxine binding globulin
TBI traumatic brain injury
TBLM total body non-osseous lean mass
TBME tert-butyl-methyl-ether
TBO trendione
TBOH trenbolone
TBV total blood volume
TBW total body water
TBX thromboxane
TC total cholesterol
TC testosterone cypionate
TC target compounds
TC trapping column
T/C testosterone/cortisol ratio
TCA tricarboxylic acid
TCAR total carnitine
TCEP tris(2-carboxyethyl)phosphine hydrochloride
TCM traditional Chinese medicine
Tcore core body temperature
TCr total creatine
tCS transcranial current stimulation
TCT tone cue task
TD testosterone deficiency
TD testosterone deconate
TD total distance
tDCS transcranial direct current stimulation
153
TDI tissue Doppler imaging
TDI tolerable daily intake ¨
T2DM type 2 diabetes mellitus
tDNA transgenic DNA
TDS testosterone deficiency syndrome
TE testosterone
TE time to exhaustion
TE testosterone enanthate
T/E testosterone to epitestosterone ratio epitestosterone
T/E testosterone/epitestosterone (ratio)
TEAC trolox equivalent antioxidant capacity
TEE total energy expenditure
TEF thermic effect of food
TEG thrombelastographic
TEI total energy intake
TEM transmission electron microscopy
TEMPO tetramethylpiperidinyl-1
tEPO transgenic EPO
TER terbutaline
TES testosterone
TEST testosterone
TFA total fatty acids
TFAA trifluoroacetic acid
TFC total flavonoids content
TFAM mitochondrial transcription factor A
TFM trifunctional folate-metabolizing enzyme
TF-SPME solid phase microextraction in thin film geometry
TG triglyceride
TG testosterone glucuronide
TG triacylglycerol
TGF transforming growth factor
TG/EG testosterone glucuronide to epitestosterone glucuronide ratio
TGSH total glutathione
TH tyrosine hydroxylase
tHB total hemoglobin
tHb-mass total hemoglobin mass
THC tetrahydrocannabinol
THCA 11-nor-delta9-tetrahydro-cannabinol-9-carboxylic acid
THC-COOH 11-nor-9-carboxy-delta9-tetrahydrocannabinol
THC-OH 11-hydroxy-delta9-tetrahydrocannabinol
THCV tetrahydrocannabivarin
THG tetrahydrogestrinone
THE cortisol, tetrahydrocortisone
THF tetrahydrocortisol
THG tetrahydrogestrinone
THP tetrahydroprogesterone
THP tetrahydropyranyl ether
THS desoxy-tetrahydrocortisol
TI testosterone isocaproate
TIBC total iron binding capacity
TIC total ion current
t.i.d three times a day
tIGF total insulin-like growth factor
154
TIM tracer interaction methodology
TIMP tissue inhibitor of metalloproteinases
tITP transient isotachophoresis
TKD taekwondo
TLC thin layer chromatography
T/LH testosterone to luteinizing hormone ratio
Tlime running to volitional exhaustion
TM trajectory method
TMA trimethylamine
TMAO trimethylamine-N-oxide
Tmax time to exhaustion
TMB tetramethylbenzidine
TMD total mood disturbance
TMg total magnesium
TMIS trimethyliodosilane
TMN hypothalamic tuberomammillary nucleus
TMS transcranial magnetic stimulation
TMS trimethylsilyl
TMSIh trimethyliodosilane/methyl-N-trimethylsilyltrifluoroacetamide
TMX tamoxifen
TMZ trimetazidine
TNF tumor necrosis factor
TNFR tumor necrosis factor-alpha receptor
TNL thermoneutral conditions
TOF time of flight
TOF/MS time-of-flight mass spectrometry
TOP topical
TP testosterone propionate
TP testosterone pellets
TP total protein
tPA tissue plasminogenactivator-antigen
TPB theory of planned behavior
TPC total phenolics content
Tp-e end of the electrocardiographic T wave
TPH tryptophan hydroxylase
TPN total parenteral nutrition
TPP testosterone phenyl propionate
TPP total plasma protein
T-PRO total protein
TQ-MS/MS triple quadrupole mass spectrometry
TRA theory of reasoned action
TRALI transfusion related acute lung injury
TRAP total radical-trapping antioxidant potential
TR-CAF time-release caffeine containing supplement
TRE time to running exhaustion
TRE trenbolone
TRE trehalose
Tre rectal temperature
Trec rectal temperatures
TREN trenbolone-enanthate
TRF transferrin
TRIM transfusion associated immunomodulation
TPR total peripheral resistance
155
TRP time release capsule
Trp tryptophan
TRS thermogenic supplement
TRT testosterone replacement therapy
TRYP tryptophan
TS transferrin saturation
TS taurine supplementation
TS testosterone suspension
TS total-sprint
Ts team sports
Tsat transferrin saturation
TSB tryptic soy broth
TSEC tissue-selective estrogen complex
TSH thyroid stimulation hormone
Tsk skin temperature
tSOD total superoxide dismutase
TSP total suspended particulate matter
TST tail suspension test
TST total sprint times
TST testosterone
TST testosterone supplementation therapy
TT total testosterone
TT Tribulus terrestris
TT time trial
Ttail tail skin temperature
TT/C testosterone/cortisol ratio
TTE time to exhaustion
TTF time to fatigue
Tty tympanic temperature
TU testosterone undecanoate
TUD testosterone undecanoate
TUE therapeutic use exemption
TUEC therapeutic use exemptions committee
TVP textured vegetable protein
TW total work
TWC total work completed
TWD total work done
TWI total water intake
TYMP tympanic temperature
TYR tyrosine
TXB thromboxane B
TZD thiazolidinedione
UA uric acid
UACS unsupplemented ambulatory control subjects
UAE ultrasound assisted extraction
UAMA upper arm muscle area
UAMS unmodified acid/alcohol-modified cornstarches
Ub ubiquitin
UBISP upper-body intermittent sprint performance
UCI International Cycling Union
UCLA University of California at Los Angeles
Ucol urine color
UCP uncoupling protein
156
UDP uridine diphosphophate
uCr urinary creatinine
UEFA Union of European Football Associations
uEPO urinary erythropoietin
UER ultra-endurance runners
UG UDP-glucuronosyltransferases
uGF urinary growth hormone
UGT uridine diphospho-glucuronosyl transferases
uhEPO urinary erythropoietin
UHPLC ultra-high pressure liquid chromatography
UHPLC/MS ultra high-performance liquid chromatography-mass
spectrometrometry
UHPLC-MS/MS ultra-liquid chromatography-tandem mass spectrometry
UHPLC-ESI-MS/MS ultra-high pressure liquid chromatography coupled to electrospray
ionization tandem mass spectrometry
UHPLC-TOFMS ultra-high-performance liquid chromatography electrospray
time-of-flight mass spectrometry
UHPLC-Q-Orbitrap-HRMS ultrahigh-performance liquid-chromatography-
quadrupole-orbitrap high resolution mass spectrometry
UHPSFC ultra-high-performance supercritical-fluid chromatography
UK United Kingdom
UL upper limits
uIGF-1 urinary insulin-like growth factor-1
UNESCO United Nations Educational, Scientific and Cultural Organization
UNSR urea nitrogen synthesis
Uosm urine osmolality
UPLC upper-body power and lower-body strength
U-PLC unfolded partial least-squares
UPLC ultra performance liquid chromatography
UPLC-MS ultra-performance liquid chromatography-mass spectrometry
UPLC-MS ultra-performance liquid chromatography-mass spectrometry
UPLC-MS-MS ultra-performance liquid chromatography-tandem mass
spectrometry
UHPLC/TOFMC ultra-high performance liquid chromatography combined with time-
of-flight mass spectrometry
UHPLC-QTOF-MS ultra-high-pressure liquid chromatography coupled to a
quadrupole time-of-flight mass spectrometry
UPLC-QqQ-MS ultrahigh-performance-LC-electrospray ionization-triple-
quadrupole-MS
UPLC-UV ultra-performance liquid chromatography with ultraviolet detection
URCR urinary creatine
URCRN urinary creatinine
URI upper respiratory infection
URT upper respiratory tract
URTI upper respiratory tract infection
US ultrasonography
USADA United States Antidoping Agency
USATF USA Track and Field
USDA US Department of Agriculture
USG urine specific gravity
Usg urine specific gravity
USMS United States Masters Swimming
USOC US Olympic Committee
157
USP unique selling proportion
USP United States Pharmacopeia
USPIO ultrasmall superparamagnetic particles of iron oxide
UTI urinary tract infections
utPr urinary total protein
UTPT Unique Trait Personality Theory
UUN urine urea nitrogen
UUPS underperformance syndrome
UV ultraviolet
UVB ultraviolet B
UV-vis ultraviolet and visible spectroscopy
UWP undenatured whey protein
%VA percent voluntary activation
VAS visual analog scale
VAT visceral adipose tissue
vBMD volumetric bone mineral density
VC vitamin C
VC vascular conductance
VCO2 carbon dioxide production
VCT Vogel conflict test
VCT visual cue task
VCAM vascular cell adhesion molecule
VDR vitamin D receptor
VDRE vitamin D response elements
VE vaginal estrus
VE ventilation
VEGF vascular endothelial growth factor
VEGFA vascular endothelial growth factor A
VEpeak peak ventilation
VF ventricular fibrillation
VHIR very high intensity running
VISA-A Victorian Institute of Sport Assessment – Achilles
VISA-P Victorian Institute of Sport Assessment – Patella score
VJ vertical jumps for height
VL vastus lateralis
VLCAD very long chain acyl-CoA dehydrogenase
VLDL very low-density lipoprotein cholesterol
VLH ventrolateral hypothalamus
VLT velocity at lactate threshold
VM Valsalva manouver
vMALDI vacuum matrix-assisted laser desorption ionization
VMHVL ventromedial hypothalamus
VMN ventromedial nucleus
VMS vitamin/mineral supplements
VO vaginal opening
Vo velocity
VO₂ oxygen consumption
VO2peak peak oxygen consumption
VO2max maximal oxygen uptake
4-VP 4-vinylpiridine
VPDP Vienna PeeDee Belemnite
V2R vasopressin type 2 receptor
VRM visual recognition memory
158
VSM vibrating sample magnetometer
VSMC vascular smooth muscle cells
VT ventilatory threshold
VT tidal volume
VTA ventral tegmental area
VTE venous thromboembolic events
Vv volume density
vWF von Willebrand factor
W power output (Watt)
VVA vulvovaginal atrophy
WAA whey amino acids
WADA World Anti-Doping Agency
WAADS World Association of Anti-Doping Scientists
WAC Wingate Anaerobic Capacity
WADC World Anti-Doping Code
WAnT Wingate anaerobic test
WanT Wingate test
WAS weight-altering supplements
WBC white blood cells
WBE wastewater-based epidemiology
WBPT whole-body protein turnover
WBGT wet bulb globe temperature
WBPB whole body protein balance
WBPT whole-body protein turnover
WBPTO whole-body protein turnover
WBS whole-body strength
WBSF Warner-Bratzler shear force
WCID whole column imaging detection
WDEIA wheat-dependent exercise-induced anaphylaxis
WE walking exercise
WG Wingate test
WG weeks of gestation
WGA wheat germ agglutinin
WGH wheat gluten hydrolysate
WGO wheat germ oil
WHO World Health Organization
WHR waist-to-hip ratio
WIH water-induced hyperhydration
WISP WNT1-inducible-signaling pathway protein
WM weight management
Wmax maximum power output
WM weight-maintenance
WM white matter
WMD weighted mean difference
WMP Wingate power test on a cycle ergometer maximum
WOC World Championships
WOMAC Western Ontario and McMaster University's Arthritis Index
vVO2max minimum speed associated with VO2max
vVO2peak velocity that elicited VO2peak
WP whey protein
WPC whey protein concentrate
WPH whey protein hydrolysates
WPI whey protein isolate
159
WPP Wingate paek power test on a cycle ergometer maximum
WR world records
WRA women of reproductive age
WT wild type
WT wrist temperature
VWR voluntary wheel running
WWTP wastewater treatment plants
XA xanthurenic acid
XCS cross-country skiing
XIC extracted ion current
XLHb cross-linked hemoglobine
XO xanthine oxidase
XRD X-ray diffraction
YAS yeast androgen receptor reporter system
YMRS Young Mania Rating Scale
YOG Youth Olympic Games
Yo-Yo IR2 Yo-Yo intermittent recovery test level 2
YPHV years from peak height velocity
YSR Youth Self-Report
ZH zilpaterol hydrochloride
ZMA zinc monomethionine aspartate
ZMA zinc/magnesium aspartate
Zn zinc
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BACKGROUND FOR TODAY’S DOPING PROBLEMS
The use of doping is linked with the history of sports. Doping abuse escalated until the mid
sixties when government and sports authorities responded with antidoping laws and drug
testing. Today, the details of substances detected in controls give a good indication on the
importance of doping use. Three classes of pharmaceuticals account for most of the positive
controls. They are anabolic steroids, stimulants and narcotics. Their use can be related with
the goal of the athletes. Anabolic steroids are mainly used in sports such as bodybuilding or
weight lifting in order to develop strength. Stimulants are used in sports were speed favors
performance. All the products that enhance blood oxygen transportation are used in
endurance sports, their efficacy is not scientifically demonstrated, but their use does result in
real risks. Several studies have evidenced the medical problems resulting from prolonged
doping. Doping control is impaired by the fact that many products now used, e.g. EPO or
rhGH, are not detectable. Regular medical examination of athletes could help prevent use of
doping [01001].
Medal-winning athletes are the undisputed icons of society. As role models, they are also
expected to show impeccable character and behaviour. Society rewards them with
admiration and dedication, in no way objecting to exorbitant financial gains by their idols;
governments and companies consider athletes' triumphs as advertisements for their politics
and products respectively. In addition to exhaustive training, this places athletes under
tremendous pressure, not only to excel in their discipline, but also to resist the temptation to
use any illicit means, e.g. drugs, to enhance performance. “Play true” is the motto of the
World Anti-Doping Agency (WADA), appealing to all athletes to refrain from using illicit drugs,
but without the elaborate worldwide network of doping controls and sanctions against doping
(www.wada-ama.org), WADA's call for fairness would remain without echo. However, doping
is not reserved for the small squad of elite athletes, it has spread from the idols at the top to
all rank and file participants in sports, from adolescents to seniors. Not only classical sport
disciplines are involved; the phenomenon is similarly widespread among bodybuilders, so
that the drugs used have been collectively classified as “appearance and performance-
enhancing drugs”s (APEDs) and summarised in the WADA Prohibited List 2015 (www.wada-
ama.org). Although doping has been practiced since antiquity, often with placebo or toxic
effects, really effective APEDs only became available with the rise of modern pharmacology,
and in particular, following the isolation and synthesis of testosterone and AASs.
Testosterone came into clinical use shortly after its synthesis in 1935 and its first
documented use for doping was by German rowers in 1952 (to maintain their marital duties
during exhausting training) and by Russian weight lifters in 1954 to enhance their power.
Since then AASs lead the lists of APEDs worldwide and among these testosterone is used in
almost 50 percent, be it in the 4500 doping-positive samples collected by WADA worldwide
in 2012 or be it among black market substances confiscated by customs and police. As all
licensed testosterone and AAS preparations are available only by prescription, the drug
sources remain obscure. In part, these substances are no longer or have never been on the
open market. There have been instances doctors prescribing AASs especially under
pressure from bodybuilders, who undertake any risk to become champions. Surveys among
fitness centre clientele revealed that up to half of APED users obtained the drugs with or
without prescription from physicians or pharmacies. Labs in Eastern Europe, Asia and South
America producing multitudes of AASs offer them for sale on the internet which, next to gyms
and fitness studios, has become the major source of AASs. When counted in November
2011, there were 328 000 internet pages accessible under the search term “steroids for sale”!
Furthermore, AASs may be added to food supplements – often undeclared on the label or
found in phytopharmaka and animal organ extracts. For example, musk pods used in
Chinese traditional medicine contain 16 different, undeclared AASs, as discovered in doping
161
control. Finally, secret but official programmes of sport organisations or states may provide
AASs and other APEDs to their athletes, as demonstrated by the systematic doping
programme of the former German Democratic Republic (GDR) in the 1970s and 1980s,
which became evident after the collapse of that règime in 1989 [150001].
Doping in sport is a widespread problem not just among elite athletes, but even more so in
recreational sports. In scientific literature, major emphasis is placed on doping detection,
whereas detrimental effects of doping agents on athletes' health are seldom discussed.
Androgenic anabolic steroids are well known for their positive effects on muscle mass and
strength. Human growth hormone also increases muscle mass, although the majority of that
is an increase in extracellular fluid and not the functional muscle mass. In recreational
athletes, growth hormone does not have major effect on muscle strength, power or aerobic
capacity, but stimulates anaerobic exercise capacity. Erythropoietin administration increases
oxygen-carrying capacity of blood improving endurance measures, whereas systemic
administration of beta-adrenergic agonists may have positive effect on sprint capacity, and
beta-adrenergic antagonists reduce muscle tremor. Thus, there are certain drugs that can
improve selective aspects of physical performance. However, most of the doping agents
exert serious side-effects, especially when used in combination, at high doses and for a long
duration. The extent of long-term health consequences is difficult to predict, but likely to be
substantial, especially when gene doping is considered. One review summarises the main
groups of doping agents used by athletes, with the main focus on their effects on athletic
performance and adverse effects [15002].
Amateur athletic competition must occur on an even playing field. To that end, performance
enhancing agents are prohibited in amateur athletic competition so that only athletic skills
can determine outcomes. Although performance enhancing agents, especially
amphetamines and anabolic steroids, were used throughout the past century by Olympic
athletes, it was not until 1967 that the International Olympic Committee Medical Commission
listed specific drugs as banned substances, and athletes were disqualified for taking such
agents. Yet the rules are specific, if a drug is not listed as a banned substance, the athlete
should not and cannot be disqualified for having taken such agent. This brings one to the
crux of the problem in athletics; the spirit and letter of the law. The letter of the law is exact,
the spirit of the law is much broader. It is no longer a question of the pleasure of competition,
or the glory of athletic victory, but now endorsements/appearances/results are major
economic factors. When athletes were purely amateurs, there was a greater willingness to
accept the spirit of the law. When athletes and coaches realised that a non-banned
substance would result in performance enhancement, these were not always used. But not
all athletes, coaches, or countries were honest. Perhaps the greatest stain on amateur
athletics and sports physician credibility was the national “doping” programme developed by
East Germany, which included unethical experimentation on minors and the specific
research intent of finding means of administration of substances that would evade
international control [05003].
More than two thousand years ago, naked athletes competed at the Olympic games in
ancient Athens for eternal fame and an olive branch. Today, most athletes run, jump or swim
not only for fame and honour but also for money – after all, a gold medal is the ticket to
lucrative advertising contracts. Not surprisingly, professional sports now resemble high-tech
races in which any technological trick is used to gain milliseconds to set the next record. This
involves not only designing faster bobsleds, racing bikes or shark-skin swimsuits but also
pushing the physical abilities of athletes by using the latest medical and biological research
[07001].
Since ancient times, unethical athletes have attempted to gain an unfair competitive
162
advantage through the use of doping substances, even though the methods and drugs have
varied over time. Nowadays a list of doping substances and methods banned in sports is
published yearly by the World Anti-Doping Agency, and what is on that list is defined as
doping irrespectively if enhance performance or not. A substance or method might be
included in the list if it fulfills at least two of the following criteria:
- enhances sports performance

- represents a risk to the athlete's health
- violates the spirit of sports.
This list, yearly updated to reflect new developments in the pharmaceutical industry as well
as doping trends, enumerates the drug types and methods prohibited in and out of
competition. Among the substances included are steroidal and peptide hormones and their
modulators, stimulants, glucocorticosteroids, beta2-agonists, diuretics and masking agents,
narcotics, and cannabinoids. Blood doping, tampering, infusions, and gene doping are
examples of prohibited methods indicated on the list. From all these, hormones constitute by
far the highest number of adverse analytical findings reported by the antidoping laboratories.
Although to date most are due to anabolic steroids, the advent of molecular biology
techniques has made recombinant peptide hormones readily available. These substances
are gradually changing the landscape of doping trends. Peptide hormones like erythropoietin,
human growth hormone, insulin, and insulin-like growth factor I are presumed to be widely
abused for performance enhancement. Furthermore, as there is a paucity of techniques
suitable for their detection, peptide hormones are all the more attractive to dishonest athletes
[08001].
The human competitive nature is not only innate but essential in evolutionary and survival
terms. In the sporting arena, this manifests as an all-consuming drive to win. The enormous
financial gains successful sportsmen and -women can accrue and the political imperative for
a national side to achieve have led some professional athletes to resort to cheating to win.
The use of performance-enhancing substances has become increasingly sophisticated, and
there are concerns that the athlete who wins no longer has the best physiology but the best
pharmacologist [05001].
Athletes use substances to produce pleasure, relieve pain and stress, improve socialization,
recover from injury, and enhance performance. Therefore, they use some substances in
substantially higher rates that nonathletes. Despite these higher rates of use, rates of
addiction may in fact be lower in athletes. One article reviewed the prevalence and patterns
of use, health and performance effects, and preventive and treatment interventions for
alcohol, tobacco, stimulants, and steroids. Each substance is considered from the differing
perspectives of abuse/addiction and performance enhancement models. Similarities and
differences between college and professional athletes are discussed. Finally, suggestions for
future research are made [05385].
The national attention and economic gains that come with success in professional sports has
produced tremendous pressure on modern athletes to win at all costs. In 1994, it was
completed a survey of 198 Olympic level athletes. They were asked, if they were guaranteed
of winning an Olympic medal and not getting caught would they take a banned substance.
One hundred and ninety five of 198 athletes answered yes. When presented with the same
scenario, with a guarantee of winning every competition for the next five years, but they
would eventually die from adverse effects of the substance, more than 50 percent of the
athletes reported that they would still use the substance [07002].
Over the last 20 years systematic doping has become a major threat for elite sport. So far,
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there is no clear information about the daily practice of doping. Repeated scandals and
recent personal statements have added to our knowledge. Several more recent doping
agents like Erythropoietin (EPO) and, probably, growth hormone (GH) enhance performance
in a highly effective way and, together with the well-known anabolic steroids (AAS), belong to
the major doping categories. The introduction of EPO has really changed the paradigm in
endurance sports allowing a good middle class athlete to become a champion. It is evident
that doping practices are influenced by the possibilities of the anti-doping control system.
Unethical, criminal medical doctors play a decisive role in the ongoing practice of major
doping. Apart from the already mentioned substances AAS, EPO and GH several novel
drugs appear on the horizon. They are highly effective and there is no doubt that they will be
used in attempts to improve performance. During the last years, doping control systems have
also been improved: EPO can now be detected in urine samples and the detection of AAS
has also become much more sensitive. However GH hormone detection is not possible at
the moment and this remains a major weakness of doping control. Other problems are the
control procedures which are far from being optimal. In the future the quality of doping
controls will be decisive and not only the quantity; controls will have to be "intelligent". The
effective fight against doping in the next years will decide about the survival of elite sport
[07003].
The list of drugs prohibited by the World Anti-Doping Agency (WADA) has grown in the last
decade. The newer entries into this list include gonadotropins, estrogen antagonists,
aromatase inhibitors, androgen precursors, and selective androgen receptor modulators. The
use of mass spectrometry has revolutionized the detection of various compounds; however,
challenges remain in identifying newer designer androgens because their chemical signature
is unknown. Development of high throughput bioassays may be an answer to this problem
[10043].
Success in sports is often defined by winning, which drives athletes to use performance-
enhancing drugs (PEDs) to gain an advantage over opponents. Over the past 20 years, use
of PEDs by Olympic and professional athletes has led to public discussion regarding
potential negative health effects and ethical implications of their use. Unfortunately, PEDs
are not isolated to professional athletes, as PED use in adolescents has increased
dramatically. Many professional organizations, including the American Academy of
Orthopaedic Surgeons, have taken a stance against PED use in sports. The AAOS believes
neither anabolic steroids nor their precursors should be used to enhance performance or
appearance, and that these substances should be banned in all sports programs.
Pediatricians and orthopedists are often the first physicians to see these young athletes. It is
critical for these physicians to recognize the significance of the problem, have the knowledge
to inform adolescents, dissuade them from future use, and provide viable alternatives for
meeting performance goals [10435].
Doping incidents infesting high prestige sport events such as the 1998 Tour de France,
which was dubbed as the “Tour of Shame” or the 2004 Athens Olympic Games with a
sudden double number of positive cases; and the reaction to them (i.e. establishing national
anti-doping agencies) indicate that these events may only be the tip of the iceberg. Whilst the
adverse analytical findings (positive results) in tests conducted by the World Anti Doping
Agency (WADA) remain low around 2 percent, other occassions have revealed an elevated
level of substance use. For example, the presence of some kind of drug or supplement was
evidenced in 45 percent of the athletes who participated and were tested in the Tour de
France 2000. However, the problem seems to be rooted more deeply. The literature supports
the assumption that the consideration of and actual use of doping starts well before the
athlete reaches his/her best career years as the prevalence of doping, particularly the use of
anabolic steroids, is well documented among adolescents and even among pre-adolescent
164
athletes where a steady increase in doping use was observed over the period of four years
from age 11 to 15 [07004].
Ergogenic aids (from the Greek, ergon, meaning work) are ingested to enhance energy
utilization in athletes. While there is no scientific evidence to support the usage of these
agents for enhancing performance in children and adolescents, using supra-physiological
doses may be associated with undesired side effects [07005].
Doping is a problem that has plagued the world of competition and sports for ages. Even
before the dawn of Olympic history in ancient Greece, competitors have looked for artificial
means to improve athletic performance. Since ancient times, athletes have attempted to gain
an unfair competitive advantage through the use of doping substances. A Prohibited List of
doping substances and methods banned in sports is published yearly by the World Anti-
Doping Agency. Among the substances included are steroidal and peptide hormones and
their modulators, stimulants, glucocorticosteroids, β₂-agonists, diuretics and masking agents,
narcotics, and cannabinoids. Blood doping, tampering, infusions, and gene doping are
examples of prohibited methods indicated on the List. Apart from the unethical aspect of
doping, as it abrogates fair-play's principle, it is extremely important to consider the hazards it
presents to the health and well-being of athletes. The referred negative effects for the
athlete's health have to do, on the one hand, by the high doses of the performance-
enhancing agents and on the other hand, by the relentless, superhuman strict training that
the elite or amateur athletes put their muscles, bones, and joints [11556].
The fact that athletes routinely use a wide array of substances is well documented as are the
potential reasons for use. Whilst performance-enhancing substances are recognised in
global as well as local anti-doping prevention programmes, other substances such as
alcohol, tobacco and psychoactive drugs constitute a somewhat neglected area in the
current idealised anti-doping educational effort. This prevailing approach creates an artificial
divide between athletes' lives as sportspersons and private individuals. In reality, athletes
constantly navigate their athletically active years on a tightrope between the different
expectations they face both as athletes (often being in the spotlight) and as ordinary citizens,
and know that failing in one part of their lives could easily result in failures in the other and
vice versa. Substances in sports are mainly used for the following reasons:
- enhancing physical capacities (e.g. enhancing endurance, strength, or recovery

between exercise sessions)
- psycho-stimulation (e.g. as a way of dealing with psychological stress)
- improving physical appearance (e.g. for achieving a lean figure)
Contemporary sport legislation recognises two types of substances used in sports: non-
controlled substances, such as the majority of nutritional supplements, and products that
contain prohibited substances (the use of which is often referred to as doping). Nutritional
supplementation is defined as a preparation intended to provide nutrients, such as vitamins,
minerals, fibre, fatty acids or amino acids, which are otherwise missing or not consumed in a
sufficient quantity in the athlete's diet. It is generally accepted that substance use and misuse
(SUM) in sports is more common in physically demanding sports (e.g. weightlifting or cycling)
than in sports that require advanced specific motor skills (e.g. diving, sailing, table tennis or
curling). However, to our knowledge, such generalisation is not sufficiently supported by any
systematic comparative analyses of SUM across a variety of sports. Instead, the association
of doping with particular types of sports has mostly come from anti-doping testing and the
consequential public perception about doping in certain sports such as professional cycling,
track and field or weightlifting. The majority of sport activities take place outside of controlled
environments, leading to substance use without medical advice or supervision. The
165
mismatch in targets in the anti-doping prevention and deterrence programmes coupled with
the limited concern over substances such as alcohol and social drugs raises questions about
the suitability of the current anti-doping policy. Whilst both arms of WADA's anti-doping effort
represent heroic measures to keep doping out of sports, laboratory statistics shows no
significant change between 2003 and 2009 with the proportion of adverse and atypical
findings ranging between 1.50 and 2.12 percent. Self-reports, alternative analyses and
epidemilogic estimations indicate that the actual prevalence of doping is greater than this
official statistic and ranges up to 40 percent. Although it is difficult to make a direct
comparison between the latter and the WADA laboratory report, a recently published report
evaluating 7,289 blood samples from 2,737 track and field athletes in the athlete testing pool,
using the Athlete Biological Passport approach, estimated the prevalence of blood doping to
be at 14 percent overall and between 1 and 48 percent for sub-populations, which supports
the results from the epidemiologic studies. Anti-doping policy focuses on preventing selected
substance use in situations where such behaviourhas been deemed to result in increased
athletic performance giving an unfair advantage. Drugs such as anabolic steroids that have
long lasting effects and are considered “training drugs” are prohibited both in and out of
competition. Other substances, such as alcohol, marijuana and opiates, have only an in situ
effect on performance and, thus, are only prohibited in competition. Furthermore, the
detection-based doping policy sanctions athletes if there is evidence of a prohibited drug in
their body whilst completely disregarding whether the substance found has any performance-
enhancing effect on the individual. This narrow view fails to address health concerns that
might arise from SUM that happens outside the regulated domain. The main pillars of the
current anti-doping approach are fair play, level playing field and equal chance; only those
substances that violate these principles are considered with health being secondary. The
detection- and sanction-based approach to prevent doping reinforces the priority given to
protecting the sport instead of protecting the athletes' health. Future anti-doping policies
should address the gaps that currently exist between the testing pool and all athletes
including emerging (thus not yet selected for the testing pool) athletes and those training and
competing at the sub-elite level; A holistic approach to substance use and misuse that
considers athletes' substance use behaviour as a whole should be used in order to prevent
doping and preserve not only the integrity of sports but also the athletes' health. Critical
analysis of one of the three pillars of the doping ban, namely the protection of the health of
athletes, points to the health risks inherently present in elite level sports along with the
widespread use of acceptable substances that can also pose health risks. Furthermore, that
excessive alcohol or social drug use does not pose infringements upon the anti-doping rules
if their use happens outside of competition is a concerning phenomenon among athletes and
gym patrons. Both experts and athletes concerned agree that tailored and innovative ways
are needed to deliver relevant information on performance-enhancing and illicit drugs to
athletes and key stakeholders [11557].
Antidoping laws are undoubtedly focused on ensuring fair competition, however, there are
occasions, when honest athletes discover medical diagnoses through failed antidoping tests.
The relevant sporting bodies must do everything they can to try and ensure that difficult
medical circumstances are swiftly communicated to an athlete and sympathetically managed,
rather than attempting to first process an antidoping rule violation and seeking medical
assistance second. However, an athlete choosing to continue in elite sporting competition in
spite of a cancer diagnosis, complex medications and treatments, may face ethical and
antidoping procedural challenges. Competitive sport requires regular physical activity with a
huge array of physical and psychological benefits, associated with and resulting from
cardiorespiratory fitness, which will benefit athletes in these situations. The benefits of
regular physical activity for people living with cancer and undergoing chemotherapy are
considerable, leading to improved clinical outcomes and better quality of life [14014].
166
Sport and exercise have always played an important role in human life, either as leisure
practices, pedagogical practices, for health care, or as part of military training. It was noted in
1994 that the functions of sports include: (1) dimensions of egalitarianism, (2) the
introduction of the use of predefined rules imposed on practitioners, and (3) the experience
of equality and social justice. The commodification and politicalizing of sports, competitive
events, and athletes and the decrease in “sportsmanship” as a norm, value, and process are
relatively new in the history of athletics. Among the various sports events, the most popular is
the Olympics. The Olympic Games of the modern era were organized by Pierre de Coubertin
and the first modern Games were held in April 1896 in the city of Athens, Greece. The
Olympic Games have become a means for dissemination of ideals and political regimes, as
in the 1936 Games, which were used to display Hitler's Nazism and during the Cold War,
when the United States and the Soviet-Union sought to showcase sporting events as a
political and social force. Since the 1984 Los Angeles Olympic Games, the events have
succumbed to the power of money, making the Games one of the most effective modes of
spreading a brand name and/or product, when victory by an athlete is tied to the successful
representation of the brand's sponsor. With various forms of pressure (political, social,
financial, personal, or media) requiring athletes to perform well, there has been a huge
increase in the creation and implementation of methods and substances used in order to
improve an athlete's performance and reduce his/her recovery time, resulting in the winning
of more medals and satisfying the demands of their sponsors. Therefore all of these
pressures have and continue to transform the athlete into an instrument of the needs,
agendas, and goals of a range of influential individual and systemic stakeholders without
discernment as to where the ethical, moral, social norms, and health limits lay. The
prohibited substances and methods used, and which have changed over time, are called
doping; both listed substances and the processes used [14425].
Despite the high prevalence of performance-enhancing drug (PED) use, media attention has
focused almost entirely on PED use by elite athletes to illicitly gain a competitive advantage
in sports, and not on the health risks of PEDs. There is a widespread misperception that PED
use is safe or that adverse effects are manageable. In reality, the vast majority of PED users
are not athletes but rather nonathlete weightlifters, and the adverse health effects of PED
use are greatly underappreciated. This scientific statement synthesizes available information
on the medical consequences of PED use, identifies gaps in knowledge, and aims to focus
the attention of the medical community and policymakers on PED use as an important public
health problem. PED users frequently consume highly supraphysiologic doses of PEDs,
combine them with other PEDs and/or other classical drugs of abuse, and display additional
associated risk factors. PED use has been linked to an increased risk of death and a wide
variety of cardiovascular, psychiatric, metabolic, endocrine, neurologic, infectious, hepatic,
renal, and musculoskeletal disorders. Because randomized trials cannot ethically duplicate
the large doses of PEDs and the many factors associated with PED use, it is needed
observational studies to collect valid outcome data on the health risks associated with PEDs.
In addition, it is needed studies regarding the prevalence of PED use, the mechanisms by
which PEDs exert their adverse health effects, and the interactive effects of PEDs with sports
injuries and other high-risk behaviors. It is also needed randomized trials to assess
therapeutic interventions for treating the adverse effects of PEDs, such as the anabolic-
androgen steroid withdrawal syndrome. Finally, it is needed to raise public awareness of the
serious health consequences of PEDs [14426].
Historically, media coverage concerning AAS has focused disproportionately on athletes

(from elite professionals to high school students) seeking a competitive edge. In reality, at
least four out of five AAS users are not competitive athletes but rather men who desire what
they perceive to be an “enhanced” appearance. In the 2010s, however, it is decribed that
illicit AAS use was declining among adolescents – potentially due to the success of
167
education and numerous prevention campaigns targeting high school athletes [14427].
Drug abuse occurs in all sports and at most levels of competition. Athletic life may lead to
drug abuse for a number of reasons, including for performance enhancement, to self-treat
otherwise untreated mental illness, and to deal with stressors, such as pressure to perform,
injuries, physical pain, and retirement from sport. One review examined the history of doping
in athletes, the effects of different classes of substances used for doping, side effects of
doping, the role of anti-doping organizations, and treatment of affected athletes. Doping goes
back to ancient times, prior to the development of organized sports. Performance-enhancing
drugs have continued to evolve, with "advances" in doping strategies driven by improved
drug testing detection methods and advances in scientific research that can lead to the
discovery and use of substances that may later be banned. Many sports organizations have
come to ban the use of performance-enhancing drugs and have very strict consequences for
people caught using them. There is variable evidence for the performance-enhancing effects
and side effects of the various substances that are used for doping. Drug abuse in athletes
should be addressed with preventive measures, education, motivational interviewing, and,
when indicated, pharmacologic interventions [14612].
Claims championing exotic substances that produce healing or ergogenic powers have been
around for centuries. The competitive, peer-pressured environment enveloping today's
athletes and adolescence makes these groups particularly susceptible to the uproar
surrounding the current ergogenic aid market. Presently, it seems that rumor and anecdotal
information overwhelms the available scientific data. While there is evidence that some
touted ergogenic aids do indeed enhance performance, there are many unanswered
questions about product safety, efficacy, and long-term consequences. A working knowledge
of specific ergogenic aids is essential for the treating physician in order to best advise
patients and athletes as to the possible benefits and risks of any substance they may be
using [03001].
The aim of one review was to provide an update on drug abuse by athletes, their mode of
action and the technical difficulties of detection. The most common doping agents are the
anabolic steroids (AS), testosterone derivatives modified to take advantage of the anabolic
rather than the androgenic properties of the hormone. However, there are numerous side-
effects that discourage their use. Several other substances and hormones, GH and rhEPO
are currently used alone or combined to enhance performance. The diversity in nature of the
substances used requires a constant alertness of physicians to detect drug abuse in sports.
Doping is not limited to the professional athletes. It seems to be a generalized phenomenon
that reflects modern society's concept of success. Therefore, the campaign to eradicate
doping must also focus on individual responsibility. Since modern-day doping is strongly
related to hormonal preparations, endocrinologists may play a pivotal role in providing
information, protecting athletes' health and, moreover, retaining the ethical value of sport.
The impressive development of sport (from Latin "disporto") in our society during the last few
decades has wide-ranging social, educational and health ("ad salutem servandam")
repercussions. Sport may alleviate the pressure that modern society exercises on the mind
and body, it enforces self-discipline and ameliorates endurance. "Therefore, sport can bring
both fun and fitness to our life". However, the commercialization of sport has progressively
changed this spirit as the desire to win at any cost has overcome all other considerations
[03002].
Nowadays, doping in sport is more multifaceted than ever before with numerous standpoints
and opinions coming from all possible conceivable perspectives. Spearheaded by the
consistently recurring question as to whether athletes should generally be allowed to utilize
168
doping practices, juridical, medical, and philosophical as well as ethical aspects have been
challenging and the (in)efficiency of the existing doping control system have been presented,
underlining the complexity of modern sports drug testing, one core element of which is the
annually issued Prohibited List as established by the World Anti-Doping Agency (WADA) [14
[14715].
Doping in sport is a widespread problem not just among elite athletes, but even more so in
recreational sports. In scientific literature, major emphasis is placed on doping detection,
whereas detrimental effects of doping agents on athlete's health are seldom discussed.
Androgenic anabolic steroids are well known for their positive effects on muscle mass and
strength. Human growth hormone also increases muscle mass, although majority of that is
an increase in extracellular fluid and not the functional muscle mass. In recreational athletes,
growth hormone does not have major effect on muscle strength, power or aerobic capacity,
but stimulates anaerobic exercise capacity. Erythropoietin administration increases oxygen
carrying capacity of blood improving endurance measures, whereas systemic administration
of beta-adrenergic agonists may have positive effect on sprint capacity, and beta-adrenergic
antagonists reduce muscle tremor. Thus, there are certain drugs that can improve selective
aspects of physical performance. However, most of the doping agents exert serious side
effects, especially when used in combination, at high doses, and for long duration. The
extent of long-term health consequences is difficult to predict but likely to be substantial,
especially when gene doping is considered. This review summarises main groups of doping
agents used by athletes, with main focus on their effects on athletic performance and
adverse effects [14716].
Semantics and definitions of doping
The International Olympic Committee's (IOC) definition of doping is the "use of an expedient
(substance or method) which is potentially harmful to athletes' health and/or capable of
enhancing their performance, or the presence in the athletes' body of a prohibited substance
or evidence of the use thereof or evidence of the use of a prohibited method". There is no
mention of intent or of how the substance entered the body. If the substance is in the
athlete's body, then he or shr is responsible. That is the basis of sanctions for testing positive
for a prohibited substance. Sir Arthur Porritt, first chairman of the IOC Medical Commission,
noted: "To define doping is, if not impossible, at best extremely difficult, and yet everyone
who takes part in competitive sport or who administers it knows exactly what it means. The
definition lies not in words but in integrity of character". Such agents are also known as
"performance enhancing substances" (PES) or "performance enhancing drugs" (PED). The
American Academy of Pediatrics defines these agents as: "...any substance when taken in
non-pharmacological doses specifically for the purposes of improving sport performance. A
substance should be considered performance enhancing if it benefits sports performance by
increasing strength, power, speed or endurance (ergogenic) or by altering body weight or
body composition [10001].
The WADA has defined doping in their World Anti-Doping Code (the “Code”). Under the
Code, a violation of one or more of the following rules is considered doping and may result in
sanction:
- the presence of a prohibited substance or its metabolites or markers in an athlete's

bodily specimen
- the use or attempted use of a prohibited substance or a prohibited method
- possession of prohibited substances and methods
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- administration or attempted administration of a prohibited substance or prohibited
method to any athlete
- assisting, encouraging, aiding, abetting, covering up or any other type of complicity
involving an anti-doping rule violation or any attempted rule violation.
-
A substance or method is considered for inclusion on the WADA's prohibited list if the WADA
determines that the substance or method meets any two of the following three criteria:
- medical or other scientific evidence, pharmacological effect, or experience that the

substance or method has the potential to enhance or enhances sport performance.
- medical or other scientific evidence, pharmacological effect, or experience that the
use of the substance or method represents an actual or potential health risk to the
athlete.
- determination by the WADA that the use of the substance or method violates the
spirit of sport as described in the “Introduction to the Code”
-
The current emphasis of prohibition also appears to be based on four factors:
1. Substances within the athlete's body.

2. Methods that enhance oxygen transfer through blood doping or artificial measures.
3. Altering collected body fluid samples.
4. Genetic manipulation.
The interpretation of the Code is a legal one and largely untested. The broad interpretation of
the principles behind the Code would seem to be related to any substance or method that
(potentially or actually) enhances sport performance, becomes a health threat to the athlete,
or is against the spirit of the sport. If the motivation, and indeed, the imperative, of an athlete
is to constantly seek creative ways to improve the sporting performance, what then is the
spirit of sport, and what actions are deemed to have violated that spirit? Using a
biopsychological perspective, issues of what are acceptable levels of naturally occurring
endogenous compounds, what is a method that is not considered doping, and what is the
spirit of sport may be discussed especially regarding unnatural amounts of natural
substances?Evidence-based medicine requires large sample sizes and preferably
randomised-controlled trials to provide statistical evidence of significant effects. The issue of
doping, however, is as much an interpretation of the law, as it is about the scientific and
statistical evidence. The Code states that “...regardless of whether the expectation of
performance enhancement is realistic..”. [12015].
The WADA characterises spirit of sport under the Code as:
1. ethics, fair play and honesty;

2. health;
3. excellence in performance;
4. character and education;
5. fun and joy;
6. teamwork; dedication and commitment;
7. respect for rules and laws;
8. respect for self and other participants;
9. courage;
10. community and solidarity.
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While these objectives are noble and worthy principles for sport, their abstract nature
sometimes creates confusion when applied to the day-to-day realities that athletes face. The
Code cites specific issues in the context of the spirit of sport, but these add to the confusion.
The WADA states in the Code that the [121015]:
“… use of genetic transfer technology to dramatically enhance sport performance should be
prohibited as contrary to the spirit of sport even if it is not harmful...”
and that
“… the potentially unhealthy abuse of certain substances without therapeutic justification
based on the mistaken belief they enhance performance is certainly contrary to the spirit of
sport regardless of whether the expectation of performance enhancement is realistic...”
Performance-enhancing drugs (PEDs) are pharmacologic agents that athletes and

nonathlete use to enhance performance. The term doping refers to the use of PEDs in
competitive sports. It is define nonathlete weightlifters as individuals whose goal is to
become leaner and more muscular, often simply for personal appearance, and not to
participate in formal sports competitions. There are several categories of PEDs that are
currently popular among nonathlete weightlifters and athletes. Lean mass builders, the most
frequently used PEDs, are generally promyogenic (anabolic) drugs that increase muscle
mass or reduce fat mass. By far the most prevalent illicit drugs in this category are AASs,
which are the primary focus of this report. Among nonathlete weightlifters, the use of AASs
represents a higher proportion of overall PED use than that of all other categories of PEDs
combined. Historically, the term AAS reflected the view that androgenic and anabolic effects
of androgens could be dissociated and that, in comparison with testosterone, some
androgens were more anabolic than androgenic. However, a large body of data emerged in
the late 1990s that revealed that the selectivity of androgen receptor signaling could be
mediated at multiple levels of the steroid hormone interactome that encompasses (in addition
to the androgen receptor) an interacting web of chaperone proteins, a repertoire of 300 or so
coactivators and corepressors, elements of the chromatin, effector proteins, and transcription
factors that bind specific regions of the androgen-responsive genes [14426].
In addition to AASs, nonathlete weightlifters and athletes also use human GH (hGH) and
IGF-1 because these PEDs have recently become available on the black market at reduced
cost. Similarly, some nonathlete weightlifters use the hormone insulin for its potential
anabolic effects. Finally, some nonathlete weightlifters use clenbuterol, a beta-adrenergic
agonist that is thought to possess possible anabolic properties. Clenbuterol and other illegal
stimulants, such as amphetamine, and some hormones, such as thyroid hormones, also
have thermogenic (fat-burning) properties that make them popular among nonathlete
weightlifters [14426].
Competitive athletes tend to use several other categories of PEDs in addition to AASs. For
example, some competitive bodybuilders use diuretics (e.g. furosemide and thiazides) to
improve muscle definition onstage. Some boxers or wrestlers use diuretics to reduce body
weight so they can compete in a lower weight class. Diuretics may also dilute the urine,
which can reduce the concentration of the PED below the limit of detection. Blood boosters
(erythropoietins, other erythropoiesis-stimulating agents (ESAs), and transfusions) increase
endurance in events such as cycling, long-distance running, and skiing. Athletes also may
combine AASs and erythropoietins to train harder and recover faster. Masking drugs reduce
the ability to detect a banned substance. For instance, epitestosterone can mask the
detection of testosterone use and tranquilizers (benzodiazepines and opiates) reduce anxiety
in events that require steady nerves (such as archery), and opiates can mask pain during
competition [14426].
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The World Anti-Doping Agency (WADA), an international agency that oversees the
implementation of the antidoping policies in all sports worldwide, maintains a list of
substances (drugs, supplements, etc) that are banned from use in all sports at all times,
banned from use during competition, or banned in specific sports. WADA's Anti-Doping
Program is based on the WADA Code, a universal document that contains comprehensive
guidelines for best practices in international and national antidoping programs. WADA also
publishes the doping violation thresholds for banned substances [14426].
Relevant policy documents
Recent international developments have served to solidify the international approach to

doping in sport. The development of the World Anti-Doping Agency (WADA) has resulted in
new, coordinated efforts to address this important sport issue. An array of new efforts and
initiatives has been initiated by the new agency. The Sydney and Salt Lake City Olympics
were characterized by intensive efforts to minimize doping. The antidoping environment is
evolving rapidly, and several profoundly important developments will take place in the
immediate future. To outline the challenges, opportunities, and changing circumstances of
the current antidoping environment so that sport medicine practitioners might understand the
context in which a variety of new initiatives and approaches will develop. At the same time, to
ensure that practitioners understand the importance of appropriately developed and
delivered antidoping policies, programs, and procedures, and the need for their
harmonization. To ensure that sport medicine practitioners appreciate the need for a
comprehensive approach to doping control, i.e. programs that include much more than drug
testing. A review of relevant policy documents derived from a variety of sport and antidoping
organizations; selected references drawn from MEDLINE; and materials prepared by
colleagues drawn from the international antidoping community. The increased global effort to
address doping is welcome. It will require that several critical issues be addressed that will
test the resolve of all involved [02001].
A background for understanding performance-enhancing drugs in sports
Performance-enhancing drug use is a prevalent problem in sports. It is a problem that has

captured the world's attention as the media highlights story after story of athletes who have
transformed their bodies over a short period of time, those who have simply defied the aging
process in an attempt to prolong a career and those whose careers have been tarnished
because of drug use. The baseball investigations and the Mitchell Report of 2007 opened the
US’s eyes and gave a glimpse of a secretive underground world. This "world" is much more
intelligent and sophisticated than it is given credit for. It was the goal of one article to
increase the awareness of the medical provider about the types of steroids and other
medications used, the influence these substances have on the athletes, and how and why
they use them [12018].
Judging cheaters in different domains
The present study examines how individuals judge others who use performance-enhancing
drugs in two different domains – the athletic domain and the academic domain.
Approximately 1,200 males in their freshman year of college completed a questionnaire that
included two scenarios. One scenario described an athlete who misused anabolic steroids to
help him succeed at a sporting event. The other described a college student who misused
Adderall to help him succeed on his midterm exams. Participants rated the extent to which
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they thought the target had cheated and the extent to which they felt the substances were
necessary for success. Results showed participants believed the athlete was more of a
cheater than the student, and this difference got larger as past prescription stimulant misuse
increased. Results also demonstrated that participants felt Adderall was more necessary
than anabolic steroids for bringing about success. Contributions to the literature on zero-sum
and non-zero-sum domains are discussed. Implications for future research and efforts to
prevent substance misuse are described [12019].
Overviews
Doping is a phenomenon which in the past years through the various incidences in for
example professional cycling has come more and more into the focus of the public interest.
Whilst in the young past the problems were to define the term "doping" exactly, today's
problem is to prevent adolescents and children of doping. This may partly be achieved by
carrying out controls and serious sanctions for doping violations. However, scientific research
has also proved that doping usage can be avoided by broad specific prevention measures. In
general, the earlier the athletes dope the higher the risk to become addicted later on in life to
other legal or illegal drugs. To get closer to a doping-free sport it is imporant to analyse the
prevalence of doping regarding youth-, competitive-, high performance and recreational
sports o find more specific ways for prevention. Moreover it may be rewarding to examine
also other aspects of doping abuse, risks of addiction, the legal situation, current strategies in
the fight against doping to enhance chances of further doping prevention opportunities. By
means of this data an all-embracing view should be given over the current situation,
problems and prospects [10002].
The drive toward success in sports and the need for a cosmetically acceptable appearance
have driven many adolescents to take a wide variety of doping substances. The consumption
of these chemicals in the hope and hype of improved sports performance, fueled by the
easing of government restrictions on their proof of safety and efficacy, has resulted in an
explosion of so-called ergogenic products available to the youth. Agents that have been used
include anabolic steroids, anabolic-like agents, designer steroids, creatine, protein and amino
acid supplements, minerals, antioxidants, stimulants, blood doping, erythropoietin, beta-
blockers, and others. The use of these agents has considerable potential to cause physical
and psychological damage. Use and misuse of drugs in this sports doping process should be
therefore be discouraged. Clinicians providing sports medicine care to youth, whether
through anticipatory guidance or direct sports medicine management, should educate their
young patients about the hype and hyperbole of these products that may keep them out
instead of in the game at considerable financial cost to the unwary consumer [10003].
Testosterone is the principal male sex hormone. As with all natural steroids, it is
biosynthesized from cholesterol. Phase I metabolism employs some very specific enzymes
and pathways. Phase II metabolism and excretion follow more general patterns. The effects
of testosterone are twofold: anabolic and androgenic. Because of its anabolic effects,
testosterone is frequently abused in sports. Because of its endogenous nature, testosterone
doping is difficult to detect. The standard procedure is based on the evaluation of the urinary
steroid profile. Conspicuous samples then are submitted to compound-specific 13C/12C
analysis. Synthetic and endogenous steroids differ in this measure. However, numerous
xenobiotic compounds have been derived from testosterone. The modifications typically aim
at a reduction of the androgenic properties while maintaining the anabolic potential. Most of
these compounds have been withdrawn from the legal market but are despite that found to
be illicitly added to otherwise inefficient nutritional supplements. These products represent a
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major problem to doping control. Recently, also clinical trials with selective androgen
receptor modulators have been started in a hope to get more anabolism and less androgeic
effects [10004].
Sport plays a major role in the lives of many people, both for active participation and as
entertainment. Sport is now a huge nationally and internationally based industry. The desire
to win has led some athletes to resort to the use of performance enhancing drugs. With huge
financial rewards now available in some sports the pressure to excel has grown. Some have
argued that drug use should be given free rein, however most people are of the view that it is
athletic prowess that should be applauded not the efficacy of various performance enhancing
drugs. Apart from the obvious aspects of equality and fair play, the use of drugs is associated
with significant health risks. In the 1960's the use of stimulants in sports such as cycling led
to the death of at least one cyclist. Since 1968 the International Olympic Committee (IOC)
has required all Olympic Games' host cities to provide laboratory facilities for the analysis
and detection of performance enhancing drugs. There are in 2004 29 IOC accredited
laboratories throughout the world that routinely test samples from athletes for the presence of
such drugs. The purpose of one tutorial review wais to give an overview of drug testing
procedures, including those that were used at the last summer Olympic Games in Sydney
2000, and the incorporation of the latest developments in analytical chemistry technology in
the drug testing process. More recently, developments in biotechnology mean that the use of
whole new classes of drugs are banned in sport, often requiring new methodologies and
techniques for their analysis. The contest between those who wish to cheat and those who
wish to maintain fair play in sport is an ongoing one [04001].
Little symptoms and signs
Abuse of doping subsrances has become increasingly widespread among athletes also at
sub competitive and recreational level, due in part to the lack of controls in form of doping
tests. Hypertension and the many other side effects related to the illicit use of prescription
drugs pose a substantial but often underestimated threat to public health. The symptoms are
recognized late and are then mostly repressed or misjudged. Since the abuse is concealed
to the doctor when help is finally sought, it might result in extensive and expensive tests that
can seldom lead to an effective treatment. Two case reports were presented to elaborate on
this issue [10005].
Socio-psychological background to doping
Sporting competition in the society has become the spectacle that mobilises and brings
together the greatest number of people throughout the world, with the corresponding cultural
and economic influence that this implies. As a result, the desire for athletic prowess has led
sportspersons to undergo intense training programs and to consume substances that
improve their performance, at times having recourse to doping techniques. At present,
doping is the result of a combination of social, individual, physiological and cultural factors,
which affect not only professional, but also amateur sportspeople. In order for the control and
eradication of doping to be efficient, it is necessary to understand the problem and the
substances that are most employed, amongst which special mention is merited by hormonal
substances due to the complexity of detecting them and their possible repercussions on
health [06012].
Elevate health risks in young athletes
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Young athletes may use many products and techniques in an attempt to increase competitive
edge in sports. The doping techniques that were previously seen in elite adult athletes are
now being noted in increasingly competitive elementary, middle, and high school male and
female athletes. The risk of significant morbidity and mortality associated with the use of
these products is substantially increased when other risk factors are present. The risk for
heat-related illness and possible heat-related mortality is higher in physiologically immature,
overweight, and poorly conditioned young athletes. These are the same athletes who may be
more likely to use stimulant or anabolic steroid products in attempts to catch up on training
and conditioning regimens, improve their competitive advantage, or improve their physiques.
The risk for heat-related incidents is higher in young athletes who are predisposed to these
events because of a family trait or a previous heat-related adverse event in their own medical
histories. Combinations of these factors (eg, high osmotic dietary supplements, stimulants,
pre-existing medical factors, adverse ambient conditions) may significantly increase a young
athlete's chances of a serious, potentially fatal event. Similarly, the risk of cardiac-related
sudden death in a young athlete is significantly increased by the use of stimulants such as
methamphetamine. As is the case with heat-related adverse events, the risk of cardiac-
related morbidity and mortality may be significantly increased when other variables are
present, such as the presence of other medications and pre-existing medical factors. As
athletic competition becomes increasingly intense for younger athletes, pediatricians need to
be aware of the possibility that their young patients are using ergogenic aids that may
increase the risk for sudden death significantly. Pediatricians should be aware of the
products available to these young competitors, and of the co-factors that substantially
increase the risk of morbidity and mortality in this population [03005].
Risk for drug dependence
The paper presents the health hazards of the major doping substances and raises some
questions about the relationship between doping and addictive behavior. Current definitions
of doping and addictive behavior are examined. The paper's goal is: 1- to assess the risks of
neurotoxicity and overall toxicity of doping substances: stimulants, narcotics (seldom used as
doping substances), and hormones, and assess their addictive potential; 2- to present
available data on drug-dependent patients with a record of early prolonged and intensive
physical activity or athletic practice. Some doping substances present high risks for health at
large doses, but usually low addictive potential and neurotoxicity. Dependency on doping
substances and drift towards dependency to addictive drugs, if any, are therefore determined
by genetic and environmental factors. A significant susceptibility to drug dependence has
been observed in some cases of very intensive and competitive practice. Over-
representation of intensive and competitive athletic antecedents among some drug-
dependent patients could be accounted for in either of two ways. On the first account, the
causal factor is a sensation-seeking character trait, with a likely genetic component, which
predisposes the individual to the use of drugs or doping substances, as the opportunities
arise. On the second account, the sudden interruption of intensive practice, and of the
associated organic stress and hypersensitization of the hedonic pathway, creates a weaning
syndrome and leads to the search for relief through drugs. Further exploration of this
hypothesis is called for [03006].
To move borders …
Human performance, defined by mechanical resistance and distance per time, includes
human, task and environmental factors, all interrelated. It requires metabolic energy provided
by anaerobic and aerobic metabolic energy sources. These sources have specific limitations
175
in the capacity and rate to provide re-phosphorylation energy, which determines individual
ratios of aerobic and anaerobic metabolic power and their sustainability. In healthy athletes,
limits to provide and utilize metabolic energy are multifactorial, carefully matched and include
a safety margin imposed in order to protect the integrity of the human organism under
maximal effort. Perception of afferent input associated with effort leads to conscious or
unconscious decisions to modulate or terminate performance; however, the underlying
mechanisms of cerebral control are not fully understood. The idea to move borders of
performance with the help of biochemicals is two millennia old. Biochemical findings resulted
in highly effective substances widely used to increase performance in daily life, during
preparation for sport events and during competition, but many of them must be considered
as doping and therefore illegal. Supplements and food have ergogenic potential; however,
numerous concepts are controversially discussed with respect to legality and particularly
evidence in terms of usefulness and risks. The effect of evidence-based nutritional strategies
on adaptations in terms of gene and protein expression that occur in skeletal muscle during
and after exercise training sessions is widely unknown [08002].
The creed of the Olympics states: "The important thing in the games is not winning but taking
part. The essential thing is not conquering, but fighting well". As noble a goal as this is, it has
little to do with the reality of the modern sports world. Athletes today are expected and
encouraged to seek every possible way to improve their performance, including specialised
training, hi-tech design of equipment and apparel, scientific and medical support, including
the use of nutritional supplements [08003]. Being a high performing athlete is nowadays a
profession that requires dedication, long-term commitment and sacrifice [08004], whereas a
great concern to sport governing bodies is the chemical alteration of athletic performance.
The intriguing question is then what compels athletes to risk their health or reputation for
outstanding sports performance, how high price the athelete is willing to pay, and what
factors make athletes vulnerable to doping and at which point of their careers [08005]. New,
more powerful and undetectable doping techniques and substances are now abused by
professional athletes, while sophisticated networks of distribution have developed.
Professional athletes are often the role models of adolescent and young adult populations,
who often mimic their behaviors, including the abuse of drugs [08006].
Breaking of borders
To explore how current substance use, including the use of sports supplements and illicit
drugs, may impact upon a person's future intentions to use anabolic-androgenic steroids
(AAS) 214 exercising males (mean age, 30 years; range, 17-61 years) recruited from 5
gymnasia in Sydney, Australia, completed a web-based survey. The survey contained
questions relating to sport supplement use, illicit substance use, reasons for currently not
using AAS, and reasons for intending to use AAS in the future. Participants completed a
structured interview schedule that included questions regarding licit and illicit substance use,
reasons for non-AAS use, and, where appropriate, reasons for intended future AAS use.
Sixteen percent of the sample indicated that they would use AAS in the future. Reasons for
future AAS use included increasing muscle size (80 %), improving appearance (74 %), and
increasing strength (57 %). Four-fifths (80 %) of the sample reported use of sports
supplements, with vitamins and protein supplements commonly reported (83 % and 67 %,
respectively); more than one-third (36 %) reported use of creatine in the past 6 months. Half
(52 %) of the sample reported use of illicit substances in the preceding 6 months, with
amphetamines and cannabis commonly reported (66 % and 62 %, respectively). Significant
predictors of intending to use AAS included past 6-month use of creatine and knowing AAS
users. The authors concluded that the use of sport supplements and/or illicit substances may
remove barriers for the future use of such drugs as AAS [09011].
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Much to win
Athletes are rewarded for winning at virtually every level of competition. Second place is
viewed as the "first loser". A coach's job security is directly related to his team's success, not
that they are simply "fighting well". Given this reality, it is not surprising that athletes and
coaches will sacrifice and risk a great deal in order to obtain a competitive edge and enhance
performance at all costs. Performance enhancement in Olympic and professional sport has
now become a medical, ethical, and legal problem for modern athletes and athletic
organizations. This is primarily due to the amount of money associated with winning in
today's sports industry. Multimillion dollar contracts, appearance fees, international
endorsement and sports merchandising represent a billion dollar industry that offers today's
athletes, their sponsors and entourage previously unheard of financial gains. Driven by the
millions of dollars now routinely available for winning a sporting event, unethical pharmacists,
medical professionals, trainers and sports organizations have worked secretly, and at times
without their athletes' consent, to develop sophisticated doping programs where performance
is optimized, often at the risk of the athletes' health. Now, these same doping programs are
moving out of the professional sports market to our youth and other at-risk populations at
alarming rates [08006].
Modern sports and the media's misplaced fixation on fame, fortune and winning at all costs
have unintentionally created a growing market for doping substances. These substances,
once only abused by elite athletes, are clearly spreading into schools and health clubs
worldwide. They are being accepted by a whole new generation of young customers who see
reports daily in the newspapers of sports icons accused of abusing drugs only to continue
playing, breaking records and claiming fortunes. These same performance-enhancing drugs
are also abused by adolescents and weekend athletes and non-athletes who have wider
behavioral and health risk problems. In addition, these drugs are now being abused by male
and female adolescents for cosmetic purposes in an attempt to achieve the "cut" and sexy
look promoted by the media [08006].
Ergogenic potential of AAS
It was in 2005 described physiological changes that occurred in a Tour de France cyclist as
he matured from 21 to 28 years of age during the period of 1992 to 1999. This cyclist has
recently admitted to using performance-enhancing drugs: erythropoietin, red blood cell
reinfusion, testosterone, cortisone, and human growth hormone. The main physiological
improvements he displayed over this 7-year period during which the author was testing him
were an improved gross mechanical efficiency and a reduced body weight. It is also worth
noting that four of the five laboratory-based physiological testing sessions were performed in
the precompetitive season or with reduced training, although one session was conducted at
the end of the competitive season. It is not possible to know the extent to which his drug use
might have improved his gross mechanical efficiency because there have not been direct
studies conducted to the author’s knowledge. Erythropoietin and/or red blood cell reinfusion
would seem to be taken acutely during the competitive season to boost blood volume during
a race, although it is conceivable that erythropoietin could be taken as a training aid.
However, it seems unlikely that this cyclist had elevated blood volume at the time of
physiological laboratory testing. Furthermore, it is unlikely that an elevated blood volume
would improve gross mechanical efficiency, because studies that have acutely infused red
blood cells into athletes have not reported changes in efficiency measured from open circuit
spirometry. Since publication of the 2005 paper, there have been several reports of
champion athletes displaying improved efficiency of movement. The world record holder in
the women’s marathon, Paula Radcliffe, displayed a remarkable 15 percent improvement in
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running economy between 1992 and 2003. Therefore, there is growing evidence that
mechanical efficiency can improve with chronic training. However, we cannot be absolutely
certain that the improved gross mechanical efficiency and reduced body weight displayed in
the subject of the 2005 paper was not somehow influenced by his reported drug use [0021].
Although AAS have been used in sports for ergogenic purposes for decades, many believed
that the improved performance seen with AAS was due to their influence on motivation and
aggression. It took landmark studies from Bhasin et al to prove that testosterone dose-
dependently increases muscle mass, maximal voluntary strength, and power and that these
improvements are correlated with circulating testosterone concentrations. There are multiple
mechanisms that lead to this anabolic response. Androgens are known to increase fractional
muscle protein synthesis and increase the size of both type I and type II muscle fibers.
Studies have also shown that testosterone directs the pluripotent mesenchymal stem cell
toward myogenic lineage rather than adipogenic lineage. Some clinical trials of testosterone
replacement in hypogonadal elderly men have also shown improvement in muscle strength.
Because androgens improve maximal voluntary muscle strength, it is understandable that a
high rate of androgen use is seen among weight lifters and other power athletes. However,
the use of androgens in endurance events, e.g. bicycling, is not based on scientific evidence
because androgens have not been shown to improve whole body endurance. There are two
types of androgen doping: direct and indirect. Direct doping involves exogenous
administration of both natural and synthetic androgens. Indirect doping refers to using
compounds that increase the production of endogenous testosterone (estrogen receptor
antagonists or aromatase inhibitors), androgen precursors (dehydroepiandrosterone (DHEA),
androstenedione), and gonadotropins [13003].
Androgenic-anabolic steroids (AAS) are synthetic derivatives of the male hormone

testosterone. They can exert strong effects on the human body that may be beneficial for
athletic performance. A review of the literature revealed that most laboratory studies did not
investigate the actual doses of AAS currently abused in the field. Therefore, those studies
may not reflect the actual (adverse) effects of steroids. The available scientific literature
describes that short-term administration of these drugs by athletes can increase strength and
bodyweight. Strength gains of about 5-20 percent of the initial strength and increments of 2-5
kg bodyweight that may be attributed to an increase of the lean body mass, have been
observed. A reduction of fat mass does not seem to occur. Although AAS administration may
affect erythropoiesis and blood haemoglobin concentrations, no effect on endurance
performance was observed. Little data about the effects of AAS on metabolic responses
during exercise training and recovery are available and, therefore, do not allow firm
conclusions. The main untoward effects of short- and long-term AAS abuse that male
athletes most often self-report are an increase in sexual drive, the occurrence of acne
vulgaris, increased body hair and increment of aggressive behaviour. AAS administration will
disturb the regular endogenous production of testosterone and gonadotrophins that may
persist for months after drug withdrawal. Cardiovascular risk factors may undergo deleterious
alterations, including elevation of blood pressure and depression of serum high-density
lipoprotein (HDL)-, HDL2- and HDL3-cholesterol levels. In echocardiographic studies in male
athletes, AAS did not seem to affect cardiac structure and function, although in animal
studies these drugs have been observed to exert hazardous effects on heart structure and
function. In studies of athletes, AAS were not found to damage the liver. Psyche and
behaviour seem to be strongly affected by AAS. Generally, AAS seem to induce increments
of aggression and hostility. Mood disturbances (e.g. depression, [hypo-]mania, psychotic
features) are likely to be dose and drug dependent. AAS dependence or withdrawal effects
(such as depression) seem to occur only in a small number of AAS users. Dissatisfaction
with the body and low self-esteem may lead to the so-called 'reverse anorexia syndrome' that
predisposes to the start of AAS use. Many other adverse effects have been associated with
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AAS misuse, including disturbance of endocrine and immune function, alterations of
sebaceous system and skin, changes of haemostatic system and urogenital tract. One has to
keep in mind that the scientific data may underestimate the actual untoward effects because
of the relatively low doses administered in those studies, since they do not approximate
doses used by illicit steroid users. The mechanism of action of AAS may differ between
compounds because of variations in the steroid molecule and affinity to androgen receptors.
Several pathways of action have been recognised. The enzyme 5-alpha-reductase seems to
play an important role by converting AAS into dihydrotestosterone (androstanolone) that acts
in the cell nucleus of target organs, such as male accessory glands, skin and prostate. Other
mechanisms comprises mediation by the enzyme aromatase that converts AAS in female
sex hormones (estradiol and estrone), antagonistic action to estrogens and a competitive
antagonism to the glucocorticoid receptors. Furthermore, AAS stimulate erythropoietin
synthesis and red cell production as well as bone formation but counteract bone breakdown.
The effects on the cardiovascular system are proposed to be mediated by the occurrence of
AAS-induced atherosclerosis (due to unfavourable influence on serum lipids and
lipoproteins), thrombosis, vasospasm or direct injury to vessel walls, or may be ascribed to a
combination of the different mechanisms. AAS-induced increment of muscle tissue can be
attributed to hypertrophy and the formation of new muscle fibres, in which key roles are
played by satellite cell number and ultrastructure, androgen receptors and myonuclei
[04002].
Precursors of testosterone
One article reviewed the recent literature on the use of anabolic-androgenic steroids (AAS)
for performance enhancement. Recent studies utilizing supraphysiologic doses of
testosterone have demonstrated increases in strength and improvements in body
composition, despite earlier assertions by the medical community that steroids were
ineffective as ergogenic aids. Although data that support the theory of conversion of
prohormones, such as androstenediol, to testosterone in the body is available, support for
testosterone precursors alone as ergogenic aids is lacking. Drug testing laboratories are
utilizing new techniques that analyze carbon-13 levels of urinary steroids to detect
exogenously administered steroids as well as the use of urine-manipulating agents.
Investigations that seek to refute athletes' various claims for positive drug tests are ongoing.
The recent discovery, characterization, and development of a urine test for tetra-hydro-
gestrinone, a designer steroid, has brought the issue of performance enhancement once
again into the public spotlight. Increasing attention is also being paid to the long-term effects
of AAS abuse, as more authors characterize the changes to hematologic, hepatic, lipid, and
hormone profiles as a result of years of steroid use. Although the understanding of AAS and
testosterone precursors as performance-enhancing drugs continues to advance, there are
likely to be more revelations as scientific investigations continue [04003].
Testosterone/epitestosterone (T/E ratio)
To circumvent the detection of synthetic androgens, athletes have resorted to doping with
testosterone. Hence, the detection of illegal use depends upon distinguishing between
endogenous and exogenous testosterone. There are two main detection methods available.
Epitestosterone (17alpha-hydroxy-4-androsten-3-one) is a 17-epimer of testosterone that is
also secreted by the Leydig cells of the testes. It was first described in 1947 as an androgen
metabolite; however, the first direct evidence of its urinary excretion was seen in the 1960s. It
is biologically inactive, and there is no interconversion between testosterone and
epitestosterone. Although its production rate is less than 5 percent of testosterone, its urinary
excretion is 33 percent that of testosterone. It is mainly excreted in the urine as a
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glucuronide; however, a small amount is also excreted as a sulfate. Due to its rapid
clearance, excretion rates of testosterone and epitestosterone are similar, making the urinary
T/E ratio approximately 1. Because there is no interconversion between the two compounds,
administration of exogenous testosterone results in an increase in the T/E ratio.
Measurement of T/E ratio in a large number of athletes has shown that generally the ratio
remains below 6. This initially led the IOC to adopt the cutoff threshold of 6 to consider the
test as positive. However, it was later recognized that genetic differences in various
populations may influence T/E ratio. Some athletes have low endogenous epitestosterone
production rate; hence, their T/E ratio always exceeds 6. To the contrary, a deletion
polymorphism in uridine diphosphate glucuronyl transferase 2B17, an enzyme that facilitates
epitestosterone excretion, lowers T/E ratios (especially in Asian populations). Considering
these genetic variants, the IOC recently lowered the T/E cutoff ratio to 4. Hence, any value
above 4 is considered suspicious. To complicate matters, some athletes may mask the use
of exogenous testosterone by coadministration of epitestosterone. In such cases, the
exogenous use of epitestosterone is detected by elevated concentrations of its urinary
metabolites. Generally, in athletes not using AAS, the T/E ratio remains fairly constant.
Hence, monitoring T/E ratio serially in the same athlete is occasionally performed to detect
any change that may suggest illicit use [13003].
Isotope ratio mass spectrometry (IRMS)
If the T/E ratio is abnormal, the WADA requires additional confirmation by gas
chromatography combustion IRMS, which involves measurement of 13C/12C isotope ratio in
testosterone. This method is based on the fact that the percentage of 13C (a naturally
occurring isotope of carbon) in endogenous testosterone (synthesized in the body from
carbon sources derived from animals and plants) is higher than in synthetic testosterone.
Hence, synthetic testosterone has a lower 13C/12C ratio. During the IRMS, the steroids are
separated by gas chromatography and oxidized to carbon dioxide in a combustion chamber.
The ratio of 13CO2/12CO2 is monitored in an isotope ratio mass spectrometer. A lower 13C/12C
ratio suggests exogenous testosterone administration. In addition to testosterone, IRMS is
also used to distinguish endogenous from exogenous nandrolone, dihydrotestosterone, and
DHEA [13003].
.
Synthetic androgens
The availability of synthetic androgens has not only provided more options for the athletes
but at the same time circumvents their detection by the doping authorities because chemical
signatures of many of these compounds are not readily available. Norbolethone has been
credited as the first designer androgen that was identified in the 1960s. In 2002, its chemical
signature was identified after it was detected in an athlete’s urine. In 2003,
tetrahydrogestrinone was identified. It is a derivative of gestrinone (a progestin); hence, it is
both a potent androgen and progestin. In 2005, a third designer androgen was identified as
desoxymethyltestosterone. In addition to these androgens, SARMs have recently gone to
clinical trials and have the potential for abuse in various sports. Various bioassays are being
employed in the detection of these designer androgens [13003].
Androgen bioassays
Bioassays are functional assays that are employed to determine the bioactivity of a
compound. Hence, they measure potency of a substance. The unit of bioactivity is defined as
the lowest concentration at which a functional response is measured. This unit is then
compared with known bioactivity of a standard compound (such as testosterone or
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dihydrotestosterone). The main advantage of these bioassays lies in their ability to detect
even those androgens whose chemical structure is unknown, hence making this an ideal tool
to detect novel designer androgens (unlike RIA and mass spectrometry that require the
chemical signature of the steroid to be known). Bioassays can be divided into in vivo and in
vitro assays. The former involves evaluation of in vivo responses of androgenic stimulation
such as the growth of capon comb or changes in weight of androgen-depended tissues
(levator ani, seminal vesicles, etc.) in castrated male rats. These assays are time consuming
and expensive. Hence, in vitro assays are used more commonly because they are faster,
relatively inexpensive, and can simultaneously screen a large number of test samples. The in
vitro assays are of three types [13003]:
- Receptor binding assays: These assays evaluate the ability of a substance (test
sample) to bind to the androgen receptor in the presence of a known radioactive
ligand for the receptor. Hence, the displacement of the radioactive ligand from the
androgen receptor by the test sample correlates with its bioavailability.
- Cell proliferation assays: These assays involve measurement of bioactivity of an
unknown substance by its ability to proliferate androgen-dependent cell lines, e.g.
human prostate cancer cell line (LNCap). Although sensitive, these assays are not
entirely specific for androgens.
- Reporter gene assays: In these assays, the bioactivity of a compound is measured by
the degree of expression of the reporter gene. These assays can be performed in
either yeast (usually Saccharomyces cerevisiae) or mammalian cells. In short, the
process entails choosing a host cell line that does not express an endogenous
androgen receptor. The next step involves introduction of two plasmids into the cell
line: an expression plasmid for constitutive expression of androgen receptor in the
cells and a reporter plasmid in which the androgen response element sequences
drive the expression of a truncated form of firefly (Photinus pyralis) luciferase. The
latter serves as the reporter gene. The amount of luciferase expression is
proportional to the bioactivity of the compound being tested in the sample and is
measured by luminometry. These assays have now been optimized for high
throughput screening. Hence, a large number of samples can now be tested with
reporter gene assays.
Indirect androgen doping
Indirect doping refers to strategies employed by athletes that result in a sustained increase in
endogenous testosterone production and is tailored to circumvent the banning enforced on
the administration of natural or synthetic androgens by WADA. These strategies include
[13003]:
- estrogen blockers such as estrogen receptor antagonists (antiestrogens) or

aromatase inhibitors
- androgen precursors such as DHEA and androstenedione
- gonadotropins.
Estrogen blockers
The rationale for using estrogen antagonists and aromatase inhibitors stems from the fact
that estrogen is a more powerful negative regulator of hypothalamic-pituitary-gonadal axis
than testosterone itself. This is evident in men with congenital aromatase deficiency where
both androgens and gonadotropins are elevated and exogenous estrogen replacement
suppresses them. Although less than 1 percent of daily testosterone produced is aromatized
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to estradiol, it has 100-fold higher molar potency than testosterone. Hence, the reflex rise in
endogenous testosterone as a result of estrogen blockade may result in myotrophic effects,
although the rise in serum testosterone concentrations is modest (50-65 %). Hence, both
estrogen receptor antagonists and aromatase inhibitors have been put on the list of banned
drugs by WADA. The original antiestrogens were the nonsteroidal drugs like clomiphene and
tamoxifen that bind to both estrogen receptor alpha and beta. Since then, newer nonsteroidal
agents have become available such as raloxifene, toremifene, and droloxifene. The newest
steroidal estrogen analog that has come to market is fulvestrant. Aromatase is an enzyme
that is a product of CYP19 gene and is responsible for converting testosterone to estradiol.
Aromatase inhibitors completely and irreversibly inhibit this enzyme, which results in a
decrease in estradiol synthesis. Aminoglutethimide was the first steroidal drug in this class.
Since then, more specific and potent steroidal aromatase inhibitors have become available
such as testolactone, atamestane, and exemestane. The nonsteroidal aromatase inhibitors
include anastrozole, letrozole, and vorozole. Recently, mass spectrometric methodologies
have become a formidable tool in identifying both estrogen blockers and aromatase
inhibitors. Hence, a sensitive and robust assay is now available to circumvent indirect doping
by these agents [13003].
Androgen precursors
This class of agents includes drugs like androstenedione and DHEA. Androstenedione is a
prohormone that is produced in the gonads and the adrenals in both sexes. It is synthesized
from DHEA and is converted to testosterone by 17beta hydroxysteroid dehydrogenase. On
the other hand, DHEA is predominantly secreted by the adrenal glands. Both these
compounds were sold over the counter in an uncontrolled fashion as dietary supplements for
almost two decades under the Dietary Supplement Health and Education Act. It was only
recently that the US Congress added androstenedione to the list of banned steroids. To the
contrary, the IOC prohibited the use of these agents more than a decade ago (DHEA in
November 1996 and androstenedione in December 1997). Androstenedione at a dose of 300
mg results in a significant increase in serum testosterone levels, whereas a dose of 1500 mg
results in an increase in lean body mass and muscle strength. Similarly, one study showed
that DHEA at high doses (100 mg) also results in an increase in muscle strength. Hence, the
banning of both these prohormones by the IOC is justified because they do carry ergogenic
potential. Each of these hormones results in an increase in T/E ratio, which is then followed
by confirmation of 13C/12C ratio with IRMS. In the case of androstenedione, another mode of
confirmation is by measuring its metabolite, 4-hydroxyandrostenedione, by mass
spectrometry or IRMS [13003].
Gonadotropins
Another form of indirect doping is the use of gonadotropins such as LH or human chorionic
gonadotropin (hCG), both on the WADA’s list of prohibited drugs for male athletes. The latter
is a dimeric glycoprotein containing an alpha- and a beta-subunit that is normally produced
by the human placenta. Its alpha-subunit is similar to other glycoprotein hormones such as
FSH, LH, and TSH. It undergoes glycosylation with sialic acid residues that prolongs its half-
life and makes it a long-acting analog of LH. Clinically, recombinant LH or hCG is used to
stimulate spermatogenesis and endogenous testosterone production in men who have
central hypogonadism and desire fertility. Not only are hCG and LH expensive, requiring
injections several times per week, but the evidence that they improve muscle strength is
sparse. A small randomized placebo-controlled study of older men showed that hCG therapy
resulted in an increase in serum testosterone levels and lean body mass; however, it failed to
improve upper or lower body strength (61). In addition to its presumed ergogenic effects,
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subjects abusing AAS also use hCG to avoid detection of exogenous testosterone by
stimulating endogenous testosterone production and preventing testicular atrophy. Because
hCG doesn’t alter T/E ratio, this methodology is not useful in the detection of indirect doping
with hCG. The detection of hCG is initially performed with immunoassays, and if positive,
confirmation is carried out with immunoextraction and mass spectrometry. hCG is not
banned in female athletes because there is no evidence suggesting that it improves muscle
mass and strength. Furthermore, there is an ethical dilemma because positive hCG may
reveal underlying pregnancy and is therefore considered an invasion of privacy. Detection of
LH is accomplished by immunoassays [13003].
GnRH is synthesized in the hypothalamus, and it is transported from there via the
hypothalamic-pituitary portal venous microcirculation to stimulate the gonadotrophs, resulting
in LH and FSH production. For this, GnRH must be secreted in a pulsatile fashion (peaks
every 60–90 min). Continuous administration of GnRH desensitizes the gonadotrophs,
resulting in down-regulation of GnRH receptors. This results in suppression of testosterone
to castrate levels (after an initial flare) and forms the basis of therapy for prostate cancer.
Pulsatile administration of GnRH via pumps is performed in clinical settings in men who have
a hypothalamic cause of hypogonadism; however, serum testosterone levels achieved are
generally within the therapeutic range. Hence, GnRH administration doesn’t result in
sustained production of supraphysiological testosterone levels. For these reasons, GnRH
and its analogs are not banned by WADA. However, mass spectrometry-based assays have
been developed to detect the use of exogenous GnRH. Under normal circumstances, only
fragments of GnRH may be detectable in the urine. Hence, a urinary concentration of intact
GnRH of at least 20 pg/ml is considered unambiguous evidence for surreptitious use of the
hormone [13003].
For decades, scientists have been trying to dissect out the anabolic and androgenic
properties of androgens. The discovery of SARMs was a result of the constant quest for
achieving tissue selectivity mainly so that the anabolic effects of these compounds on muscle
and bone can be achieved without any risk to the prostate. This tissue selectivity is of
tremendous importance in the elderly who have a higher incidence of prostate-related
events. This was finally accomplished in 1998 when it was developed the first SARM. This
compound was derived from androgen receptor antagonists like flutamide and bicalutamide.
Over the years, four groups of SARMs have been synthesized. These include
arylpropionamides, bicyclic hydantoins, quinolines, and tetrahydroquinolines. Based on their
anabolic properties in musculoskeletal tissues, in January 2008, WADA added SARMs to its
prohibited list. Because SARMs possess enormous structural heterogeneity, their detection
remains a daunting task. However, most of the available SARMs can be detected either by
liquid or gas chromatography tandem mass spectrometry [13003].
Boosting in paraolympic
“Boosting” is defined as the intentional induction of autonomic dysreflexia (AD) by athletes

with a spinal cord injury (SCI) at or above the level of T6 for the purpose of improving sports
performance. Boosting has been shown to confer up to a 10 percent improvement in race
time. Additionally, to compete in a hazardous dysreflexic state, whether intentional or
unintentional, would present an extreme health risk to the athlete. For these reasons, the
International Paralympic Committee strictly bans the practice of boosting, and has developed
a protocol to test for its presence. Testing was performed at three major international
Paralympic events. Education regarding the dangers of AD was provided to athletes and
team staff. Testing was conducted on athletes from the relevant sport classes: Athletics
(wheelchair racing classes T51/T52/T53) and Handcycling (H1). Key parameters included
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the athlete's demographics (gender, country of origin), classification and blood pressure
measurements. An extremely elevated blood pressure was considered to be a proxy maker
for AD, and a systolic blood pressure of ≥180 mm Hg was considered a positive test. A total
of 78 tests for the presence of AD were performed during the three games combined. No
athlete tested positive. The number of athletes tested, by classification, was: 6 in Athletics
T51, 47 in Athletics T52, 9 in Athletics T53 and 16 in Handcycling H1. Of those tested, the
average systolic and diastolic blood pressures were 135 mm Hg (range 98-178) and 82 mm
Hg (range 44-112), respectively. All athletes were compliant with testing. No athletes were
withdrawn from competition due to the presence of AD. Testing for the presence of AD in
paralympic athletes with SCI prior to competition has been carried out for the first time at
three major international paralympic competitions. There have been no positive tests thus
far. Knowledge gained during these early testing experiences will be used to guide ongoing
refinement of the testing protocol and the development of further educational initiatives
[13056].
In vitro models to study effects and mechanisms in doping
A particular field of analytical chemistry applied to forensic toxicology is represented by the

anti-doping analysis, where biological samples (urine and in some instances blood) collected,
either "in competition" or "out of competition", from athletes ruled for national/international
sport federations, are analyzed to detect the putative use of prohibited substances and
methods. Together with the official anti-doping activity to test the athletes (i.e. who engages
in competitive sport) for the non-physiological enhancement of sport performance, it is
mandatory to activate a new strategy of doping control, that should necessarily comprise a
deep and exhaustive toxicological evaluation of the entire spectrum of doping substances
and methods. An outline of the present status and of the future trends of the antidoping
research is here presented, showing that most of the new tasks could greatly benefit from an
approach based on in vitro methods, ranging from specific toxicity studies to the possible
detection of new forms of doping [03003].
Combinations with nutritional supplements
The use of performance-enhancing substances by athletes is nearly as old as sport itself.

There are two primary categories of substances available to modern athletes: anabolic
androgenic steroids (AAS) and nutritional supplements. All AAS and many of the nutritional
supplements are used to increase testosterone levels in the body, thereby enhancing the
athlete's ability to build lean muscle mass. Other nutritional supplements are used to
increase the amount of energy available for workouts or competition. Although steroids are
available in the US via physician prescription, nutritional supplements are widely available to
all consumers with relatively scant regulation. Steroids are associated with a variety of side
effects that can lead to physical changes, psychological disturbances, morbidity, and even
mortality. The side effects of nutritional supplements are not as well studied but are
presumed to be similarly dangerous. However, for many athletes at all levels facing pressure
to excel, the potential benefits of taking these substances appear to be outweighing the
associated risks. Increased testing at all levels is recommended [03004].
Possible lack of effects of doping
Doping is a serious issue bedevilling the sporting arena. It has consequences for athletes'
careers, perception of sports in the society and funding of sports events and sporting
organisations. There is a widespread perception that doping unfairly improves results of
athletes. A statistical study of information on best lifetime results of top 100 m sprinters
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(males better than 9.98 s, females 11.00 s), over the period of 1980-2011 was conducted.
Athletes were divided into categories of “doped” (n=17 males and 14 females), based on self
admission, the confirmed detection of known doping agents in their bodies or doping
conviction, and “non-doped” (n=46 males and 55 females). No significant differences
(unpaired t-test) between dopers and non-dopers were found in their average results: male
“dopers” 9.89 s identical with 'non-dopers' 9.89 s, females 10.84 s and 10.88 s respectively.
Slopes of regressions of best results on dates for both 'dopers' and 'non dopers' were not
significantly different from zero. This indicates that no general improvement as a group in
100 m sprint results over a quarter of a century occurred irrespective of doping being or not
being used. Since there are no statistical differences between athletes found "doping" and
the others, one of the following must be true: (1) "doping" as used by athletes so detected
does not improve results, or (2) "doping" is widespread and only sometimes detected. Since
there was no improvement in overall results during the last quarter of the century, the first
conclusion is more likely. Objectively, various "doping" agents have obvious physiological or
anatomical effects. These may not translate into better results due to the clandestine use of
doping that prevents its scientific structuring. Perception of the effectiveness of doping
should be reconsidered. Policy changes may be required to ensure the continued fairness
and equity in testing, legislation and sports in general [13022].
Cognitive doping
A growing concern in today’s society is the consumption of substances to increase physical

or cognitive performance. For example, the use of drugs such as anabolic steroids in
professional sports has long been a concern. In order to combat physical doping in
professional sports, the World Anti-Doping Agency (WADA) annually lists banned
substances. Besides illicit or banned drugs, athletes also consume legal and freely available
substances such as analgetics, caffeine, and other ergogenic aids (e.g. creatine, vitamins,
minerals, carbohydrates, proteins), which may also improve physical performance. Cognitive
doping can include illicit substances (e.g. cocaine) and prescription drugs (pharmacological
neuroenhancement) such as stimulants (e.g. methylphenidate and amphetamines),
antidepressants, beta-blockers, or modafinil, which are primarily designed and used for the
treatment of diseases. Prevalences for the use of such cognitive-enhancing substances
range from 1.2 to 35 percent among German and American students, and are estimated to
be 20 percent among readers of the journal Nature, 20 percent among surgeons, and 5
percent among office workers in Germany. Besides illicit and prescription drugs, the use of
legal and freely available substances such as ginkgo biloba or caffeinated drinks (e.g. coffee,
energy drinks) are also a matter of debate although their ergogenic potential is still unknown.
It is of particular concern that these nutritional supplements have been shown to fail tests of
safety, purity, and quality of ingredients and may contain prohibited substances. One study
assessed, for the first time, prevalence estimates for physical and cognitive doping within a
single collective of athletes using the randomized response technique (RRT). Furthermore,
associations between the use of legal and freely available substances to improve physical
and cognitive performance (enhancement) and illicit or banned substances to improve
physical and cognitive performance (doping) were examined. An anonymous questionnaire
using the unrelated question RRT was used to survey 2,997 recreational triathletes in three
sports events in Germany. Prior to the survey, statistical power analyses were performed to
determine sample size. Logistic regression was used to predict physical and cognitive
enhancement and the bootstrap method was used to evaluate differences between the
estimated prevalences of physical and cognitive doping. 2,987 questionnaires were returned
(99.7%). 12-month prevalences for physical and cognitive doping were 13 and 15 percent,
respectively. The prevalence estimate for physical doping was significantly higher in athletes
who also used physical enhancers, as well as in athletes who took part in the European
Championship in Frankfurt compared to those who did not. The prevalence estimate for
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cognitive doping was significantly higher in athletes who also used physical and cognitive
enhancers. Moreover, the use of physical and cognitive enhancers were significantly
associated and also the use of physical and cognitive doping. The use of substances to
improve physical and cognitive performance was associated on both levels of legality
(enhancement vs doping) suggesting that athletes do not use substances for a specific goal
but may have a general propensity to enhance. This finding is important for understanding
why people use such substances. Concerning a potential gateway for cognitive doping, the
prevalence estimate for cognitive doping was significantly higher in athletes who used
cognitive enhancers than those who did not. Since we do not know which type of substance
was used first by the athletes – legal and freely available or illicit substances – these data do
not strongly support the gateway theory that the use of cognitive enhancers is the first step
for cognitive doping. Consequently, more effective prevention programs against substance
abuse and doping could be developed [13023].
Doping may be defined, broadly, as the use of unauthorised means to increase performance
in sport. Doping is most commonly associated with the use of drugs. It was discussed some
recent advances in neuroscience that suggest that the skills and abilities underpinning sports
performance can be enhanced using technologies that change the activity of the brain.
These factors may include motor learning, enhanced muscular strength or reduced fatigue,
or even changes to mental state or concentration. The devices needed to generate these
effects are already available, and are currently in use in laboratories or clinics to produce
short- or long-term changes in performance. It was argued that brain stimulation, or
neurodoping, will become a key technology for the future of sport and sports medicine. It will
be suggested that neurodoping may have different uses in different sports, and it was argued
that each sport must determine whether neurodoping should be considered as cheating, or
should be considered a legitimate aid to training or performance [13024].
Brain stimulation techniques
Two main brain stimulation techniques are available. Transcranial magnetic stimulation
(TMS) involves the discharge of brief magnetic pulses through a stimulating coil held against
the subject’s head. This rapidly-changing magnetic field induces electric currents in the brain
tissue near to the centre of the coil. The immediate effect of this is to generate action
potentials in those cells, followed by a refractory period as the cell recovers. The fire-
andrecover pattern is most visible when a TMS pulse is triggered over the hand area of
primary motor cortex: muscle activity of the contralateral hand, measured with electromyo-
graphy, shows a burst (called the motor-evoked potential) followed by relative quiescence
(called the silent period). Recent developments in the application of TMS have involved
temporally patterning the pulses delivered by the stimulator to induce both inhibitory and
excitatory effects in the target brain area (called thetaburst stimulation). These effects outlast
the stimulation phase by several tens of minutes, with the possibility of longer-term
reorganisation of brain activity if the stimulation is applied at regular intervals. Transcranial
current stimulation (tCS) comes in two common variants. Transcranial direct current
stimulation (tDCS) involves passing a weak electric current from a negative electrode
(cathode) to a positive electrode (anode). The magnitude and polarity of the electric field at
the brain surface near the electrodes determines its effect: cells in the vicinity of the anode
will tend to increase in excitability, through a process thought to involve a modulation of the
resting membrane potential of the cells; conversely, cells near the cathode become less
active through the same process. Transcranial alternating current (tACS) uses a similar
principle, except that the current alternates at a specific frequency. Researchers typically
apply tACS with a frequency related to functionally-relevant oscillatory brain potentials, such
as might be seen with electroencephalography. tCS has a number of advantages over TMS.
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The technology is cheaper and more portable. Indeed, wireless tCS stimulators are now
commercially available, and websites exist that give instructions for home-made tCS
stimulators. TMS is, however, a more focal technique, with a relatively small area of the brain
being affected by the sti13ulation, whereas the electric field induced by tCS spreads across
the whole brain surface [0024].
What can be done with brain stimulation?
It is foreseen two domains where neurodoping may potentially change performance in sport.
These divide into immediate gains from increasing cortical excitability versus longerterm
gains from stimulation during training. In the ‘‘acute’’ phase following stimulation, participants
have demonstrated enhanced motor skills including: improved time-to-fatigue, response time,
and tremor suppression. The effect of tDCS is maximal shortly after the end of stimulation
and declines over roughly a 20- to 60-min period, depending on the stimulation parameters.
The effects of theta-burst TMS last for a similar length of time, but with the peak of effect
some 5 min after the end of stimulation. So, it is possible to envision a time when an athlete
might take a ‘‘hit’’ of stimulation before shooting a pistol or setting off on a ski slalom. A
second use of neurodoping might be in skill acquisition. Skills learned in the context of
anodal tDCS are acquired more rapidly, and reproduced more accurately, than those learned
without. Sports performance at the highest levels require good technique and good timing.
These are skills learned during training, so enhancing the efficiency of learning during the
training phase will be of greater benefit at competition time. It has been suggested that an
athlete could use these techniques to make training more efficient and thereby gain an
advantage. It is possible that neurodoping will add little to the performance of elite athletes.
Most studies of brain stimulation recruit non-expert, healthy participants from the community
of the laboratory (in practical terms, university students) and test in conditions where
performance is likely to be changed but not reach its maximum. Elite athletes who are
already performing close to the physical limits of the human body may not gain from the
potential benefits of brain stimulation. Further research is needed to explore whether
neurodoping and elite performance are compatible [13024].
Can it be detected? What are the risks?
There is no known way to detect reliably whether or not a person has recently experienced
brain stimulation. A modern technique for analysing brain composition is magnetic resonance
spectroscopy (MRS), which can detect changes in the concentration of neurotransmitters
and related metabolites. Theta-burst TMS appears to affect inhibitory processing by gamma-
amino butyric acid (GABA) while not affecting excitatory processing involving glutamate.
Anodal and cathodal tDCS modulate GABAergic and glutamatergic processing differently.
When looking specifically at the brain region targeted by TMS or tDCS, the changes in brain
chemistry are of the order of 10 percent in metabolite concentration, and require carefully
controlled conditions to pick out the signal from the noise. Several factors means that MRS is
unlikely to be of practical utility in detecting neurodoping [13024].
Ethical issues
There is an argument that human enhancement of any type is not wrong in sport or in any
other context. It has been argued that enhancing performance with drugs is analogous to an
act of creativity whose only limit should be the safety of the participant. So, by this argument,
regulating pharmacological enhancers places an unnecessary constraint on the limits of
human achievement. Allowing drug enhancement would simply add another option for
athletes who wish to choose among all the available means of improving their overall
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performance. While the analogy with the kind of neuroenhancement is not perfect,
nonetheless brain stimulation offers a potential adjunct for immediate performance or for
training and should be considered as if it were another form of drug doping. A related
question about the use of human enhancement is whether the performance shown by the
person is ‘‘authentically’’ theirs. If a neuro-enhanced sprinter is faster in reacting to the
starter’s pistol, is that advantage hers or should the stimulator be given the credit? It was
recently argued that brain training with tDCS enhances the person’s own latent cognitive
abilities and increases the efficiency of a training programme. Thus, brain stimulation
mediates a person’s ability but does not enhance it in the strictest sense. A third argument is
practical: as discussed above it is not possible, as far as is known, to determine whether a
person has or has not had brain stimulation. Each sport determines its own rules. It would be
suggested that each sport determines whether neurodoping poses a risk to its ethos. For
example, performance in a sport such as pistol-shooting would be greatly improved by
tremor reduction, so governing bodies should decide whether shooters should be prevented
from using tACS during or immediately before competing to reduce tremor, just as beta-
blockers are banned in many sports. Conversely, a tennis player’s performance in a match is
heavily influenced by the probability of regularly getting the first service in, which is a skill
learned in training and therefore potentially susceptible to neurodoping [13024].
Effect of international results during the anti-doping era
World records in running as indication for doping in the elite
If recent improvements in athletic performance have been driven by doping, then improved
doping control might be reflected by a leveling off or declining performances in sports where
doping is thought to be ubiquitous. In recent analyses of major cycling races including the
Tour de France, Giro d’Italia, Vuelta A España, the average speed has been leveling off or
declining, since the introduction of improved techniques to detect use of exogenous EPO in
2005. However, the analysis of cycling is confounded by varying race distances, yearly
changes in course, and weather. Endurance running eliminates many of these confounding
factors. The tracks and courses are identical from year to year. The major cycling "Grand
Tours" have shown an attenuation of performance over the last decade. This has been
interpreted as circumstantial evidence that newer anti-doping strategies have reduced the
use of performance-enhancing drugs. To examine this idea under more controlled conditions,
speed trends for world class 5000 m, 10000 m, and marathon performances by men from
1980 to 2013 were analyzed. We obtained comprehensive records from the International
Association of Athletics Federations, Association of Road Racing Statisticians, and the Track
and Field All-time Performances database webpages. The top 40 performances for each
event and year were selected for regression analysis. For the three distances, we noted
cumulative performance improvements in the 1990s thru the mid-2000s. After the peak
speed years of the mid 2000s, there has been limited improvement in the 5000 m and 10,000
m and world records set during that time remain in place today, marking the longest period of
time between new records since the early 1940s. The world record for the 5000 m was set in
2004 while the 10000 m world record was set in 2005; these records stand today, which is
the longest gap between world records since the 1940s. The number of performances below
the 2012 Olympic A qualifying standard for the 5000 m and 10,000 m also appears to have
leveled off since the middle 2000 s. Similarly the number of athletes breaking 2 10 00 for the
marathon has also leveled off. 2 10 00 was chosen as a comparable standard for the
marathon because this time is considered generally similar to or slightly slower than the A
standards for shorter distances based on various empirical point tables, scoring systems and
time conversion programs. Furthermore, year alone explained a large percentage of the
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variation in the speed trends. Consistent with these overall model estimates, the 5000 m and
10000 m had significant increases in speed during the 1990s whereas the marathon showed
an increase over the entire three plus decades. In particular, the 5000 m the speed trend
levels off starting around 2000. The marathon and 10000 m times do not show this as a
pronounced tendency. With the 5000 m and 10000 m, there is a pronounced “outlying” effect
of the top performance from 1995 to late 2010. The marathon, however, displays no
attenuation of the increased speed over the epoch sampled and the relative speed of the
fastest annual time does not appear to be an outlier. The degree to which the fastest times
were relatively fast (compared to other years) was observed during the 2000s in the 5000 m
and 10000 m distances. The regression spline analyses supported these findings that the
fastest relative times for the 5000 m and 10000 m varied over the epoch. While the speed
trends for 5000 m and 10000 m track results parallel those seen in elite cycling, the
marathon trends do not [14701].
The speed and performance trends for top 5000 m and 10000 m distance running performers
on the track show a period of increased speed among the fastest runners to the mid-2000 s
with an attenuation of speed in either all (5000 m) or the fastest performances (10000 m)
after this period of time. For the marathon, all indices of speed show a nearly linear increase
in speed with an increased number of elite performances over the three plus decades was
sampled. It was believe there are a number of possible explanations for these findings. First,
the findings for the 5000 m can be interpreted as consistent with the hypothesis that
improved drug testing has limited the ability of elite athletes to manipulate their oxygen
transport systems with EPO (or other techniques to improve oxygen transport during
exercise) since the middle 2000 s. These observations are also broadly consistent with
recent speed trends in elite cycling races. This interpretation can also be applied to the
10000 m results, but only when considering the fastest times. By contrast, the data for the
marathon shows continued improvements in running speed during the same time period
along with more total elite performances and world records. These observations challenge
the idea that the speed leveling seen in the 5000 m on the track and in the so-called “Grand
Tours” of cycling is due primarily to better drug testing and the reduced use of performance
enhancing drugs. A second possible interpretation is that world class performances are
leveling off and reaching a physiological upper limit as has been postulated for equine and
canine athletes. In the case of the marathon a number of empirical estimates and
physiological modeling suggest the record is relatively slow in comparison to the 5,000 m
and 10,000 m times and is merely catching up by comparison. In this context, it is interesting
to note that top speeds have not fallen for the shorter races but only leveled off [14701].
The third element of any interpretation focuses on the changing financial incentives in
professional distance running. Prize money for top marathon performances has increased.
Specifically, in 1980 the highest total payout for any marathon was USD 50,000; just over
two decades later the first million dollar race was run. These incentives could be attracting a
stronger pool of competitors to “move up” and focus on the marathon and forgo record
setting attempts at shorter distances. This could lead to more competitive races among top
runners at the major marathons. Second the highest profile marathon races are now being
staged in a way designed for world record attempts that include the use of pacers. Along
these lines, the use of pacers has been wide spread for races on the track for many years,
and many top athletes have bonus plans and other financial incentives from sponsors that
reward fast times at the shorter distances. There are a number strengths and limitations to
this study. A major strength of our data set and analysis is that it includes standardized
distances and courses with numerous competitive opportunities at the shorter two distances
when environmental conditions are likely to be optimal. By contrast, a limitation to our
analysis is that we have no idea if improved approaches to training or equipment (shoes and
tracks) might have contributed to the trends we report. However, we favor the interpretation
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that the entire epoch we have analyzed has been relatively stable from a technical
perspective. This includes widespread use of high volume and high intensity training,
widespread availability of synthetic tracks, and adequate footwear. Additionally, while ideas
about training have been refined it is not known if how uniformly these have been adopted by
elite athletes, especially the East Africans. This perspective contrasts to the major
improvements in equipment for cycling that includes use of advanced materials and
improved aerodynamic designs to construct faster bikes [14701].
A final concern whenever the topic of doping is raised is discussed relates to what might be
called the continuous “cat and mouse” game between those trying to enforce the rules with
improved testing and those trying to circumvent them. This has engendered speculation that
micro-doses of EPO can be titrated by athletes in a way to achieve high levels of
performance and yet avoid a positive drug test. There is also widespread speculation about
the use less or undetectable compounds and so-called designer performance enhancing
drugs. Advocates of these points of view have argued that while doping is considered
widespread the number of positive tests in major competitions is quite low. The counter
argument is that the low number of positive results demonstrates that the testing is working
and deterring doping. The lack of hard data on the true incidence of doping and how it has or
has not been influenced by improved testing is unknown and a major limitation to any
discussion on this topic. However, it is clear that anonymous questionnaire based surveys
suggest the true incidence of doping it is much higher (14-39 %) than about 2 percent rate of
positive tests suggests. This is clearly an area of sports sociology that requires increased
attention. It should also be noted that the sociology surrounding the doping phenomenon
along with the ongoing incentives to dope are complex. In this context, strategies beyond
testing alone will be required to improve the efficacy of doping control [14701].
Prediction of further improvements (year 2002)
The limiting factors of top athletic performance and the psycho-physiological mechanisms
involved remain controversial. The aim of one study was to attempt a prediction of world
records (WR) for the next ten years in five athletic track and field and events. Our prediction
has been produced by means of computer-aided mathematical models. In short, polynomials
that could best approximate the WR of the last decades have been calculated and projected
over the period 2000-2010. The predicted values for the year 2010 point to an improvement
rate of the WR considered varying between 0.2 and 10.3 percent, depending on event and
gender. Those values could be influenced by the use of better sports equipment, better
nutrition and training and especially by the impact of doping and of anti-doping measures
[02009].
100 meter and 5000 meter
The introduction of doping substances and methods in sports triggers noticeable effects on
physical performance in metric sports. Here, we use time series analysis to investigate the
recent development in male and female elite sprinting performance. Time series displaying
the average of the world's top 20 athletes were analyzed employing polynomial spline
functions and moving averages. Outstanding changes in performance over time were
statistically analyzed by Welch's t-test and by Cohen's measurements of effect. For validation
we exemplarily show that our analysis is capable of indicating the effect of the introduction of
in- and out-of-competition doping testing on women's shot put as well as the effects of the
market introduction of erythropoietin (EPO) and the introduction of EPO and continuous
erythropoiesis receptor activator (CERA) testing on 5000 m top 20 male performances. Time
series analysis for 100 m men reveals a highly significant drop by more than 0.1 s from 2006
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to 2011 with a large effect size of 0.952. This is roughly half of the effect size that can be
found for the development of the 5000 m performance during the introduction of EPO
between 1991 and 1996. While the men's 200 m sprinting performance shows a similar
development, the women's 100 m and 200 m sprinting performances only show some minor
abnormalities. It was discussed why the striking sex-specific improvement in sprinting
performance is indicative for a novel, very effective doping procedure with insulin-like growth
factor-1 (IGF-1) being the primary candidate explaining the observed effects [13068].
Human upper performance limits in the 100-m sprint remain the subject of much debate. The
aim of this commentary is to highlight the vulnerabilities of prognoses from historical trends
by shedding light on the mechanical and physiological limitations associated with human
sprint performance. Several conditions work against the athlete with increasing sprint
velocity; air resistance and braking impulse in each stride increase while ground-contact time
typically decreases with increasing running velocity. Moreover, muscle-force production
declines with increasing speed of contraction. Individual stature (leg length) strongly limits
stride length such that conditioning of senior sprinters with optimized technique mainly must
be targeted to enhance stride frequency. More muscle mass means more power and thereby
greater ground-reaction forces in sprinting. However, as the athlete gets heavier, the energy
cost of accelerating that mass also increases. This probably explains why body-mass index
among world-class sprinters shows low variability and averages 23.7 ± 1.5 and 20.4 ± 1.4 for
male and female sprinters, respectively. Performance development of world-class athletes
indicates that about 8 percent improvement from the age of 18 represents the current
maximum trainability of sprint performance. However, drug abuse is a huge confounding
factor associated with such analyses, and available evidence suggests that we are already
very close to "the citius end" of 100-m sprint performance [150023].
100 meter in Olympic Games
physical performance in metric sports. Here, we use time series analysis to investigate the
factor-1 (IGF-1) being the primary candidate explaining the observed effects. It is known that
human growth hormone (hGH) has been abused in professional sports since the 1980s,
disregarding its appearance on the World Anti-Doping Agency (WADA) list for banned
substances. For modern elite athletes, especially for cheating sportsmen and women,
growth-promoting effects play an important role and can be induced by various substances.
One study demonstrates that hGH administration in recreationally trained athletes results in
statistically significant improvements in sprint capacity. IGF-1 is known for evoking growth-
promoting effects and for its direct anabolic impact on skeletal muscle. At least some of the
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hGH deployed anabolic actions are exerted through triggering the generation of IGF-1.
However, so far no clinical trials have demonstrated a beneficial effect of IGF-1
administration on athletic performance. IGF-1 acts in endocrine, autocrine, and paracrine
modes on skeletal muscle. Exogenous administration of IGF-1 causes powerful anabolic
actions similar to the effects of hGH. Due to limited experience with long-term administration
of exogenous IGF-1, only side-effects from short-term usage have been documented in
clinical trials. Next to edema, arthralgia, headache, and jaw pain especially hypoglycemia
should be mentioned. Furthermore, it is appealing to use IGF-1 in combination with other
drugs like hGH, insulin, and anabolic steroids, as the combined application most likely
provides an enormous potential to improve performance. Consequently, IGF-1 is included in
the WADA list of banned substances, but its abuse by elite athletes is assumed to be lower
than for hGH, because of its lower availability due to the lack of a natural resource from
which IGF-1 could be harvested. Nevertheless, IGF-1 will be more attractive to use than
hGH, since testing for hGH is improving. Preparations containing IGF-1 were first approved
for the American market in August 2005 by the US Food and Drug Administration (FDA) for
the treatment of growth failure in children. Therewith the possibilities to acquire and abuse
IGF-1, despite the lack of a natural source, have been increasing even more. The results
from the time series analysis disclose a time decreasing trend in male short-distance running
that started around 2005/2006. Over 100 m, elite athletes underwent a large effect (0.952) in
the last five years that is clearly displayed by both approaches: spline and moving average.
The last six years over 200 m entailed an effect size of 0.481. In women's track and field
sprinting, an effect is also detectable over 100 m. However, the moving average is at the
same level in the beginning and the end of the regarded interval and over 200 m there is no
effect noticeable after 2004. These explicit developments can be discussed to be associated
with doping, but it is important to mention the existence of other possible or additional
explanations. Simultaneously with the time decreasing trend over 100 m for men, we noted
that more athletes from the West Indies entered the annual top 20 lists. Thus changing
conditions concerning training for these populations of athletes could contribute to the stated
developments. When taking doping into consideration as a plausible explanation, several
different substances have a strong enough physiological effect and therefore could be
related to this development. HGH may still be used as presently no robust long-term testing
method for the detection of hGH administration exists. Unfortunately it is also possible that
new synthetic ‘designer steroids’ have been developed that cannot be detected by the tests
currently available. Additionally the list of other substances that could possibly improve
sprinting performance is long. Next to hGH, IGF-1 and ‘designer steroids’ another example
would be oestrogen receptor antagonists. However, it was argued in the following that
among the substances that were recently introduced to the market, IGF-1 is a very important
candidate that could explain the observed development in sprinting performance The
mechanism of IGF-1 with its beneficial actions concerning physical performance
presupposes its abuse, alone and in combination with other agents, in track and field. 2008
is the year in which we first noted a significant change over 100 m, but IGF-1 was approved
in 2005. Interestingly, we detected the first sign of an EPO effect in 1992, although it entered
the American market in 1989. In one opinion, athletes first need to get acquainted with drugs
they want to abuse. Its handling is usually only known for medical purposes. So the doping-
related application of the drug and its incorporation in the training process take some time to
be optimized. Additionally, examination of the number of athletes that ran the 20 fastest
times of each year over 100 m shows a fall from ten in 2005 to four in 2006 and eleven
athletes ran the fastest times in 2011. It was believed that this progression illustrates a slow
integration: IGF-1 abuse started with a small amount of athletes and now is integrated in the
world elite of track and field sprinters. Induced by the effect detected over the women's 100
m, it was suspected female athletes to be using IGF-1, too. Even so, it seems to be less
beneficial for women, because there is no parallel effect over 200 m. Furthermore, athletes
usually try to deliver top performances in Olympic Games years. Over the women's 100 m
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and 200 m, 2008 – the year of the Olympic Games in Beijing – is the minimum. In contrast,
the men have their minima in 2011 and there is a local maximum in 2008 over 200 m. A
study showed that women responded with a smaller increase in IGF-1 levels, although they
received a larger dose of hGH compared to men. So far it is not known whether treatment
with IGF-1 leads to sex differences in the related physiological response. However, sex
differences can be found for serum levels of IGF-1. IGF-1 would not be the only doping
substance with different effects depending on sex. For example, our analysis of women's and
men's shot putt shows greater effects for female shot putters. Considering the whole period
from the introduction of anabolic steroids to the beginning of out-of-competition controls, it
was computed an effect size of 3.482 for men from the earlier effect onset in 1961 to 1988
and 5.154 for women from 1966 to 1988. Franke and Berendonk mentioned that enormous
effects, especially in female athletes, were noticed in the 1970s during the systematically
organized doping efforts of the German Democratic Republic. Anabolic steroids and EPO
are the best-known doping substances and the portrayed examples account for enormous
effect sizes. Anabolic steroids had an effect of 3.177 over ten years in the women's shot putt
and EPO 1.925 over a five-year period in the men's 5000 m. Both were abused in a variety of
sports disciplines. Nowadays blood doping, including potential abuse of EPO, is widespread
among athletes regardless of the progress in testing. This is well in line with our notion that
the introduction of EPO testing had no profound effect. In comparison, out-of-competition
testing for anabolic steroids led to a clear decrease in performances displayed through the
arithmetical mean of the 20 best athletes of each year. The effect sizes that was computed
for short-distance running from 2001 to 2011 are not as large, but still enormous enough to
animate other athletes to misuse IGF-1. In our opinion, IGF-1 could definitely also be
effective concerning track and field in the jumping disciplines, although analysis of long-jump
and high-jump did not display any recent significant developments, but future examination
might. Thus, the official introduction of the recently developed test procedure for IGF-1 could
prevent the expansion of the abuse of IGF-1 in professional sports [13069].
Contrary to that statement, when analyzing men’s 100 m performance at the Olympic level,
the winners (average 86.36 kg) have outweighed the rest of the finalists (77.72 kg) dating
back thirty years. This phenomenon is also consistent when comparing the medal winners
(80.45 kg) versus non-medal winners. Contrary to the claims made by the author’s there
have been statistically significant improvements over the last quarter century in elite
performance of the 100 m dash, both in the men’s and women’s division. A separate one-
way ANOVAs compared the 100 m finals times for men and women from the Olympic
Games over the past 20 years with Bonferroni post hoc analysis when appropriate. There
were significant differences observed for men and women. Post hoc analysis revealed men’s
times from 2012 were significant lower than 1992 and 2000; with 2008 also being lower than
1992. Women’s times from 2012 were lower compared to 2000. This holds true for not only
the medal winners of the Olympic Games, but also of the participants in the finals as a whole
[13070].
It is believed that the remarkable improvements observed in the 100 and 200 m are mostly
due to the performance of one single athlete, Usain Bolt, who has repeatedly broken the
world record of these sprint disciplines in 2008 and 2009, by lowering the limits by notable
coefficients of 0.984 and 0.993, respectively. Since the dramatic drop of the polynomial lines
is almost entirely due to the performance of one athlete, it cannot be attributed to an entire
group of athletes. This phenomenon has been recently defined as the Usain Bolt effect, and
has been attributed to stature and reduction in stiffness as a consequence of the increased
contact time and lower step frequency, which both result in an advantage in relative power
development and mechanical efficiency. This would not support the theory of improvement
by doping but – rather – the well known possibility of “extreme outliers” that seldom occur in
a normal distribution of athletes, and may remarkably account for an improvement in records.
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A second important aspect is that Usain Bolt, the athlete who has dramatically improved both
100 and 200 m world records, has never been found positive during anti-doping controls,
either in- or out-of-competition, by whatever anti-doping authorities. Until opposite evidence
can be provided, this is the only reliable proof that we have that the world records were
broken by a fair athlete. Then, it is also questionable to assert that the use of hGH and/or
IGF-1 explains the effect on 100 m performance between 2006 to 2011, since it has been
clearly acknowledged that athletes have been abusing hGH for its anabolic effects since the
early 1980s, whereas the first test was not introduced until 2004. Accordingly, it is much
more likely that the abuse of hGH had been commonplace before 2006, and not afterwards.
An identical consideration can be made on the potential abuse of IGF-1, since this substance
appeared much earlier than 2006 on the black market [13071].
Influence on world records in running by doping and anti-doping testing
Improvements in track and field sports have been attributed to factors such as population
increase, drugs and new technologies, but previous research has found it difficult to
distinguish the contributions from specific influences. Here it is shown how this is possible by
means of a performance improvement index based on useful work done combined with
modelling of the annual top 25 performances. The index was set to 100 in 1948 and showed
that, by 2012, it had increased in running events to between 110.5 and 146.7 (men's 100 m
and marathon). Underlying global effects accounted for the majority of all improvements
(16.2 to 46.7) with smaller influences attributable to an influx of African runners (3.6 to 9.3),
and a 4-year oscillation that arose from staging of the Olympic Games (±0.2 to ±0.6).
Performance decreased with the introduction of compulsory random drug testing (-0.9 to -
3.9) the World Anti-Doping Agency (WADA; -0.5 to -2.5) and fully automated timing (-0.6 to -
2.5). Changes in elite sporting performance since the 1890s are attributable to societal
changes caused by the industrial revolution and globalisation superimposed on millennia of
human evolution [14001].
Is doping-free sport a Utopia?
The impressive legend of 7-time Tour de France winner Lance Armstrong has died, replaced
by an equally impressive legacy of shameless lying and cheating on a grand scale, team
doping orchestration and discipline on and off the road, and conspiracy to fool the world
while earning tens of millions of dollars. But, the harsh reality is that the doping-control
system did not catch arguably the biggest, boldest, and most brazen drug cheat in the history
of sport. Hundreds of analytical doping tests performed over nearly a decade in state-of-the-
art laboratories failed to reveal his charade. And the same system failed to catch any of his
teammates as long as they were on his team. Riders have confessed under oath how easily
the tests could be manipulated. Doping testing failed miserably. A federal investigation
compiling 1000 pages of evidence and testimony from 26 different cycling teammates and
support staff finally caught Armstrong. All of this evidence is now public. If one of the world’s
most visible athletes evaded detection despite “500 doping tests” (probably the real number
was about half that) over all those years while the whole world watched and the doping-
control officials took blood and urine samples, why should we have any confidence that the
situation is better today? In numbers, the power-to-weight ratio for the best cycling climbers
in the world on their hardest, most decisive climbs rose from about 5.8 W/kg in the late 1980s
and early 1990s quite rapidly to ≥6.3 W/kg by the mid-1990s and remained there to the mid-
2000s, before beginning to fall again to 5.8 to 6.0 W/kg in the most recent tours. This
anecdote suggests that EPO plus consumption/injection of anabolic agents facilitating
accelerated recovery may have been “worth” as much as a about 10 percent increase in
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sustainable power over climbs of 30 to 40 minutes. The Festina doping scandal of 1998
seemed to scare the peloton straight in 1999, because climbing power (otherwise)
inexplicably dropped about the same 10 percent, before rising again in 2000 and thereafter.
That is a measurable, chronologically preciseblip that adds some contrast to the doping
fingerprint. With the exception of doping-positive Alberto Contador, the fastest climbs in Tour
history were performed during the 1996-2006 time window, with slower climbing times since
by the best climbers and the Tour de France winners in the last 2 years [13028].
Armstrong’s doping violations were detected by antidoping laboratories on several

occasions, but these results appear to have been systematically swept under the rug at a
higher level. A broomshaped checkbook may have been used, as evidenced by large
“financial contributions” by Armstrong to the International Cycling Union (UCI). This would
mean not that doping testing systematically failed but that the system was corrupt. This is a
second small piece of evidence that the drug labs can go a long way toward keeping cycling
and all sports cleaner, provided that the sport-governing bodies they serve are 100 percent
clean. A third source of encouragement amid all the gloom is that the culture of collective
“tight lips” among athletes has collapsed. In addition to a substantial increase in positive tests
since 2006, a large share of a generation of top cyclists has confessed their doping
practices. A long-standing code of silence has now been broken. Effective doping on the
immense scale uncovered with Team US Postal (and subsequent versions of Team
Armstrong) exemplifies how critical the cooperation of athletes, sport scientists, doctors, and
coaches is to achieving the combined goals of extensive and effective doping on one hand
and detection avoidance on the other. Good sport science and the open dissemination of
research and best-practice methodology in all aspects of athlete preparation have probably
contributed to making the race to the top of the international podium more expensive. The
top-20 medal-winning countries are winning a smaller share of the total medals in the
Olympics, from 90 percent in 1992 down to 75 percent in 2012. This is great for sport and in
part a result of better sport-science support in a larger number of countries, all along the path
from talent identification to physiological and technical development, to performance peaking,
to race management. The difference between a gold medal and finishing out of the medals is
smaller than ever before. Good sport science can make the tiny difference between gold and
fourth place on a given day. At the world-class level, the performance-enhancing effects of
doping clearly exceed what cutting-edge sport science can achieve with further optimization
of training programs, recovery methods, etc. For the sake of sports, it must be hoped that
good sport science, and not just good chemistry, matters more in the future [13028].
Arguments for allowing doping
Drug abuse
Limited drugs testing was trialed at the Mexico Olympics and, during the 1970s, track and
field and other sports introduced penalties for those who fell foul of the new rules. Antidoping
policies have since been adopted by every major sport. As a measure of their effectiveness,
consider the Tour de France results 52 years after Simpson’s death. The winner of the race
Alberto Contador was subsequently banned for 2 years and stripped of his 2010 Tour title for
a doping violation. The third placed rider was Lance Armstrong. The lesson is unambiguous:
doping control has failed. A more rational approach, one that is more congruent with the
reality of sports in the twenty-first century and one that would make sports immeasurably
safer is to permit doping. Competitors could then be treated as rational decision-makers,
capable of evaluating evidence and making informed choices – rather than internees. After
the Simpson tragedy, cycling’s governing body the UCI (Union Cycliste Internationale) might
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have sought a way of distinguishing the illegal recreational substances that have little or no
relevance to sport from the performance enhancing materials that were thought to promote
athletic performance. They could have accepted that the substances used by athletes were a
response to the changing demands of professional sport and sought policies that attempted
to safeguard the welfare of competitors without trying to extirpate the use of what later
became known as performance enhancing substances. Instead, it continued to lump them
together under the rubric of “drugs” – a term that still evokes images of crack-addicted
mothers who sell their babies and murderous Medell´ın cartel dealers operating in a
continent of violence [14428].
Sponsors
Commercial sponsors has increasingly used sports as marketing vehicles, inviting some to
coin the term corporatization to describe the hijacking of what was once a wholesome
competitive endeavor. So reliant were many sports on the largess from sponsors, that a
withdrawal could have been ruinous. Brands such as McDonalds and Coca-Cola paid
generously to have their names associated with a pursuit that resonats health, cleanliness,
and purity. How would they have reacted if sport allowed “drugs”? When sports governing
bodies resolve to clamp down harder on drug users, it is for the benefit of its sponsors more
than either the competitors or the fans [14428].
Antidoping policy is expensive, ineffectual, unproductive, futile, and a denial of the athletes’
freedoms. Athletes from across the spectrum have made their intentions signally clear: they
will continue to defy the most stringent tests and stay ahead of the curve, always leaving
testers lagging in their quest to eliminate doping. A sensible response and one that would
render sport safer and consistent with competition today is: to remove the banned
substances list and allow athletes to make informed and intelligent choices as to whether or
not they wish to take performance-enhancing substances. After all, sport practically incites
competitors; let me explain. Typically, athletes use dope not to cheat but to remain
competitive. Andrea Petróczi and Eugene Aidman submit: “Athletes today are expected and
encouraged to seek every possible way to improve their performance, including specialized
training, hi-tech design of equipment and apparel, scientific and medical support, including
the use of nutritional supplements.” Among the reasons cited for taking performance-
enhancing drugs (not recreational) are: perceived external pressure, suspicion that rivals are
using something, painkilling, meeting the physical demands of training. Most competitors
would prefer to compete drug-free; many are still prepared to use drugs, provided the
substance is undetectable. Some do not see drugs in sport as being a problem and accept
that drugs are part of their training regime. It has been concluded that athletes typically
agonize over whether to dope, trying to anticipate what their rivals will do. Their findings find
support from the Olympic discus thrower Werner Reiterer, who, in his (2000) autobiography,
reflects on how he found himself caught on the horns of the same dilemma: “The minority of
athletes who are natural are at a disadvantage,” Reiterer believes. “You must adapt to an
environment as it is, not as you think it should be.” His adaptation was to use. Doping
practices grow out of habitual engagement in a range of acceptable performance
enhancement (PE) practices, such as physiotherapy, advanced nutrition, training techniques,
specialized equipment and apparel” [14428].
The dilemmas of rational calculators
It has also been shown the culture and mentality of competitors, who strive to achieve and
habitually face the decision about whether to engage in “functional drug use,” which “refers to
a strategic use of substance to achieve a set goal (i.e. to improve a function or skill)”. As
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such, functional use should not be confused with “experimental, recreational, or dependent
use (abuse/addiction).” It has been approached highlights the “vulnerability” of athletes as
they progress through a sports life-cycle: at various stages, they make key choices,
commitments about goals, investments in training and comparisons, asking themselves
questions such as, “have you got what you hoped for?” “has the plan worked?” and “what is
next?” At every stage, influences from coaches, friends, fans, and perhaps the media shape
decision-making. Remember the background: a highly competitive and possibly
remunerative, win-oriented culture in which supplements and pharmaceuticals are habitually
used. Not even the most stringent antidrugs policy can remove this. Competitors, like most
humans, are “rational calculators who, with the benefit of time and reflection, make decisions
designed to be of net benefit to themselvs. Most athletes’ decisions to use banned
substances are presumably rational.” Viable is an interesting choice of word: it suggests that
the response was regarded as workable, a feasible way out of a tough predicament. So what
are athletes thinking when they make their decisions? The deterrent effect of legal sanctions
and the disgrace involved is not nearly so effective as WADA and sports governing
organizations apparently assume. The proliferating number of undetectable or designer
drugs means that laws against drugs are impossible to fully implement and “unenforceable
laws are less able to convey the moral or social threats required to inhibit behavior” [14428].
The ban on performance-enhancing drugs in sport reflects society’s view that performance-
enhancing drug use in sport is both morally wrong and potentially harmful to the individual.
But, this is easily changed: if the laws against drug use did not exist, then athletes would
know they could use performance-enhancers with impunity and would not believe they were
engaging in an act that violated morality. While the governing bodies continue to outlaw
pharmaceutically produced substances, athletes are forced to engage in clandestine
arrangements, procuring “doping agents” from unknown sources and taking them in amounts
and for periods that may prove detrimental to their health. A morally honest policy would
permit doping, but invite athletes to disclose whatever substances they have used. Sports
organizations could then commission research and advise athletes on what is most effective
and in what quantities and at what intervals it can safely be ingested. Remember: antidoping
policies were initially designed to protect the health of athletes [14428].
Fair play
There are no moral absolutes in sport. Definitions of cheating and fair play are protean: they
change perpetually. Today’s athletes use air-inflated soles on their spikes, rather than flats;
they run on fast artificial surfaces, not cinders; they wear aerodynamic body suits rather than
baggy shirts. And, of course, they train; this would have been tantamount to cheating in the
early nineteenth century. Polyurethane swimwear was fair, at least until 2009 when FINA
(Fédération Internationale de Natation), aquatic sports’ governing federation, declared it
unfair and illegal. Even today, there are mystifying contradictions [14428].
Diffuse borders
In December 2013, the football player Jan Vertonghen, then playing for London’s Tottenham
Hotspur, used a legal technique, known as PRP (platelet-rich plasma): a sample of his blood
was taken to remove the platelets and then injected them back into his injured ankle. Tiger
Woods and Rafa Nadal have also used this “blood spinning” technique. But the injection of
their own oxygenated blood into athletes before an event, a process known as blood doping,
is a punishable violation. There appears to be little consistency. It is contentedly allowed,
even recommend some types of aids to today’s athletes, yet instantly condemn those
athletes if traces of a banned substance are detected. Yet the inconsistencies multiply. If an
197
Olympic archer uses contact lenses to assist his or her performance, we let it pass. If he or
she takes a beta-blocker to steady their nerves, we suspend them. The hypocrisy of track
and field, in particular, is apparent every season when athletes are given huge cash
incentives to break records or remain unbeaten on the Diamond League circuit, yet denied
the right to maximize their athletic effort. The kind of “dope” favored by athletes is not some
sort of magic elixir: it merely enables them to train harder and for longer and so become
more adept at a discipline [14428].
Economical incentives
The joy of competing for competition’s sake has ceded place to awinner-takes-all mentality,
cultivated by professionalism. This has been made possible by corporate sponsorship on a
scale that makes the World Bank envious. One of the reasons corporate sponsors turned to
sport from the mid-1980s was because rock stars and movie actors were too prone to
embarrassing transgressions. That must be a question that weighs heavily on the minds of
sport’s administrators. Somehow, they must persuade sponsors and the public that the
pharmaceutical materials typically taken by athletes are not drugs, at least not in the way
they are popularly conceived. The alternative is to persist in the self-defeating search for
evermore sophisticated and comprehensive tests to detectnsubstances that probably do not
even have a name at the moment. Already, the costs of detection are punishingly high. There
will come a point at which the kind of surveillance and inspection required to monitor athletes
will be just too expensive; the corporations will have to be persuaded that radical change is
the only way forward. This provides little comfort to idealists who still hark after the amateur
ideals. They may abominate the prospect of their children aspiring to achieve in a profession
in which doping is extensive. It is sensed that parents today are no longer dissuading their
kids from going into sport. The lure of lucre has made it appear to be a feasible and
rewarding career. Would they think twice if they anticipated their offspringwould soon be
using performance enhancers, if only to remain competitive in an environment in which
athletes have carte blanche? [14428].
Drugs available
Many of the products on the banned list are available over the counter and most are
accessible with a prescription. They are no dirtier than the kind of products the nation is
habituated to taking every day. Those parents who insist they would not, need to remind
themselvs that the future they deplore is actuallynalready with us. Sanctimoniously denying it
by claiming, “we are winning the war on drugs” remains a deception. This is no policy of
surrender, only an acknowledgement that, like every other feature of culture, sport changes
and develops [14428].
198
DOPING AND ANTI-DOPING HISTORY
Semantics
Throughout history, athletes have sought to improve their performance through dietary and
medical help. Doping is the use of banned pharmacological substances with the unique aim
of improving physical performance despite serious adverse effects on health. The word
"doping" derives from "dope", a term describing a primitive alcoholic drink used as a
stimulant in South African ceremonial dances. Since success in sport is associated with fame
and financial rewards, the use of banned, performance-enhancing agents is highly tempting.
Concerns about ethics and controversy over the safety of doping among elite athletes first
appeared during the 1920s and 1930s [03002].
Overviews
Though we may still sing today, as did Pindar in his eighth Olympian Victory Ode, "… of no
contest greater than Olympia, Mother of Games, gold-wreathed Olympia…", we must sadly
admit that today, besides blatant over-commercialization, there is no more ominous threat to
the Olympic games than doping. Drug-use methods are steadily becoming more
sophisticated and ever harder to detect, increasingly demanding the use of complex
analytical procedures of biotechnology and molecular medicine [13001].
The foundation of amateur athletics is the Olympic triad: (a) creed, (b) oath, and (c) motto.
(a) “The most important thing in the Olympic Games is not to win but to take part, just as the
most important thing in life is not the triumph but the struggle. The essential thing is not to
have conquered but to have fought well.” (b) In the 2000 Olympics, the reference to non-use
of drugs was added to the Olympic oath. “In the name of all competitors, I promise we shall
take part in these Olympic Games, respecting and abiding by the rules which govern them,
committing ourselves to a sport without doping and without drugs, in the spirit of true
sportsmanship, for the glory of sport and the honor of our teams.” (c) The Olympic motto is:
“citius, altius, fortius” or “swifter, higher, stronger” [05003]
The practice of enhancing athletic performance through foreign substances was known from
the earliest Olympic Games. In 1967, the International Olympic Committee (IOC) established
a Medical Commission responsible for developing a list of prohibited substances and
methods. Drug tests were first introduced at the Olympic winter games in Grenoble and at
the summer games in Mexico City in 1968. In February 1999, the IOC convened the World
Conference on Doping in Sport in Lausanne, Switzerland. The Lausanne Declaration on
Doping in Sport recommended creation of an International Anti-Doping Agency. The World
Anti-Doping Agency (WADA) was formed in Lausanne, Switzerland on the basis of equal
representation from the Olympic movement and public authorities. One of the mandates of
WADA was to harmonize the Olympic antidoping code and develop a single code applicable
and acceptable for all stakeholders. The world antidoping code developed by WADA
included creation of several international standards (IS). The purpose of each IS was
harmonization among antidoping organizations. The ISs were developed for laboratories,
testing, the prohibited list, and for therapeutic use exemptions (TUE). The objective of one
manuscript was to present a brief history of doping in sport and describe creation of WADA in
1999. The components of the World Anti-Doping code (in particular, the Therapeutic Use
Exclusion program or TUE) is described. The WADA code defines a TUE as "permission to
use, for therapeutic purposes, a drug or drugs which are otherwise prohibited in sporting
199
competition." Experiences of the Canadian Centre for Ethics in Sport Doping Control Review
Board are presented because this national TUE committee has been operational for over 12
years. The challenge of developing a rigorous global antidoping program requires
acceptance of doping as a problem by sport organizations, athletes, and public authorities.
Individual stakeholders must be prepared to preserve the values of sport, which means free
from doping. This will require vigilance by all interested parties for the benefit of elite athletes
and society overall [04004].
In fact, doping is older than organized sports. Ancient Greek Olympic athletes dating back to
the third century BC used various brandy and wine concoctions and ate hallucinogenic
mushrooms and sesame seeds to enhance performance. Various plants were used to
improve speed and endurance, while others were taken to mask pain, allowing injured
athletes to continue competing. Yet, even in ancient times, doping was considered unethical.
In ancient Greece, for example, identified cheaters were sold into slavery [14612].
The use of drugs to enhance physical performance has occurred since the beginning of
recorded time. Ancient Greeks ate mushrooms and sesame seeds to enhance performance,
and Roman gladiators used stimulants to increase endurance. In modern sports,
documentation of the abuse o fperformance enhancing drugs appeared in the early1900s,
when athlete singested stimulants (cocaine, amphetamines, ephedrine, and strychnine) to
alleviate fatigue and increase focus. Anabolic-androgenic steroids (AAS) are now the most
common illicit drugs used to enhance performance at the modern Olympic Games alongwith
stimulants, primarily by weigh tlifters and athletes in track-and-field. The AASs are a group of
synthetic derivatives of testosterone with bot hskeleta lmuscle building (anabolic) and
masculinizing (androgenic) effects. In 1889,physiologist Charles E.Brown Sequard reported
improvement in avarietyof his body functions (strength, intellect, and force of urine stream)
following the injection of an extract of testicles from the dog and guinea-pig. The primary
natural male hormone, testosterone was first isolated from the testis of bulls in1935 by
Davidetal. Butenandt and Hanisch and Ruzicka et al independently synthesized testosterone
in the sam eyear, and both chemists received the Nobel Prize in 1939 for their work. Most of
the AASs were developed during the1950s when chemists attempted unsuccessfully to
separate the anabolicand androgenic properties of thesetestosterone derivatives.
Nandrolone, the19-noranalog of testosterone was the first anabolic steroid with sufficient
dissociation o fandrogenic and anabolic properties to justify introduction into clinical practice
during the 1950s. Dr. John Ziegler, an American physician-weight lifter, administered AASs
to 3 future American weight lifting champions after learning of th success of AAS-using
Russian weight lifters at the 1954 World Championships. In 1958, the US Food and Drug
Administration (FDA) approved the use of methandrostenolone (Dianabol) for the treatment
of hypogonadism, resulting in the increased availability of this steroid. By the mid-1960s, the
use of AASs to enhance performance in sports spread, particularly among weight lifters and
other strength athletes. An estimate done was that a third of the US track-and-field athletes
in the 1968 pre-Olympic training camp were using AAS. From 1966 until the collapse of the
German Democratic Republic in 1990 ,hundreds of East German physicians ands cientist
sperformed doping research and administered prescription drugs as well as unapproved
experimental drug preparations to adult and adolescent athletes of both sexes. In 1963, the
Council of Europe defined doping in sports as a result of the death of a Danish cyclist at the
1960 Olympics, the death of a UK cyclist at the Tour de France, and th eprevalence of
potentially life-threatening drugs in sports. In 1964, the International Olympic Committee
(IOC) unanimously voted to ban doping in sports. By 1967, the IOC established a Medical
Commission with responsibilities to prohibit doping, to develop the Olympic Movement Anti-
Doping Code, and to formulate a list of prohibited substances. In 1974, the IOC banned the
use of AASs, and testing for AASs byimmunoassay screening and gaschromatography-mass
spectrometry confirmation began in 1976. In 1984, the use of testosterone was also banned.
200
From the 1960s through the 1980s, the German Democratic Republic established a
systematic doping program for thousands of their athletes that included the use of
parenteralpreparations of epitestosteronepropionate to avoide dection of illicit AASs. In 1988,
the IOC stripped Ben Johnson of his Olympic gold medal and world record in the 100-meter
dash for usingan AAS. In the same year, the distribution o rpossession of AAS swith intent to
distribute without a valid prescription became a felony, when US Federal Food, Drug, and
Cosmetic Act (FFDCA) was a mended as part of the Anti-Drug Abuse Act. In 1990, the
Anabolic Steroids Control Act defined an AAS as any drug or hormonal substance chemically
and pharmacologically related to testosterone (other than estrogens, progestins, andcortico-
steroids) that promotes muscle growth. These synthetic compounds became DEA schedule
III drugs as defined by the US Controlled Substances Act. Later, this act was amended b y
the Anabolic Steroid Control Act of 2004; on January 20, 2005, the amended Controlled
Substance Act added both anabolic steroids and prohormones to the list of controlled
substances, making possession of the banneds ubstances afederal crime. In response to
continuing demand for illicit AASs, designer AAS appeared as a means to avoid detection of
these illicit drugs. An example was the synthesis of tetrahydrogestrinone from the palladium-
charcoal catalyzed hydrogenation of gestrinone by the Bay Area Laboratory Cooperative, an
American nutritional supplement company. However, analyses and legal action resulted in
the banning of several athletes as a result of the use of these synthetic steroids.
Subsequently, major league baseball revamped their AAS policy calling for a 50-game ban
for first-time offenders (up from 10days), a 100-game penalty for second-time offenders (up
from 30 days), and a life time ban for a third positive test. Previously, a baseball player could
be suspended for life only after the fifth positive test [13002].
The earliest records of doping in sport come from the Ancient Olympics games when athletes
are reported to have taken figs to improve their performance. With the advent of modern
pharmacology in the 19th century, many athletes began to experiment with cocktails of drugs
to improve strength and overcome fatigue. As this practice was not illegal, there are good
records of the lengths athletes would go to in order to win. Alongside the benefits, came the
dangers and following several fatalities, a code to ban performance enhancing drugs was
gradually developed. Growth hormone was first isolated from the human pituitary gland in the
1950s. Its anabolic effects were soon recognised and athletes had begun to abuse it by the
early 1980s, at least a decade before it was used therapeutically by adult endocrinologists. A
number of high profile athletes have admitted using growth hormone. Detection of its abuse
has been challenging and the lack of an effective test has undoubtedly encouraged its
abuse. Only now are methodologies being developed that should stem this tide [09001].
Doping is as old as sport itself. The word itself is likely derived from the Dutch word, dop, the
name of an alcoholic beverage made from grape skins used by Zulu warriors to enhance
their prowess in battle. In the 20th and 21st centuries, various agents have been used:
alcohol, caffeine, strychnine, amphetamines and then after World War II anabolic/androgenic
steroids and more recently some of the peptide hormones including insulin, erythropoietin,
human GH, and a host of other growth factors. Anabolic/androgenic steroids remain a
mainstay in the performance enhancement drug arena given that they are really the only
major class of steroids that are unequivocally anabolic with salutary effects on athletic
performance. The arms race will continue as long as designer steroids are produced, tested
in vitro, and then, for the more difficult parameter, that they and their metabolites will not lead
to a positive test at the doping control laboratory [11554].
Sporting associations have stated that the fundamental aims of doping controls and anti‐
doping policies are to [06101]:
- uphold and preserve the ethics of sport

201
- safeguard the physical health and mental integrity of the players
- ensure that all competitors have an equal chance.
The word doping is probably derived from the old Dutch word dop, which was the name of an
alcoholic beverage made of grape skins used by Zulu warriors to enhance their prowess in
battle. Ancient Greek athletes are known to have used special diets and stimulating potions
to fortify themselves. Strychnine, caffeine, cocaine, and alcohol were often used by cyclists
and other endurance athletes in the nineteenth century. Thomas Hicks ran to victory in the
Olympic marathon of 1904 in Saint Louis with the help of raw eggs, injections of strychnine,
and doses of brandy administered to him during the race. The term “doping” progressed into
mainstream use in the early twentieth century, originally referring to drugging of racehorses.
The practice of enhancing performance through foreign substances or other artificial means,
however, is as old as competitive sport itself. By the 1920s it had become evident that
restrictions regarding drug use in sports were necessary. In 1928 the International Amateur
Athletic Federation became the first international sport federation to ban the use of doping
(use of stimulating substances). Many other international federations followed suit, but
restrictions remained ineffective as no tests were performed. The death of Danish cyclist
Knud Enemark Jensen during competition at the Olympic Games in Rome 1960 – the
autopsy revealed traces of amfetamine and nicotinyl tartrate – increased the pressure for
sports authorities to introduce drug tests. In 1966 the International Cycling Union and the
Fédération Internationale de Football Association (FIFA) were among the first international
sports federations to introduce doping tests in their respective world championships. In the
following year the International Olympic Committee (IOC) instituted its Medical Commission
and set up its first list of prohibited substances. Drug tests were first introduced at the
Olympic Winter Games in Grenoble and at the Olympic Summer Games in Mexico in 1968
after the urgency of anti‐doping work had been highlighted by another tragic death, that of
cyclist Tom Simpson during the 1967 Tour de France. A reliable test method to detect
anabolic steroids was finally introduced in 1974 and the IOC added anabolic steroids to its
list of prohibited substances in 1976. This resulted in a marked increase in the number of
drug disqualifications in the late 1970s, notably in strength related sports such as throwing
events and weightlifting. Blood boosting or blood doping, which involves removal and
subsequent reinfusion of the athlete's blood in order to increase the level of oxygen‐carrying
haemoglobin, has been practised since the 1970s. The IOC banned blood doping as a
method in 1986. Anti‐doping work was complicated in the 1970s and 1980s by suspicions of
state‐sponsored doping practised in some countries. The most famous doping case of the
1980s concerned Ben Johnson, the 100 metre runner who tested positive for stanozolol
(anabolic steroid) at the 1988 Olympic Games in Seoul. Johnson's case focused the world's
attention to the doping problem to an unprecedented degree. In 1998 a large number of
prohibited medical substances were found by the police in a raid during the Tour de France.
The scandal led to a major reappraisal of the role of public authorities in anti‐doping affairs.
As early as 1963, France had been the first country to enact anti‐doping legislation. Other
countries followed suit, but international cooperation in anti‐doping affairs was long restricted
to the Council of Europe. In the 1980s there was a marked increase in cooperation between
international sports authorities and various governmental agencies. Before 1998 debate was
still taking place in several discrete forums (IOC, sports federations, individual governments),
resulting in differing definitions, policies, and sanctions. One result of this confusion was that
doping sanctions were often disputed and sometimes overruled in civil courts. The Tour de
France scandal highlighted the need for an independent international agency, which would
set unified standards for anti‐doping work and coordinate the efforts of sports organizations
and public authorities. The IOC took the initiative and convened the World Conference on
Doping in Sport in Lausanne in February 1999. Following the proposal of the Conference, the
World Anti‐Doping Agency (WADA) was established on 10 November 1999. On 5 March
2003, at the second World Conference on Doping in Sport, some 1200 delegates
202
representing 80 governments, the IOC, the International Paralympic Committee, all Olympic
sports, national Olympic and Paralympic committees, athletes, national anti‐doping
organisations, and international agencies supported the World Anti‐Doping Code as the
basis for the fight against doping in sport. The Code entered into force on 1 January 2004.
On 19 October 2005, the World Anti‐Doping Code was adopted at the 1st International
Convention against Doping in Sport by the General Conference of UNESCO at its plenary
session. Some 184 countries have signed the Copenhagen Declaration on Anti‐Doping in
Sport, the political document through which governments show their intention to implement
the World Anti‐Doping Code by the ratification of the UNESCO Convention [06002].
Drug use by athletes to improve performance is not a new practice. As early as BC 776, the
Greek Olympians were reported to use substances such as dried figs, mushrooms, and
strychnine to perform better [06003].
Anti-doping efforts started in earnest after the 1960 Olympic Games in Rome. During a team
time trial, 23-year-old Danish cyclist Knud Enemark Jensen collapsed, fractured his skull and
died. An autopsy reportedly found traces of amphetamine and a blood-vessel dilator in his
system. Although the drugs might not have caused his death, the episode forced cycling
officials to take a closer look at doping. The UCI banned some performance enhancers, and
in 1967 the International Olympic Committee established a commission to ferret out doping in
sport. The task is thankless: anti-doping agencies thwart one cheating strategy, only for
another to emerge. The 1972 Olympic Games in Munich, Germany, ushered in testing for
stimulants, but athletes had started to take anabolic steroids. A test for steroids arrived at the
next summer Olympics, in Montreal, Canada. But four years later, at the Moscow Olympiad,
athletes had moved on to undetectable, naturally occurring hormones, such as testosterone.
Anti-doping authorities now measure the ratio of testosterone in the blood to a related
molecule called epitestosterone. In response, some athletes have reportedly found ways of
regulating epitestosterone to keep the ratio in check. For cycling and other endurance
sports, human recombinant EPO fuelled a doping revolution. EPO is a natural hormone that
promotes production of oxygen-carrying red blood cells. The first synthetic, or recombinant,
version was developed by the biotechnology company Amgen in Thousand Oaks, California,
and in 1989 it was approved by the US Food and Drug Administration to treat anaemia. It
also offered cyclists an easy endurance boost that helped them to excel in gruelling stage
races. The drug is nearly identical to the hormone naturally churned out by the kidneys, so
was impossible to detect. It is also easier to administer than blood transfusions, which had
been used to the same effect. Typically, red blood cells account for 40-45 percent of the
blood, but in the heyday of EPO doping, some riders were showing up at starting lines with
haematocrits of more than 60 percent. The UCI instituted a “no-start” rule, disqualifying riders
if their haematocrits on the morning of a race were above 50 percent for men and 47 percent
for women. So cyclists began diluting their EPO-boosted blood with saline solution to keep
their haematocrits below the threshold. The drug companies that produce EPO have helped
anti-doping laboratories to develop direct tests based on subtle biochemical differences
between the recombinant molecules and the natural form. The first of these was approved for
use in 2000. But athletes increasingly obtain knock-off forms produced in China and India,
and researchers have struggled to keep up [11415].
In 1960, during the Rome Olympics, for the first time, the International Olympic Committee
Medical Commission instituted drug testing for athletes. However, the IOC began doping
enforcement during the1968 Winter Olympic Games in Grenoble, France, but the limitations
were immense due to the poor methods of analysis, so only a few stimulants and narcotics
were detected. It was only in 1968 that the IOC officially took control of testing for the use of
certain doping methods and substances [14425].
203
The use of doping agents, once restricted to professional athletes, has nowadays become a
problem of public health, since it also concerns young people and non-competing amateurs
in different sports. The use is also diffused in social life for improving physical appearance
and enhancing performance and even dietary supplements assumed to improve
performance often contain anabolic steroids. While decades ago the so-called "classical
doping agents" (like stimulants and narcotics) were used, to-day anabolic steroids are more
widely diffused. Anabolic steroids are synthetic substances prepared by introducing
modifications in the molecular structure of testosterone, the main natural androgenic anabolic
steroid that forms in testes interstitial cells. The first report concerning the use of anabolic
steroids by an athlete who searched for increased weight and power dates 1954. In 1974 the
misuse of anabolic steroids in sports was banned by the International Olympic Committee
and control tests were implemented in 1976 Montreal Olympic Games through
radioimmunoassay analysis: the technique, however, only allows for unspecific detection of a
limited number of exogenous steroids. Over the years, always new doping substances are
synthesized and, as a consequence, the list of prohibited compounds is continuously
updated and new suitable analytical methods for their detection and determination in
biological matrices are continuously required. In doping control analysis the knowledge of
steroid metabolism pathway in human body is of primary importance and the analytical
methods must permit the simultaneous detection and determination not only of the forbidden
precursor agents but also of their metabolites. In addition, the potential presence and amount
in the biological samples of species that can interfere in the analysis should be evaluated.
Also the several anabolic steroids, specifically designed to circumvent doping control, put on
the market have been incorporated in the list of the prohibited substances of the World Anti-
Doping Agency (WADA). In WADA list steroids figure in three main classes, namely anabolic
steroids, corticosteroids and substances with anti-estrogenic properties. It must be strongly
reminded that assumption of doping agents not only leads to athletes the possible failing of
doping tests but causes important health risk and WADA prohibited list establishes criteria to
highlight the alteration of the natural steroid profile caused by exogenous administration
[12001].
With doping control, comes the introduction of drugs to “cheat” the system. The reported
instance of evasive measures became prevalent after the 1980’s when the first assay to
detect testosterone in urine was developed. Despite these control measures and awareness
of the risks of sport related drug use, doping in sport remains endemic; transgressing all
levels of activity. At the elite level, infamous systematic doping scenarios and individual
cases are cited throughout sporting history. The German Democratic Republic (GDR)
government administered doping program of its athletes, particularly its female athletes,
contributed to their domination of track & field and swimming events for the two decades
spanning the 1970s and 1980s. There are also numerous highly publicised individual cases
including those of Ben Johnson (Canada) who was stripped of the Gold Medal for the 100m
at the 1988 Seoul Olympics when it was reported that he tested positive for stanozolol.
Furthermore, it is interesting to recall that of the eight runners in this infamous race, five of
the other finalists either gave positive samples or were involved in some way in doping
scandals at some stage in their careers. Other more recent examples include: Marion Jones
who won five medals at the 2000 Sydney Olympics but was stripped of all these medals,
when having been implicated by the highly publicised Bay Area Laboratory Cooperative
(BALCO) investigation, she admitted in 2007 to using performance-enhancing drugs; and
Lance Armstrong, who won the Tour de France cycling event on seven consecutive
occasions (1999-2005) but was stripped of these titles in 2013 having been investigated by
USADA and admitting to doping during a television interview by “Oprah” [150003]:
Some specified dates in doping and anti-doping history

204
The first recorded drug-related fatality occurred in 1886 when Andrew Linton died on the
Bordeaux-Paris cycle race, allegedly from an overdose of strychnine, heroin, and a
compound known as “trimethyl”. In 1967, the International Olympic Committee established a
Medical Commission and formulated an official list of prohibited substances. The first
systematic testing began at the 1972 Olympic Games in Munich with the analysis of more
than 2000 urine samples by gas chromatography (GC) with nitrogen-selective detection for
stimulants. Systematic urinary screening was introduced in 1983 at the Pan American
Games, and blood testing was used for the first time in 1994 in the Lillehammer XVII Olympic
Winter Games in an attempt to detect blood doping [05001].
Some important dates in the history of anti-doping [11416, 14017, 150003]:
Second half of 19th Century

1865 Early report of drug use in sport by canal swimmers reported in Amsterdam
1800's (last third) Cyclists of the day are reported to have used coffee “spiked” with caffeine
at the start of races and then add increasing doses of cocaine and strychnine to
the mixture during the races. Boxers reported to take strychnine tablets with a
mixture of cocaine and brandy
1879 Six day bicycle race - an 144 hour continuous event. Various doping
agentsused: caffeine based mixtures (preferred by French racers); sugar cubes
in ether (preferred by Belgian cyclists); alcohol-containing cordials; and nitro-
glycerine (used specifically by sprinters).
1886 First fatality attributed to doping reported (unconfirmed). English cyclist, Arthur
Linton, was reported to have overdosed on “tri-methyl” (presumably containing
caffeine or ether) during a 600 km cycling event held in France (between
Bordeaux and Paris). Note: Evidence to this report is however conflicting with
others reporting he died 10 years later.
1887 Amphetamines first synthesised
1800's (last third) The use of stimulants among athletes is common-place and not
concealed unless the drug combination was unique and thought to provide a
competitive advantage that the athlete did not want to share.
First half of 20th Century

1904 Thomas Hicks, the winner of the marathon in the 1904 St. Louis Olympic
Games, takes strychnine and brandy several times during the race.
1928 Doctor Wilhelm Knoll - a Swiss physician, administers a stimulant (Coramin) to
skiers at the St. Moritz Olympic Games
1928 The IAAF becomes the first federation to ban doping
1932 In the Los Angeles Olympic Games, the victories of Japanese swimmers were
rumoured to be the result of their being “pumped full of oxygen”
1933 The word doping is now part of the English language
1935 The first steroid (testosterone) isolated
1936 Rumours of testosterone injection at the Berlin Olympics among German
athletes mid 1930's
1937 Amphetamines identified as a central nervous system stimulant and became
available via prescription
1939 Ruzicka and Butenandt
1940s Widespread use of Testosterone and other AAS to treat “male climacteric” and
other medical conditions
late 1940's Amphetamines are used for the first time in professional sport
1945 First evidence of formal discussions about the viability of doping in sport
through the use of stimulants in Soviet Union
205
1948 Soviet sport established their goal of meeting or exceeding all world records
Second half of 20th Century

1950 Nandrolone (19-nortestosterone) first synthesized by AJ Birch 1950's-1960's
First reports of female anabolic steroid use relates to Soviet female track and
field athletes
1954 Soviets employ systematic use of testosterone with their weightlifters
1954 Russians use AAS for weightlifting at championship in Vienna
1956 Growth hormone (hGH) first isolated from the human pituitary gland by Li and
Papkoff
1958 Ciba Pharmaceutical Company released Dianabol (methandrostenolone) and
US sports doctor doctor John Ziegler begins experimenting with testosterone
for the US weight lifting team. Efficacy of these drugs apparently spread by
word of mouth during the early 1960s to other strength-intensive sports, from
field events to football
1959 High school sport related drug use rumours to have commenced – a physician
in Texas allegedly administered Dianabol to the high school football team
1960 In Rome Olympic Games, Knud Jensen, a 23-year-old Danish cyclist, collapsed
during competition and dies. Autopsy results revealed the presence of
amphetamines. This is the second death in an Olympic competition and the first
doping related death. Note: The first Olympic death occurred in 1912 at the
Stockholm Olympics when a marathon runner died of heat exhaustion during
the race.
1960's Anabolic steroid use became widespread.
1961 IOC form a medical committee – in response to the death at the Rome
Olympics in 1960.
1962 Mr Olympia body building contest premieres
1964 Urine samples taken from cyclists after the races during the Tokyo Games
“were actually blue in colour due to the use of various drugs.”
1965 Tests conducted on Belgian cyclists in 1965 showed that 37 percent of
professionals and 23 pe cent of amateurs were using amfetamines, while
reports from Italy showed that 46 percent of professional cyclists tested positive
for doping.
1966 First clearly documented National doping program commences – GDR doping
program documented by the STASI.
1966 The IAAF, the Union Cycliste Internationale (UCI), and the Fédération
Internationale de Football Association (FIFA) introduce urine drug tests in their
respective championships
1967 The International Olympic Committee (IOC) institutes its Medical Commission
and sets up the first list of prohibited substances
1967 First doping death televised. 29 year old English cyclist (Tom Simpson)
collapses during the 13th stage of the Tour De France. The autopsy revealed
high levels of methamphetamine in his system. Simpson was carrying a vial of
methamphetamine on him at the time of his death.
1967 The masculine appearances of a number of female track and field athletes from
the Eastern bloc countries in the mid-1960s led to speculation that they were
either hermaphrodites or men disguised as women. In response, a
chromosome test was initiated in 1967 at the European Cup
1967-1968 Doping controls first introduced – at the end of 1967 the International Olympic
Committee (IOC) votes to adopt a drug-testing policy banning the use of
specific drugs – not including anabolic steroids. This policy is released in 1968.
1968 IOC first performs drug testing at Montreal games
1968 Soccer player Jean-Louis Quadri collapses during a game in France. He is
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pronounced dead on arrival to hospital. His autopsy reveals amfetamines are in
his system.
1968 A cyclist, Yves Mottin, died from “excessive amphetamine use” two days after
winning a race
1969 First application of RIA for the measurement of steroids in biological fluids
published
late 1960's Blood doping by the reinfusion of an athlete's own concentrated oxygen
carrying red blood cells or those of a typed-matched donor, shortly before
competition is thought to have begun.
1970s Widespread use of AAS throughout elite sports. Marked increase in the number
of doping-related disqualifications after the introduction by the IOC of anabolic
steroids to its list of prohibited substances
1972 Unofficial poll taken by a 1972 Olympic Track and Field team member, Jay
Sylvester, find that 68 percent of male track and field contestants have used
anabolic steroids during their training.
1972 Ric Demont, an asthmatic, finished first in the 400 metres swimming event, but
was disqualified for using a proprietary medicine containing ephedrine
Antoinette Bevilacqua, 4th in the high jump in Atlanta, after testing positive for ephedrine,
present in ginseng, in an out-of-competition test, had her placing annulled
1973 Two tests developed by British scientists to detect for anabolic steroids. One
test is by radioimmunoassay and the other is by gas chromatography couples
with mass spectrometry. The IOC decides to adopt both tests in tandem to
ensure accuracy. These assays do not detect the use of testosterone.
1974 AAS introduced as a banned class of compounds by the IOC following the
positive screening results of the 1974 Commonwealth Games
1976 The first female athlete tests positive for anabolic steroids at the Olympics
Games and East German women emerge as a dominant force internationally.
1977 IOC meeting in Prague discusses placing "approved" drug testing labs all
around the world.
1977 American College of Sports Medicine publishes position paper stating that AAS
are ineffective for muscle gains
1978 In the World Cup Willie Johnstone of Scotland was positive for an ephedrine
compound, which he said he took to help him sleep!
1980 First assay to detect testosterone in urine to retrospectively detect doping in
sport developed by doctor Manfred Donike. Tenty percent of all athletes tested
positive, including 16 gold medallists in 1980 Olympics
1980's Significant improvement in mass spectrometry particularly GC-MS
1980s Introduction of out-of-competition testing
1981 First edition published of “Underground Steroid Handbook”
1981 First recombinant form of human hGH (rhGH) produced
1982 Caffeine and exogenous testosterone are added to the IOC doping list of
prohibited substances
1982 “Conan the Barbarian” and “Rambo” released by Hollywood
1983 Second edition of extended “Underground Steroid Handbook” appears
1983 First use of the testosterone/epi-testosterone ratio. At the Pan American games
held in Venezuela 15 athletes (including 11 weightlifters) tested positive. In
addition after these results were announced, 12 American track and field
athletes withdraw and returned home before competing.
1983 Recombinant erythropoietin produced - Patent number US 5441868 A
1982-1984 Human growth hormone (hGH) recognised to be part of doping regime for body
builders. hGH described as the “fad anabolic drug” of the Los Angeles Olympic
Games.
1984 Beta blockers used by most pentathlon competitors to reduce tremors and
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anxiety at Olympic Games. Note: bet-blockers were not banned at this time.
Post the Olympic Games, 24 members of the US men's cycling team admitted
to blood doping prior to competition.
1984 Media reports related to the 1984 Olympic Games suggest that some athletes
were given instructions on how to evade drug tests for anabolic steroids
1985 Beta blockers are added to the banned substance list and blood doping is
prohibited. There is no test at this time however to detect blood doping
1985 Bio-synthetic human growth hormone manufacturing commences at Genetech
(FDA approved).
1986 Blood transfusion banned by IOC
1986 Diuretics are added to the IOC banned substance list
1986 Statistical analysis of IOC approved lab positive results shows that anabolic
steroid comprise two -thirds of drugs detected in this year and of these two-
thirds of these positives were for nandrolone.
1987 West German Heptathlete, Birgit Dressel dies at the age of 26 years of anabolic
steroid related complications
1987 In revised position stand of American Colleges of Sports Medicine concedes
that AAS is effective for muscle gains
1988 Federal Anti-Drug Abuse Act of 1988 changes AAS distribution from a
misdemeanor to a felony
1988 Buckley and colleges report that 6.6 percent of 12th grade boys report use of
AAS
1988 Seoul Games, two gold medallist weightlifters tested positive for diuretics.
1988 Ben Johnson, winner of the 100-metre dash, tested positive for an anabolic
steroid. The follow-up investigation identified at least half of the athletes testing
positive for anabolic steroids.
1988 Linford Christie in the sprint events in Seoul had problems with
pseudoephedrine that was present in ginseng. His urinary levels of
pseudoephedrine were not above the cut-off level and he was allowed to keep
his silver medal
1980's Speculation that >12 deaths were the result of EPO during this decade.
1988 Peptide hormones are added to the IOC banned substance list
1989 Monitoring of Future Study adds AAS to its annual high school questionnaire
1989-1990 Fall of communist Europe – Berlin Wall fell in late 1989 followed by the collapse
of the GDR in 1990 and therefore the end of the GDR National doping program.
1990's Some GDR coaches found employment with other teams including within
China's sports programs. A number of Chinese athletes test positive during
1990's, including 29 track and field athletes and 19 swimmers
Early 1990s The East German state-sponsored doping program revealed
1990s DEA enforcement largely eliminates domestic illict AAS production, but has
little effect on oversees import
1990s rEPO included in the IOC’s list of prohibited substances
1990s Introduction of blood tests
1991 Anabolic Steroid Control Act of 1990 in the US becomes law, reclassifying AAS
as Schedule III controlled substances
1991 Argentinian footballer, Diego Maradona, banned for 15 months in 1991 for
testing positive for cocaine
1994 In the 1994 World Cup, Diego Maradonna tested positive, not for cocaine this
time, but for ephedrine, pseudoephedrine, norpseudoephedrine,
methylephedrine and the related propanolamine and was
1994 Doping using DHT detected in 11 Chinese athletes participating in the Asian
Games by the IOC accredited laboratory in Tokyo.
1995 World's youngest athlete tests positive for anabolic steroid use a 14-year old
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female long jumper and sprinter from South Africa
1996 GC-HRMS testing employed for the first time at the Atlanta Olympic Games.
1996 “GI Joe Extreme” action toy, with the equivalent of a 26 inch bicap and a 55
inch chest, released
1998 Baseball player, Mark McGwire, acknowledges he uses androstenedione.
Anabolic steroids were banned by the IOC and NCAA but not in professional
baseball at the time of the report.
1998 Cyclist Willy Voet of the Festina team, arrested by French customs police for
transporting performance-enhancing drugs. This arrest resulted in anextensive
investigation which exposed the extent and 30+ year history of doping use in
this sport.
1999 WADA is established
21st Century
2000 Australian Government provided a special research fund in the lead up to the
Olympics to ensure that state of the art testing available for Sydney Olympics.
Sydney Olympics – first to use the EPO testing by IEF. National Measurement
Institute of Australia’s WADA testing facility develops bases of what is now
used for the haematology module of the ABP. Population studies for IRMS
which allowed the first positive finding by IRMS during the Paralympics for
testosterone
2000 National Institute on Drug Abuse (NDA) announces national multimedia public
education program on AAS
2000 to present Increasingy frequent cases of elite athletes exposed for using performance-
enhancing drugs
2001 ASDA (now ASADA) became the first National Anti-Doping Organisation to
establish a domestic blood-testing program
1999-2005 Lance Armstrong wins the Tour de France on seven consecutive occasions
2000 Three cyclists fail the mandatory health test just prior to commencement of the
2000 Tour de France – they were not permitted to start because they had a
haematocrit >50 percent
2003 First World Anti-Doping Code first adopted
2003 Gene doping was added to the list of Prohibited Methods from 1 January
2004 Caffeine, probably the most popular drug in the world, is removed from the IOC
banned substance list, notably; research indicates that the ergogenic benefits
from caffeine ingestion may be gained from relatively low doses, including
those attained from drinking strong coffee
2004 Athens isoform assay for hGH
2004 Anabolic Steroid Control Act of 2004signed into law, expands list of prohibited
AAS and urges increased penalties
2004 The World Anti-Doping Code is adopted worldwide
2005 United Nations Educational, Scientific and Cultural Organization (UNESCO)
adopts the International Convention against Doping in Sport 2005 In letter to
House Committee of Government Reform, GAD, reports of hundereds of web-
sites selling AAS
2005 Congressional hearings on the use of AAS in baseball and other aspects of
AAS abuse
2005 Operation Gear Grinder: DEA targets eight Mexican manufacturers estimated to
sell US 5.000,000 of AAS annually in the United States
2006 Floyd Landis was stripped of his title after testing positive for synthetic
testosterone
2007 Mitchell report on AAS use in Major League Baseball generates widespread
publicity
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2007 Operation Raw Deal: DEA seizes 11.4 million dosage units of AAS in largest
seizure ever
2007 BALCO investigation – Marion Jones stripped of the five medals she won at the
Sydney Olympic games in 2000
2009 Athlete Passport Haematological variables approved
2008 The UCI is the first federation to introduce the Athlete Biological Passport
2008 to present News stories regarding use of AAS by military and by private security
contractors in Iraq and Afganistan
2008 to present News stories regarding use of AAS by law enforcement officers in many US
cities
2009 WADA Code amended
2011 Norwegian terrorist Anders Behring Breivik describes use of steroids in
preparation and execution of mass murder of 77 people
2012 Lance Armstrong retroactively stripped of his titles
2012 London Olympics biomarker assay for hGH introduced
2013 ABP Steroidal Module approved – monitors selected urinary steroid
concentrations over time in order to detect steroid doping
2013 Lance Armstrong admits to doping during his television interview with "Oprah"
and was subsequently stripped of his seven Tour de France medals
2014-2016 The Russian Doping scandal
December 3, 2014: The German TV channel ARD accuses Russian athletes for
systematic doping led by among others the chairman Valentine Balachnitjev
November 4, 2015: the former chairman of IAAF, Lamina Diack, is taken to
custody of French police for being bribed for not forward information of Russian
doping
November 9, 2015: WADA presents an investigation of systematic and state-
supported doping
November 13, 2015: IAAF suspend all Russian athletes from internation
competing
March 7, 2016: The Russian tennis player Maria Sjaparova tells the press in
Los Angeles that she has been caught for using meldonium
March 8, 2016: Gold medalist Semjon Elistratov, short track, and skating star
Pavel Kulizjnikov are tested positive for meldonium
March 17, 2016: Swimmer Julia Jefimov is caught formeldonium
March 21, 2016: Four track-and-field athletes cought for using meldonium
March 22, 2016: At least 10 Russian wrestler cought for meldonium
March 23, 2016: The Times acuses Russian swimming for systematic doping by
the doctor Sergej Portugalov
The drugs-in-sport problem first came to prominence in the 1960s with the use of
amphetamines among professional European cyclists. At the same time, steroids were
becoming widespread in the United States and Eastern Europe. As money flowed in
commensurate with an unprecedented media interest, sport began to globalise in the 1980s,
and its commercial value increased exponentially. A number of high-profile drugs scandals
occurred in the 1980s, culminating in the Ben Johnson affair in 1988. The consequent media
feeding-frenzies encouraged a number of sporting bodies to introduce anti-doping
regulations. Plagued by constant allegations of drug use in international sport, along with the
Tour de France drug crisis of 1998, the IOC led the push for the establishment of an agency
with the responsibility for managing and enforcing global anti-doping policy. WADA was born
in 1999 and has become a global force in the war on drugs-in-sport. WADA's success in
establishing an international drug code has been underpinned by three developments. First,
WADA is funded jointly by the IOC and a group of national governments. This has provided
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the agency with both capital and influence. Secondly, WADA has secured a series of
international declarations that have commended and ratified the policy code it has
developed. Thirdly, WADA policy has recently been approved by the United Nations
Educational, Scientific and Cultural Organisation (UNESCO) as an international convention.
These achievements have consolidated WADA's position as the central international agency
for regulating drug use in sport [10312].
Despite the fact that doping is not a new phenomenon in sport, enhancing performance
through artificial means has only been banned since the 1960s. Doping as a potential danger
to the modern Olympic movement was recognized in the '50s and officially acknowledged ten
years later by the creation of a list of banned substances. After an agonizing period over
athletes' amateur status, performance enhancing drugs have taken over as the major basis
for tension and concern within the Olympic movement since 1972. Researchers seem to
agree that doping is unwelcome in sport. However, opinions are divided between doping
being a serious deviance one must fight against and doping as undesirable but unavoidable
consequence of the institutionalized sport. Notably, the reason behind banning doping
initially was the growing concern about athletes' health. Doping only became established as
unethical after that point [07004].
The seriousness of the doping problem is reflected by the recent increase in organised effort
to combat doping in sport. The first step toward a globalised effort was the creation of the
Anti-Doping Code of the World Anti-Doping Agency (WADA) in 1999 as an organisational
level response to the Festina Scandal at the Tour de France, parallel to the European
Union's (EU) pledged support in the fight against doping. The first report (known as the
HARDOP report) was commissioned in 1998 and published in 1999, followed by targeted
research projects under the EU's Competitive and Sustainable Growth run under 5th
Framework Programme. The globalised effort was recently manifested in the creation of the
International Convention Against Doping in Sport by the United Nations Educational,
Scientific and Cultural Organization (UNESCO). The UNESCO convention is the first legally
binding international framework setting out the responsibilities of national governments and is
currently signed either as ratification, acceptance, approval or accession by 65 countries
[07004].
Tom Simpson 1967
July 13, 1967, on Mont Ventoux in southern France, 29 year-old British rider Tom Simpson
was lying seventh overall in Tour de France when the 13th stage of the race set off from
Marseilles. The temperature was well over 40oC (105oF) but Simpson was an experienced
competitor, having turned professional 10 years before, and would presumably pace himself.
Unexpectedly, he slowed almost to a halt, wobbled and veered to his right. Helpers, sensing
his distress, rushed to help as Simpson fell from his cycle. Simpson appeared to lose
consciousness as he fell; he never recovered and died. Three tubes were found in Simpson’s
pocket, one full of amphetamines, and two empties. The British team’s luggage was
searched and more supplies of the pills were found. At the time, the drugs element did not
cause the sensation that might be expected today: the death itself was of most concern. In
continental Europe, there was substantial and open advocacy of the use of stimulants to
alleviate the strain of long-distance cycling. There is little doubt that many of the leading
contenders in the 1967 and other tours were taking amphetamines. Seven years before, in a
less publicized tragedy, another cyclist, Knut Jensen collapsed during an Olympic race and
later died in hospital where amphetamine was found in his system (his was the second
Olympic death after Portuguese marathon runner Francisco Lazaro died from heatstroke in
1912.) Simpson’s death occasioned soul-searching among Tour organizers. It was not the
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first time they had considered the use of stimulants. A tentative attempt in the previous year
to introduce drug testing was opposed by leading cyclists, including the five-times Tour
winner Jacques Anquetil, who told the publication France-Dimanche: “Yes, I dope myself.
You would be a fool to imagine that a professional cyclist who rides 235 days a year in all
temperatures and conditions can hold up without a stimulant.” Interestingly, Simpson was not
denounced as a cheat at the time; his death opened up a rather different discourse about the
perils of drug taking rather than the morality of it [14428].
Ancient history of doping and anti-doping
The first marathon
In 490 BC, the Persian Army landed on the plain of Marathon, 25 miles from Athens. The
Athenians sent a messenger named Feidipides to Sparta to ask for help. He ran the 150
miles in two days. The Spartans were late. The Athenians attacked and, although
outnumbered five to one, were victorious. Feidipides was sent to run back to Athens to report
victory. On arrival, he screamed ‘‘We won’’ and dropped dead from exhaustion. The
marathon was run in the first modern Olympics in 1896, and in many ways the athletic ideal
of modern athletes is inspired by the myth of the marathon. Their ideal is superhuman
performance, at any cost [04005].
History of sports medicine in ancient Greece
Cheating in sport is nothing new – only its form has changed. In ancient Greece, athletes
attempted to lay their rivals low with curses, or simply turned to bribery. Fines levied on those
caught were used to build bronze statues of Zeus, which lined the entrance of the Olympic
stadium, reminding competitors of the perils of cheating [00001].
The ancient Greeks supported the humanistic ideals that people were born into aristocracy
and that one’s position in society was established through blood lines. Participants in the
Olympic Games were eligible because of their place in society. That did not stop early
reports of drug misuse. Galen, in the third century BC, reported that Greek athletes used
stimulants to enhance their physical performance. At the ancient Olympic Games, athletes
had special diets and were reported to have taken various substances to improve their
physical capabilities. The winner of the 200 m sprint at the Olympic Games of 668 BC was
said to have used a special diet of dried figs. This scenario is not so far removed from the
supplements that feature in today’s sports nutrition support programmes for athletes. Women
were excluded from these Games, although there is a suggestion that one unofficial female
participant – a Greek woman called Melpomene – “crashed” the Marathon in protest [01002].
“The story of organized athletics in the ancient world,” writes the historian of sport William
Baker, “is primarily the story of Greece”. James Longrigg opens his account of the history of
medicine in the classical world with a similar generalization: “One of the most impressive
contributions of the ancient Greeks to Western culture was their invention of rational
medicine.” It is not surprising, then, that the origins of Western sports medicine, and the
ethical problems associated with it, are to be found in ancient Greece, the civilization that
gave birth to both organized athletics and rational medicine. Initially combinations of religious
ceremony and athletic competition, athletic festivals were a significant part of Greek life for
more than a millennium. Hundreds of athletic festivals were held each year throughout
Greece and its colonies. By the fifth century BCE, four major festivals dominated the scene.
The so-called “Circuit Games” included the biennial Isthmian Games at Corinth; the biennial
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Games at Nemea; the quadrennial Pythian Games at Delphi; and, the oldest and most
prestigious of the games, the quadrennial Olympic Games. According to tradition, the first
Olympic contest was held in 776 BCE. Games-playing became a profession not later than
680 BCE, and it remained that until the end of antiquity. As professionals, athletes
specialized in particular sports, and engaged in full-time, supervised training. Municipal pride
led to public funding of athletes' training, and many cities provided athletes with cash awards
for victories and with retirement pensions. The modern distinction between professional and
amateur, which rests upon whether or not one is paid for competing, did not enter the picture.
All Greek athletes expected and accepted material rewards for victory. It was not until about
50 BCE though, that athletes finally organized themselves into formal collegia – social and
religious societies that also served the economic function of advocating on behalf of their
members [04006].
Athletic training became more methodical and systematic with the introduction of the
gymnasium in the early sixth century BCE, and the subsequent appearance of professional
trainers. According to Harris “By the fifth century BC it seems to have become normal for
every athlete of any pretensions to be trained by a professional.” Typically former athletes
themselves, trainers were expected to be experts not only in the techniques of various
sports, but also in massage, diet, physiotherapy, and hygiene. Competitors in the Olympic
Games and their trainers were required to be in Elis one month before the Games began,
where they trained under the strict supervision of the judges. The judges prescribed a
stringent regimen of exercises, and were free to disregard the training habits of individual
athletes. On the opening day of the Olympics, athletes, along with their family members and
trainers, swore an oath to Zeus that they would obey the rules of the Games, and that they
had faithfully trained in a proper manner. According to Philostratus, the judges directed the
athletes, “If you have worked in a manner worthy of coming to Olympia, and have done
nothing in an offhand or base way, proceed with good courage; but as for those who have
not so exercised, go away wherever you like.” The relationship between physicians and
trainers was close, though often bitter. The relationship was strained by professional rivalry
and, ultimately, by the fact that physicians and trainers were committed to very different
fundamental values. In the ancient world, physicians and trainers were fierce rivals in the
world of hygiene and therapy 04[006].
There is a common notion of health underlying Hippocrates and Galen's criticisms. It is the
notion of health pioneered by Alcmaeon of Croton in the second-quarter of the fifth century
BCE, onto which Empedocles grafted his four-element theory a generation later. In this view,
health is considered a balance or equilibrium (isonomia) of the various constituents of the
body, and illness is the result of imbalance. The influence of this view was enormous, as it
was adopted within the Hippocratic Corpus and subsequently endorsed by Galen, and it is at
the core of both the hygienic and therapeutic regimens prescribed by physicians in this
tradition. Though the professional trainers held an unassailable monopoly on the training and
conditioning of athletes, physicians had ample opportunity to observe, record, and analyze
the effects of the various training and dietary practices of athletes. Galen, as it is well known,
began his career as a doctor for gladiators. It was on the basis of their empirical findings that
physicians condemned the athletic lifestyle. A case in point is the analysis of the athletes'
compulsory diets, which were typically heavy in meat consumption. A heavy meat diet was
not normal in ancient Greece. The typical diet consisted of a vegetable stew, fish, and bread;
usually meat was eaten only at religious celebrations. Some athletes, though, were known to
eat up to 10 pounds of lamb per day, and Hippocratic physicians cataloged a variety of
intestinal disorders observed in athletes who were compelled to eat this much meat [04006].
The best-known attempt to reform athletic training in light of the ancient physicians' medico-
ethical critique of athletics came not from a trainer, but from the sophist Philostratus. His
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Gymnastic is the most significant source of our knowledge of Greek athletic training, and
despite the fact that this work is highly critical of contemporary training methods, Finley and
Pleket argue that Philostratus should be seen as “a defender of athletics and of trainers, in
particular against the doctors.” One of the primary targets of criticism is the trainers' rigid
enforcement of the system of the tetrad, which was developed at some point before the first
century CE. The 4-day training cycle began with a day of preparatory exercises, followed by
a day of intensive workout, a day of relaxation, and finally, a day of medium-intensity training.
By alternating periods of maximum effort with periods of relative rest, the system of the tetrad
bears an obvious resemblance to the system of interval training that became popular in the
1950s. Philostratus provides the cautionary tale of his contemporary, the Egyptian wrestler
Gerenus. Having just won at Olympia, Gerenus celebrated with friends for 2 days. When he
reported for training, hung-over and exhausted, on what was the second day of the tetrad,
his furious coach insisted that Gerenus give the all-out effort that the day's training schedule
demanded. In the midst of his workout, Gerenus collapsed and died. The trainer's mindless
adherence to schedule and disregard for the athlete's condition typified the conduct that the
physicians criticized. But again, Philostratus was not offering such tales simply to reinforce
the physicians' critique of athletics; rather, it was his hope that this work would bring about
the significant reform of training methods [04006].
The odes to athletes who died in the pursuit of victory and the epitaphs on their graves bear
witness that the athletes' attitude of “victory or death” was popularly regarded as both
reasonable and praiseworthy. A well-known case in point involves Arrichion, who died in the
final round of the pankration at the Olympic Games of 564 BCE but still won, because his
opponent signaled submission as Arrichion collapsed. Reflecting on a painting of Arrichion,
Philostratus wrote: “Though indeed it is a great thing that he already won twice at Olympia,
what has just now happened is greater: he has won at the cost of his life and goes to the
land of the Blessed with the very dust of the struggle”. The epitaph of a boxer at Olympia
reads: “Agathos Daimon, nicknamed the Camel, from Alexandria, a victor at Nemea. He died
here, boxing in the stadium, having prayed to Zeus for victory or death. Age 35. Farewell”
[04006].
Early (modern) history
The laws of antidoping are new, but the use of doping substances and methods is as old as
the history of sport. In this sense, it was reported that the Greeks ate mushrooms, believing
the mushrooms could improve the performance of athletes in competition and that Roman
gladiators used stimulants to alleviate fatigue. During the end of the 19th Century and the
beginning of the 20th Century, other illicit substances were incorporated into sport. In 1886,
Arthur Linton, a cyclist, died under the influence of stress and speed ball (cocaine + heroin)
during the Tour de France. In 1904, the first case of drug use in the modern Olympic Games
took place when Thomas Hicks, a marathoner, nearly died from a mixture of brandy and
strychnine [14425].
The use of drugs and ergogenic substances to augment athletic performance, commonly
referred to as doping, has evolved along with sporting events. Ancient Olympic athletes
consumed mushrooms, plants, and herbs in an attempt to gain a competitive edge. The
modern Olympic Games made their debut in 1896, and mixtures of cocaine, ephedrine, and
strychnine were used to enhance performance [08007].
In modern times, there is a correlation between the discovery of a drug and its use in sports.
In the 19th century, morphine was widely used in endurance sports – Welsh cyclist, Arthur
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Lindon, who died in 1896, has the dubious distinction of being the first known person to die
from such drug abuse. Strychnine was used by marathon runners in the 1904 London
Olympics; amphetamines came into their own at the Berlin Olympics in 1936; and during the
1960s at least three top athletes died from the misuse of performance-enhancing drugs.
Hormones then came on the scene and although most experts believe that hormone use is
relatively recent, history shows that this is not the case. For example, in 1939 the
Wolverhampton football team in England was trying out testosterone [00002].
The modern era of doping dates to the early 1900s, with the illegal drugging of racehorses.
Its use in the Olympics was first reported in 1904. Up until the 1920s, mixtures of strychnine,
heroin, cocaine, and caffeine were not uncommonly used by higher level athletes. By 1930,
use of perform-enhancing drugs in the Tour de France was an accepted practice, and when
the race changed to national teams that were to be paid by the organizers, the rule book
distributed to riders by the organizer reminded them that drugs were not among items with
which they would be provided [14612].
Alfons Bukowski (1858-1921) is commonly regarded as the pioneer of anti-doping research.

In 1910, he developed a method to detect alkaloids in horse saliva. One hundred years later,
this is a good moment to remember Bukowski, an outstanding Polish pharmacist, often
mistakenly represented in world literature as a Russian chemist. It is also an occasion to
mention that the real driving force in the history of doping were events related to horse rivalry
[10313].
In the 1904 Olympics, marathon runner Thomas Hicks used a mixture of brandy and
strychnine and nearly died. Mixtures of strychnine, heroin, cocaine, and caffeine were used
widely by athletes, and each coach or team developed its own unique secret formulae. This
was common practice until heroin and cocaine became available only by prescription in the
1920s. During the 1930s, it was amphetamines that replaced strychnine as the stimulant of
choice for athletes. In the 1950s, the Soviet Olympic team first used male hormones to
increase strength and power [08006].
Drugs have been in sports for a long time. In the earliest modern Olympic Games, the drugs
of choice included strychnine, heroin, cocaine, and morphine, which were probably more
harmful than helpful. The first “effective” performance-enhancing drugs, the amphetamines,
which were used widely by soldiers in the Second World War, crossed over into sports in the
early 1950s. These drugs – nicknamed la bomba by Italian cyclists and atoom by Dutch
cyclists – minimize the uncomfortable sensations of fatigue during exercise. By setting a safe
upper limit to the body's performance at peak exertion, these unpleasant sensations prevent
bodily harm. The artificial manipulation of this limit by drugs places athletes at risk for
uncontrolled overexertion. The first cases of fatal heatstroke in athletes using atoom were
reported in the 1960s. In the 1967 Tour de France, elite British cyclist Tom Simpson died on
the steep ascent of Mont Ventoux, allegedly because of amphetamine abuse. The precise
extent to which amphetamines enhance athletic performance is unknown, since, as with all
performance-enhancing drugs, there are few modern studies quantifying their effects. The
convenient absence of such information represents further evidence of a hidden problem. A
popular opinion is that la bomba can turn the usual Tour de France domestique, or support
rider, into a stage winner. Since amphetamines must be present in the body to be effective,
the sole method of avoiding the detection of their use during competition is to substitute a
clean urine sample for the doped specimen. A multitude of innovative techniques have been
developed to accomplish this swap. Cortisone, a potent but legal performance-enhancing
drug used to dampen inflammation, also reduces the discomfort of heavy daily training and
competition and lifts the mood. It is also widely abused by professional cyclists [04007].
215
The use of AS began in the 1950s among weightlifters and spread rapidly through a variety
of sports in both professionals and amateurs, men and women. By the time of the 1972
Olympics in Munich, nearly 70 percent of athletes in middle or short distance running and all
the U.S weightlifters admitted to having taken AS. The escalation of drug abuse resulted in
the inclusion of the AS on the list of banned substances for all Olympic Games since 1976
[03002]
Women in sports
The entry of women into the Olympics occurred as a result of the laissez-faire arrangements
between the International Olympic Committee (IOC) and the host cities of Paris in 1900 and
St Louis in 1904, and culminated in more formal arrangements to include women’s events in
the London Olympics in 1908. The IOC’s response was to restrict the inclusion of women to
a few events appropriate to an ideal of feminine activity but to locate them outside the official
programme. Women’s events were made official but were not given equal status with men’s
competitions until 1924. So the early Olympics sidestepped one of the more modern
controversial issues about hormones and sport by allowing only men to participate; there was
no question of gender testing in those days as men competed naked. The very exclusion of
women from the Olympic Games was itself an ethical question but undoubtedly of cultural
origin. One might argue that it was concerns about the hormone profiles of some of the more
masculine women athletes that led to the introduction of gender testing in 1968. Was it to
identify hormone abuse by women or was it to stop men participating as women? [01002].
The first doping in the Olympics
The use of performance enhancing drugs in the modern Olympics is on record as early as
the games of the third Olympiad, when Thomas Hicks won the marathon after receiving an
injection of strychnine in the middle of the race. The first official ban on ‘‘stimulating
substances’’ by a sporting organization was introduced by the International Amateur Athletic
Federation in 1928 [04005].
The Olympics 1976-1992
In 1976, the East German swimming team won 11 out of 13 Olympic events, and later sued
the government for giving them anabolic steroids. In 1992, Vicky Rabinowicz interviewed
small groups of athletes. She found that Olympic athletes, in general, believed that most
successful athletes were using banned substances [04005].
The Olympic Games in Athens
The Olympic Games in Athens were the first to follow the introduction of a global anti-doping
code. From the lead up to the games to the end of competition, 3000 drug tests were carried
out: 2600 urine tests and 400 blood tests for the endurance enhancing drug EPO. From
these, 23 athletes were found to have taken a banned substance – the most ever in an
Olympic games. Ten of the men’s weightlifting competitors were excluded. The goal of
‘‘cleaning’’ up the sport is unattainable. Further down the track the spectre of genetic
enhancement looms dark and large [04005].
Football
In Forward Arsenal, published in 1952, the Arsenal and England player Bernard Joy
described Arsenal’s use of “pep pills” before an FA Cup match against West Ham United in
216
the 1924-25 season. The fact that Joy was perfectly open about Arsenal’s use of stimulants,
and that his matter of fact style of writing is devoid of any suggestion that Arsenal might have
been cheating or doing anything improper, provide an interesting sidelight on attitudes to
performance enhancing drugs in the period before their use in sport was banned [05005].
Drug testing of athletes was first introduced at the FIFA World Cup of 1966 in England and at
the Olympic Games of 1968 in Mexico City, instigated by the deaths of athletes participating
in the Rome Olympic Games of 1960 and the Tour de France in 1967 linked to amphetamine
and nicotinyl tartrate. This early antidoping activity culminated in the creation of the 1966
FIFA antidoping regulations that comprised a list of seven groups of prohibited substances,
including narcotics and stimulants. The fundamental aims of the current antidoping policy
were developed in the late 1960s by the IOC and the IFs. They are to: (1) uphold and
preserve the ethics of sport, (2) safeguard the physical health and mental integrity of players
and (3) ensure that all competitors have an equal chance. Since then, doping controls have
been performed at most of the major sporting competitions such as the Olympic Games,
World Championships in track and field and other sports, the FIFA World Cup and major
cycling competitions. In the process, the IOC in the first instance, and subsequently the
WADA accredited laboratories, gained considerable expertise and experience in detecting
prohibited substances [14429].
Soviet and East Germany
The former Soviet Union began participating in international sport after World War II and
soon achieved a dominant position in the Olympic Games and other competitions. The
success of Soviet athletic programs led to charges of unfair practices but, because of
secrecy surrounding Soviet research in exercise biochemistry, it has been difficult to
substantiate these charges. This article presents previously restricted information regarding
the development and use of creatine supplements and blood doping in the USSR. Early work
by Olexander Palladin established the role of creatine in muscle function. In the 1970s,
Soviet scientists showed that oral creatine supplements improved athletic performance in
short, intense activities such as sprints. Subsequent studies in the West substantiated these
investigations and have led to the widespread acceptance and use of creatine supplements
to enhance muscle function and athletic performance. In addition, however, the Soviet
government supported the development of blood doping, which is banned by the
International Olympic Committee. Blood doping was pervasive in the USSR in the 1970s and
1980s, and was used by many Soviet athletes in the 1976 and 1980 Olympic Games. Open
publication and discussion may help to prevent the abuses that can come from secret
scientific research [03012].
In the 1950s, the Soviet Olympic team began experimenting with testosterone
supplementation to increase strength and power. This was part of a government-sponsored
program of performance enhancement drugs (PEDs) by national team trainers and sports
medicine doctors without knowledge of the short-term or long-term negative consequences.
Additionally, when the Berlin Wall fell, the East German government’s program of giving
PEDs to young elite athletes was made public. Many in the sporting world had long
questioned the remarkable success of the East German athletes, particularly the females,
and their rapid rise to dominance in the Olympics. Young female athletes experienced more
performance enhancement than did male athletes. Unfortunately, they also suffered
significant and delayed side effects, including reports of early death in three athletes [14612].
Physicians dope athletes for a variety of reasons that can range from unethical service to the
state to the gratifying of their own immature emotional needs. The East German doctors who
participated in the doping of thousands of young athletes, including the administration of
217
anabolic steroids to pubescent girls, functioned within a state-sponsored apparatus whose
political mission of sportive nationalism trumped medical ethics. State-sponsored doping in
West Germany expressed similar nationalist ambitions that could not be fully realized in a
democratic society. The gold medals won by East German athletes at the 1976 Montreal
Olympic Games persuaded many West German sports physicians that it was time to adopt
the use of androgenic drugs as a matter of national policy. At the Congress of German
Sports Physicians held in Freiburg in October 1976, the most prominent West German sports
physicians minimized the medical dangers of anabolic steroids and recommended that they
be administered to athletes under medical supervision. Far from being a German specialty,
however, this pro-steroid mindset can be found among sports doctors around the world.
Some physicians have issued therapeutic use exemption (TUE) certificates to athletes that
are unwarranted but allow their use of drugs that are believed to boost athletic performance
[14613].
Athletes from former East Germany who were given performance enhancing drugs for many
years and who consequently experienced longstanding health problems will receive
payments of several thousand euros, the German federal parliament decided on 13 June
2002. A special law has been passed which sets up a compensation fund of about EUR 2m
(GBP 1.3m; USD 1.9m). The fund is meant to be supplemented by the sports industry and by
national sports associations, but neither of these groups has been keen to join the initiative. It
is estimated that between 500 and 1000 men and women will apply for compensation by the
end of the year and will receive about EUR 3000 each. Currently, the association
representing athletes who have had health problems as a result of doping has about 150
members. Soon after the fall of the Berlin wall in 1989, it became apparent that many East
German athletes had had to pay a high price for the overwhelming success of the nation in
many disciplines. Continuous doping from a young age and for a very long time, mainly with
anabolic drugs, ruined their health. Doping was often done without the athlete's consent or
knowledge. East German trainers and doctors merely followed the socialist party's
instructions. The list of health problems is long: acne, hirsutism, deep voice, muscle tension,
gynaecomasty, breast cancer, bone deformation, vascular disease, and teratogenic
malformations. In some cases female athletes changed their sex as a result of the
continuous intake of male hormones. The association representing such athletes, as well as
single athletes, is not satisfied with the new law, which will come into force in 2003 [02004].
Biking
The 2003 year's Tour de France ended with no big surprises: US cyclist Lance Armstrong
won the race for the fifth time, again defeating his greatest competitor, German Jan Ullrich.
And it was a surprisingly “clean” contest, as only one athlete was found to be using the
performance-enhancing drug erythropoietin (EPO). This is clearly an improvement on the
infamous 1998 race when the whole Italian Festina team were disqualified after their coach
was found with more than 400 doping products, including EPO. Indeed, the Tour seems to
have become cleaner since then: in 2001, Spanish cyclist Txema del Olmo was the only
athlete who tested positive for EPO and who was subsequently banned from the tour. But the
Tour de France is not the only international sports contest tainted by doping. US sprinter Ben
Johnson, who rewrote the record books during the 1998 Olympic Games in Seoul, South
Korea, ran to victory with the help of a cocktail of steroids and was later stripped of his
medals. The greatest case of misuse, however, came to light after the reunification of
Germany in 1990, when investigators found that East German athletes had been
systematically doped for several years, which explained their many records and gold medals.
Some critics maintain that, without drugs, today's athletes would not be any better than their
predecessors in the 1960s, before doping became a widespread problem in competitive
218
sports. Indeed, watching the Olympic games or the Tour de France nowadays, the speed
and endurance of the competitors is incredible [03014].
IAAF banned first
Many sports organizations have come to ban the use of PEDs and have very strict rules and
consequences for people who are caught using them. The International Association of
Athletics Federations was the first international governing body of sport to take the situation
seriously. In 1928, they banned participants from doping, but with little in the way of testing
available, they had to rely on the word of athletes that they were not doping. It was not until
1966 that the Federation Internationale de Football Association and Union Cycliste
Internationale joined the International Association of Athletics Federations in the fight against
drugs, closely followed by the International Olympic Committee (IOC) the following year
[14612].
Testing
The first actual drug testing of athletes occurred at the 1966 European Championships, and
2 years later the IOC implemented their first drug tests at both the Summer and Winter
Olympics. Anabolic steroids became even more prevalent during the 1970s, and after a
method of detection was found, they were added to the IOC’s prohibited substances list in
1976. This resulted in a marked increase in the number of doping-related disqualifications in
the late 1970s, notably in strength-related sports, such as throwing events and weightlifting
[14612].
Blood doping
While the fight against stimulants and steroids was producing results, the main front in the
anti-doping war was rapidly shifting to blood doping. This removal and subsequent reinfusion
of an athlete’s blood in order to increase the level of oxygen-carrying hemoglobin has been
practiced since the 1970s. The IOC banned blood doping in 1986. Other ways of increasing
the level of hemoglobin were being tried, however. One of these was erythropoietin.
Erythropoietin was included in the IOC’s list of prohibited substances in 1990, but the fight
against erythropoietin was long hampered by the lack of a reliable testing method. An
erythropoietin detection test was first implemented at the 2000 Olympic Games [14612].
Ben Johnson
The most prominent doping case of the 1980s concerned Ben Johnson, the 100 meter dash
champion who tested positive for the anabolic steroid stanozolol at the 1988 Olympic Games
in Seoul [14612]
History of testosterone and some other anabolic steroids
The idea of designing and developing steroids with anabolic properties arose during the
1930s soon after the identification and isolation of the hormone androsterone by the German
investigator Butenandt, who collected this compound from thousands of liters of pooled
human urine derived from a number of military service volunteers. Most of the AAS used
before the 1990s were pharmacological agents approved for medicinal or veterinary use. By
the 1990s, various androgen precursors became nutritional supplements [150004].
219
The biological effects of the testes and testosterone are known since antiquity. Aristotle knew
the effects of castration and his hypothesis on fertilization is one of the first scientific
encounters in reproductive biology. Over centuries, castration has been performed as
punishment and to produce obedient slaves, but also to preserve the soprano voices of
prepubertal boys. The Chinese imperial (and other oriental) courts employed castrates as
overseers in harems who often obtained high-ranking political positions. The era of testis
transplantation and organotherapy was initiated by John Hunter in London who transplanted
testes into capons in 1786. The intention of his experiments was to prove the “vital principle”
as the basis for modern transplantation medicine, but Hunter did not consider endocrine
aspects. Arnold Adolph Berthold postulated internal secretion from his testicular
transplantation experiments in 1849 in Göttingen and is thus considered the father of
endocrinology. Following his observations, testicular preparations were used for therapy,
popularized by self-experiments by Charles-Edouard Brown-Séquard in Paris (1889), which
can at best have placebo effects. In the 1920s Sergio Voronoff transplanted testes from
animals to men, but their effectiveness was disproved. Today testicular transplantation is
being refined by stem cell research and germ cell transplantation. Modern androgen therapy
started in 1935 when Enrest Lacquer isolated testosterone from bull testes in Amsterdam. In
the same year testosterone was chemically synthesized independently by Adolf Butenandt in
Göttingen and Leopold Ruzicka in Basel. Since testosterone was ineffective orally it was
either compressed into subcutaneous pellets or was used orally as 17alpha-methyl
testosterone, now obsolete because of liver toxicity. The early phases of testosterone
treatment coincide with the first description of the most prominent syndromes of
hypogonadism by Klinefelter, by Kallmann, DelCastillo and Pasqualini. In the 1950s longer-
acting injectable testosterone enanthate became the preferred therapeutic modality. In the
1950s and 1960s, research concentrated on the chemical modification of androgens in order
to emphasize their anabolic effects. Although anabolic steroids have largely disappeared
from clinical medicine, they continue to live an illegal life for doping in athletics. In the 1970s
the orally effective testosterone undecanoate was added to the spectrum of preparations.
Recent transdermal gels and long-acting injectable preparations provide options for
physiological testosterone substitution therapy [14016].
Long before the isolation and synthesis of testosterone in the 1930s, Brown-Sequard and
later Zoth and Pregl recognized that testicular extracts could improve physical and mental
energy, as well as muscle strength. Shortly after the successful synthesis of testosterone,
Boje suggested that sex hormones might enhance physical performance. The Germans
allegedly administered AAS to soldiers going into combat. The Germans also allegedly gave
athletes testosterone in preparation for the 1936 Berlin Olympics. However, the most cited
example of systematic use of AAS in elite sports is that of the Soviet weightlifting team in the
1952 and 1956 Olympics. Dr John Ziegler, a physician associated with the US weightlifting
team, learned about the use of AAS by the Russian team at the weightlifting championships
in Vienna in 1954, and experimented with testosterone on himself and other weightlifters in
the York Barbell Club, New York. AAS use, which had been exclusive to strength-intensive
sports, spread gradually to other sports and to nonathlete weightlifting over the ensuing
decades. In particular, Ben Johnson’s positive test for stanozolol at the Seoul Olympic
Games in 1988 brought widespread public attention to AAS. The most egregious example of
state-sponsored doping was uncovered in the former German Democratic Republic after the
fall of the Communist government in 1990; classified documents revealed a comprehensive
secret state program to improve national athletic performance using PEDs with the complicity
of the state and the sports medicine physicians. Later, the relentless glare of media lime light
surrounding the detection of PEDuse by elite athletes, such as Lyle Alzado, Mark Maguire,
Barry Bonds, Floyd Landis, Marion Jones, and Lance Armstrong, has added to the allure of
PEDs and contributed to the widely-held misperception that PED use is largely limited to elite
athletes and is therefore not a widespread public health problem [14017].
220
Almost half a century before the discovery of androgens, Brown-Sequard (the father of
andrology) had recognized that the contents of testicular extracts could improve libido,
energy, and muscle strength. After synthesis of testosterone, Boje was the first to suggest
that sex hormones may enhance physical performance. Although the most well-known phase
of AAS abuse in Olympic history is that of the Soviet weight-lifting team in the 1952 and 1956
Olympic Games, it is believed that some German athletes were given androgens even during
the 1936 Berlin Olympics. The introduction of AAS among the American athletes is attributed
to Dr. John Ziegler (a physician-member of the US Weight-Lifting Team) who learned about
the use of AAS by the Russian team in 1954 during his trip to weight-lifting championships in
Vienna. Upon his return, Dr. Ziegler experimented with testosterone on weight lifters in the
York Babel Club in Pennsylvania. That is considered to be the beginning of AAS abuse in
sports in the United States, which later spread from high-intensity strength-training games to
sports such as field athletics, baseball, swimming, etc. The two common patterns of AAS
abuse are “stacking” and “cycling.” Stacking involves the use of two or more androgens in
progressively increasing doses over a short period of time. Cycling refers to the intermittent
use of AAS where use of steroids is followed by a drug holiday. The practice of “cycling” is
based on the notion that drug holidays prevent desensitization to large doses of androgen
[13003].
The first rule banning steroids from sport came in 1974 when the IOC added anabolic
steroids to their prohibited substances list. In 1983 the first endogenous steroid, testosterone,
was added. It was a further year before the detection method for testosterone was
introduced. “It’s pretty clear that steroids are worth the price of a metre at the highest levels
of sport” (Charlie Francis, Ben Johnson’s former coach, speaking at the Dubin inquiry in
1990). The early testing programmes were focussed upon competitions and led to a situation
where “only the careless or the ill advised” were to fall foul of the testing regimes of the day,
even if they had been fairly applied. Reports of manipulated sample collections, of results
being destroyed and of complicit activities preventing the revelation of the true extent of drug
misuse make it difficult to assess the actual prevalence of hormone misuse [01002].
The first report concerning the use of anabolic steroids by an athlete who searched for
increased weight and power dates 1954. In 1974 the misuse of anabolic steroids in sports
was banned by the International Olympic Committee and control tests were implemented in
1976 Montreal Olympic Games through radioimmunoassay analysis: the technique, however,
only allows for unspecific detection of a limited number of exogenous steroids [13004].
An aging Brown-Séquard eagerly reported “a decided gain in strength” after injecting himself
with the “orchitic fluid” of laboratory animals. This discovery created enthusiasm and
controversy alike. Synthetic androgens were born in the 1930s when Foss first described the
medical use of orally bioavailable methyltestosterone [14429].
Long before the isolation and synthesis of testosterone in the 1930s, Brown-Séquard and
later Zoth and Pregl recognized that testicular extracts could improve physical and mental
energy, as well as muscle strength. Shortly after the successful synthesis of testosterone,
Boje suggested that sex hormones might enhance physical performance. The Germans
allegedly administered AASs to soldiers going into combat. The Germans also allegedly gave
athletes testosterone in preparation for the 1936 Berlin Olympics. However, the most cited
example of systematic use of AASs in elite sports is that of the Soviet weightlifting team in
the 1952 and 1956 Olympics. Dr John Ziegler, a physician associated with the U.S.
weightlifting team, learned about the use of AASs by the Russian team at the weightlifting
championships in Vienna in 1954 and experimented with testosterone on himself and other
weightlifters in the York Barbell Club in New York. AAS use, which had been exclusive to
221
strength-intensive sports, spread gradually to other sports and to nonathlete weightlifting
over the ensuing decades. In particular, Ben Johnson's positive test for stanozolol at the
Seoul Olympic Games in 1988 brought widespread public attention to AASs. The most
egregious example of state-sponsored doping was uncovered in the former German
Democratic Republic after the fall of the Communist government in 1990; classified
documents revealed a comprehensive secret state program to improve national athletic
performance using PEDs with the complicity of the state and the sports medicine physicians.
Recently, the relentless glare of media limelight surrounding the detection of PED use by
elite athletes such as Lyle Alzado, Mark Maguire, Barry Bonds, Floyd Landis, Marion Jones,
and Lance Armstrong has added to the allure of PEDs and contributed to the widely held
misperception that PED use is largely limited to elite athletes and is therefore not a
widespread public health problem [14426].
Although officials have banned PEDs from Olympic competition since 1967, and the
International Olympic Committee has prohibited AAS use since 1975, it was not until 1991
that the US Congress designated AASs as Schedule III controlled substances. In 2004, the
Anabolic Steroid Control Act amended the Controlled Substances Act and expanded its
definition of anabolic steroids. The new definition, which does not require proof of muscle
growth, identified 59 specific substances (including their salts, esters, and ethers) as
anabolic steroids and listed them as Schedule III controlled substances. Most of the PEDs
that athletes and nonathlete weightlifters used before the 1990s were pharmacologic agents
approved for medicinal or veterinary use. By the 1990s, various androgen precursors
became available over the counter as unregulated nutritional supplements. Androgen
precursors are either inactive or weak androgens that the body converts into potent
androgens. These include naturally occurring precursors to testosterone such as 4-
androstenediol, 5-androstenediol, 4-androstenedione, and dehydroepiandrosterone as well
as precursors to synthetic AASs, including 4-norandrostenedione, 4-norandrostenediol, and
5-norandrostenediol, which the body converts to nandrolone. The widespread, unregulated
sale of dietary supplements on the Internet has greatly increased the number of anabolic
steroids available. Of even greater concern is the introduction of synthetic anabolic steroids
such as 17-desmethylstanozolol, methylclostebol, and methyltrienolone into the market as
dietary supplements. The Steroid Control Act of 2004 banned most of these substances.
However, we are now seeing novel synthetic designer androgens, such as
tetrahydrogestrinone and madol. Because these designer steroids have not undergone
toxicologic or safety testing in humans or animals, they potentially pose an even more
serious health risk than the more traditionally used AASs, which have received some level of
animal or human testing [14426].
The use of testosterone in sport soon appeared, because as early as 1939, some sport
enthusiasts theorized that the use of the male hormone would improve the performance of
athletes. Testosterone was first synthesized in 1935 by two different groups. The first report
of athletes using a synthesized form of this hormone was by Russian athletes in 1954 during
a weightlifting championship in Vienna. The decade of 1960–1970 was marked by many
historical events opposed to doping, such as the creation of the council, composed of 22
nations, proposing a resolution against the use of doping agents in sports. And, in 1963,
France adopted Antidoping Legislation, and two years later (1965) Belgium followed in the
footsteps of France. But the event that marked the decade was the death of cyclist Tommy
Simpson due to the nonmedical use of amphetamines during the Tour de France. This
episode led the IOC to take forceful steps to try to prevent the use and misuse of doping
substances in sports [14425].
During the decade of the 1960s, the German Democratic Republic discovered that sporting
success could both improve the self-esteem of the population and its prestige in the
222
international competitive sports arena. They began to finance studies on the use of anabolic–
androgenic steroids (AAS), invested in the discovery of young talent, and the use of
prohibited drugs. During this period, professional athletes were administered Oral-Turinabol®
pills and injections of testosterone esters and nandrolone – presenting them as being
vitamins and prophylactic measures. In 1968, the East Germans became pioneers in the
administration of androgenic hormones to female athletes. It was reported for the first time (in
1974), the development of the radioimmunoassay technique for detecting the presence of
AAS in biological samples, with the result that they were included in the lists of substances
banned by the IOC. In 1984, testosterone was analyzed during the Olympic Games in Los
Angeles, where art, technology, instrumentation, and skilled personnel were brought together
to combat the use of doping substances [14425].
On November 10, 1999, in Lausanne, Switzerland, the World Anti-Doping Agency (WADA)
was created. “The WADA aims to promote and coordinate the international fight against
doping in sport and to foster a culture of doping-free sport.” Finally, in 2003, representatives
of various governments, including Brazil, gathered in the capital of Denmark, with the
international Olympic movement, aimed at the unification of doping control policies at the
national and international levels, where participants signed the Copenhagen Declaration,
approving the World Anti-Doping Code. Since the signing of the Declaration of Copenhagen,
resulting in the adoption of the World Anti-Doping Code (2004 and later revised in 2009),
Brazil has focused on creating policies of suppression against doping. However, prior to this
event, laws did exist that regulated the use of substances aimed at improving athletic
performance [14425].
Testosterone propionate (Testoviron), the prototype of the anabolic steroids, the second
major group of potent performance-enhancing drugs, was synthesized in 1936 and appeared
in sport sometime after the 1948 Olympic Games. The subsequent synthesis of
methandrostenolone (Dianabol) in the United States in 1958 and oral chlordehydromethyl-
testosterone (Turinabol) in East Germany after 1966 marked the beginning of the “virilization”
of modern sport. The exact magnitude of benefit from the use of combined anabolic agents is
unknown. Previously secret East German records indicate that anabolic steroids alone
reduce 100-m sprinting time by as much as 0.7 second and improve performance in the 400-
m, 800-m, and 1500-m running events by 4 to 5, 5 to 10, and 7 to 10 seconds, respectively.
Equivalent benefits have been found among swimmers. Effects in throwing events are also
substantial: a gain of 2.5 to 5 m in the shot put, 6 to 10 m in the hammer throw, 8 to 15 m in
the javelin throw, and 10 to 20 m in the discus throw. Benefits are greatest in women, since
the natural secretion of testosterone in young women is negligible [04007].
During the Moscow Olympic Games of 1980, a high frequency of testosterone (T) abuse was
suspected. By that time, analytical methods to detect the administration of synthetic anabolic
steroids by gas chromatographic-mass spectrometric (GC-MS) screening procedures had
improved. Therefore, athletes switched to endogenous steroids like T. Quantitation of T as a
way to detect T abuse was inadequate because of its high metabolic turnover rate, circadian
rhythm of T excretion, and an interindividual excretion variability. Donike et al introduced the
ratio of urinary testosterone glucuronide (TG) to epitestosterone glucuronide (EG)
concentration, the T/E ratio, as an indicator of T abuse [00003].
Detection of testosterone
The history of AASs is a tale that has its roots in ancient “endocrinology.” More than 6000
years ago, farmers noted an enhanced ability to domesticate animals after castration. Years
later, the medical theories of “humoralism” developed. This doctrine was based on a theory
223
that attempted to explain diseases based on imbalances among the four humors: sanguine,
choleric, melancholic, and phlegmatic. In addition, ancient Egyptians and Romans believed
that testicles and animal penises held special healing powers. Ancient Greek athletes used a
wide variety of alleged performance-enhancing drugs, such as plant extracts and testicular
extracts. These early theories and practices marked the beginning of future discoveries. John
Hunter (1728-1793) was a Scottish surgeon who was later appointed as Surgeon General for
the British army. He made many noteworthy contributions to science, including contributions
to the understanding of digestion, fetal development, venereal diseases, dentistry, and
lymphatics. He conducted the first testicular transplant in 1786 in which he removed a testicle
from a rooster and implanted it into a hen. However, it was not until 1849 that Arnold Adolf
Berthold (1803-1861) found evidence of a “bloodstream substance” from roosters that
affected their appearance and behavior. His theory was correct, but it was not widely
accepted by his contemporaries. Berthold was a professor at the University of Göttingen, and
he performed experiments on roosters while he was a curator at a local zoo. He observed
the impacts of castration and the reimplantation of testicular tissues on roosters. Once
castrated, the roosters' combs decreased in size, they lost interest in the hens, and they lost
their aggressive male behaviors. Those effects were reversed after reimplanting testicular
tissues or extract, despite denervation. Despite these findings, other researchers did not cite
Berthold's work for nearly 50 years [07007].
Perhaps the most well-known researcher of anatomy and physiology was Charles Edouard
Brown-Sequard (1817-1894). Brown-Sequard, a prominent French physiologist and Harvard
professor, was one of the founders of modern endocrinology. He had a strong interest in
endocrinology, and he studied adrenal glands, testes, thyroid, pancreas, liver, spleen, and
kidneys. He is probably most famous for his auto-experimentation with testicular substances
(extracted from guinea pigs and dogs), the results of which were published in 1889. He
reported increased strength, mental abilities, and appetite and even claimed that the process
relieved constipation and increased the arc of his urine stream. Although no one is sure why
he experienced these effects, his experiment caused others to investigate the testicular
substance as a possible cure for various ailments, such as diabetes, tuberculosis, epilepsy,
paralysis, gangrene, anemia, influenza, arteriosclerosis, Addison's disease, hysteria, and
migraine headaches. He encouraged testing of his testosterone products by providing free
samples to physicians. Unfortunately, with such widespread use, shoddy researchers
subjected animals and humans alike to high risks for infection and inflammation. Austrian
physiologist Oskar Zoth was the first person to propose injecting athletes with a hormonal
substance, as published in his 1896 paper describing how the use of an “extract” improved
muscular strength and the “neuromuscular apparatus,” thus potentially improving athletic
performance. He and his physician partner, Fritz Pregl (1869-1930), self-injected
testosterone extracts from bulls and measured the strength of their middle fingers by plotting
them on “fatigue curves”. They won the Nobel Prize in chemistry in 1923 [07007].
Substances referred to as “chemical messengers” were discovered in 1902 by English

physiologists and professors, William Maddock Bayliss (1860-1924) and Ernest Henry
Starling (1866-1927), at University College London. Bayliss' research team was the focus of
an animal rights controversy in 1903 – the Brown Dog Affair – in which Bayliss was alleged
to have performed a live dissection of a brown dog in his laboratory. He, of course, denied
the accusation and won a civil suit, donating the money to the University for further research;
he even wrote articles promoting the humane treatment of animals. Other accomplishments
included contributions on shock, digestive system, and endocrinology; being knighted in
1922; and authoring four editions of Principles of General Physiology. Starling officially
coined the term “hormone” in 1905 when giving a Croonian Lecture (prestigious lectureships)
titled “The Chemical Control of the Functions of the Body” to the Royal College of Physicians.
The term “hormone” means “to urge on” or “impulse or arouse” in the sense of “to set in
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motion” in Greek. Years later, reports suggested that a Cambridge physiologist, William B.
Hardy, actually suggested the term “hormone” to Bayliss and Starling. In 1911, Andre Pezard
first noted a direct relationship between the amount of testicular extract injected into a rooster
and the size of his comb [07007].
An Austrian physician, Eugen Steinach (1861-1944), developed the “Steinach operation,” an

“autoplastic” treatment for the “middle-aged and listless”. The 20-minute operation involved
ligation of the vas deferens, often at the most proximal position to the testicle. This allegedly
increased testosterone production. He believed that the incision produced a “back pressure”
on the testicle, thus increasing testosterone production by the interstitial cells. He also
implanted testicular tissue grafts between the peritoneal muscles. He reported that his
patients were able to regrow hair, had better erections with less premature ejaculation, and
had increased libido. Despite little clinical evidence of his claims on “rejuvenation,” the results
of his operations, at best, likely were due to the power of suggestion; however, he performed
this procedure on some famous patients, including Sigmund Freud and William Butler Yeats.
He also discovered, by transplanting male sex glands into females and vice versa, that
guinea pigs developed sexual behaviors of the opposite sex. Later research proved that sex
hormone injections have no effect on sexual orientation but that high doses of testosterone
may increase sexual desire. In 1913 in Chicago, Victor D Lespinasse (1878-1923), a
urologist, claimed that he cured a patient who had sexual dysfunction by transplanting a
testicle from a donor. He removed the organ, made three transverse slices, and inserted
them into muscle tissue around the patient's scrotum. His most famous patient was Harry F.
McCormick (husband of Edith Rockefeller), whose case was described in The New York
Times. Five years later, the first journal of Endocrinology was published. In the 1920s, Sergio
Voronoff, a Russian-French physician and surgeon, made a fortune from removing testes
from animals (including the controversial monkey and chimpanzee gland transplants by way
of vivisection, sparking campaigns from animal rights groups and satirical cartoons and
books on the subject) and transplanting them into men. The chimpanzee tissue was not
implanted inside the scrotum but instead in the tunica vaginalis. He concluded that his
experiments with testicular transplants helped to relieve pain and provided a sense of well-
being [07007].
It was apparent to researchers that some substance circulating in the blood was responsible
for their findings; however, it was not until 1929, when a German chemist and professor,
Adolf Butenandt (1903-1995), isolated the first sex hormone, that a new path of discovery
was initiated. He isolated estrone from the urine of pregnant women and later isolated 15 mg
of androsterone (“andro” = male, “ster” = sterol, “one” = ketone) from 15,000 L of urine from a
local policemen. Over the next few years, researchers found that the hormones isolated from
the testes were more androgenic than were those isolated from urine. Perhaps the most
famous, and perhaps unethical, research of “organotherapy” occurred in the 1920s and
1930s at San Quentin prison in California where Leo Stanley transplanted the testicles from
executed prisoners into impotent prisoners. He had a limited supply, so he turned to
substituting a variety of animal gonads (from ram, sheep, goat, deer, and boar) to treat men
who suffered from senility, epilepsy, and paranoia. Over the years he performed hundreds of
operations. During the 1930s, three pharmaceutical companies each hired research teams to
isolate the testicular hormone. The term testosterone (“testo” = testes, “ster” = sterol, “one” =
ketone) was coined in 1935 by Karoly David and his research team. Ernst Laqueur isolated
testosterone from bull testes. The research team was funded by the pharmaceutical
company Organon in Oss, The Netherlands. Later that same year (on a team funded by
Schering Corporation in Berlin, Germany), Butendant and Gunicr Hanisch published “A
method for preparing testosterone from cholesterol” in a German journal. Only a week later,
Leopold Ruzicka (who synthesized androsterone in 1934) and A Wettstein published “On the
artificial preparation of the testicular hormone testosterone (andro-sten-3-one-17-ol)” in
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Helvetica Chimica Acta and applied for a patent. Butenandt and Ruzicka won the Nobel Prize
for chemistry in 1939. Butenandt spent a large part of his career studying the sex hormones
and their relationship with one another. His work laid the foundation for the production of
cortisone [07007].
In the late 1930s, experimentation using humans involved testosterone propionate (slow-
release derivative) and methyl testosterone (oral form that was slower to metabolize). Most
of the research at that time was focused on treating hypogonadism in men (inducing and
maintaining secondary sexual characteristics and treating impotence). Charles D Kochakian
discovered an increase in protein anabolic processes, thus opening the door for the
treatment of a variety of disorders by restoring tissue and stimulating growth. In 1939, it was
reported that daily topical application of testosterone by females enlarged the clitoris and
increased sexual desire. The use of synthetic testosterone skyrocketed after publication of
the book “The Male Hormon”e by Paul de Kruif in 1945, which made claims of increasing
libido and boosting athletic performance. Testosterone was a proposed treatment for
menorrhagia, dysmenorrhea, estrogen-derived breast cancers, and other breast conditions. It
was reported to help relieve pain, increase appetite, and promote a “sense of well-being.”
Despite these claims, physicians remained reluctant to begin widespread use among women
because of the virilizing side effects. Most of the profits from sale of this substance were
obtained by way of the black market [07007].
In 1849, Arnold Berthauld, a curator of a zoo in Germany, observed that castrated roosters
ceased to fight, crow and mate, that their combs and wattle regressed, and that these
symptoms were reversed by re-implantation of their testes. In 1889, Charles-Edouard Brown-
Sequard, a French physician and Harvard professor, announced that his vigor and sense of
well-being were transiently but markedly restored after injecting himself with testicular extract
from guinea pigs and dogs. These observations led to trials of animal and human clinical
research. In 1935, testosterone was identified as 17beta-hydroxyandrost-4-en-3-one
(C19H28O2), a solid polycyclic alcohol with a hydroxyl group at the 17th carbon atom
[12008].
A landmark discovery was made in 1889 when Dr Brown-Sequard announced at a scientific

meeting in Paris that he had found a substance that reversed his 72-year-old body's
ailments. He reported having injected himself with the extract of dog and guinea pig testicles
under the assumption that these organs had “internal secretions that acted as physiologic
regulators.” This bold statement was confirmed with the discovery of hormones in 1905 and
the isolation of testosterone in 1935 [06003].
The first characterized androgen was androsterone, which was isolated from urine. Shortly
thereafter, in 1935, the characterisation and synthesis of testosterone was done by
Butenandt and Ruzicka [06004]. The characterization and synthesis of testosterone resulted
in the 1939 Nobel Prize in chemistry for them [12005].
The testes as a medicinal product: organotherapy
As it was known that removal of the testes caused the clinically evidenced symptoms of
hypogonadism including impotence, prescribing ingestion of testes to remedy the symptoms
was a medical reflex inherent in organotherapy, practised since antiquity. Thus the Roman
Gaius Plinius Secundus recommended the consumption of animal testes to treat symptoms
of testosterone deficiency. Slightly more refined was the prescription of testicular extracts for
the same purpose in Arabic medicine, for example, by Mensue the Elder (777–837) in
Baghdad. Also in China, raw and desiccated testes were prescribed, documented at least in
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the twelfth century by Hsue Shu-Wei. Around the same time Albertus Magnus (1193–1280)
in Cologne, better known as a philosopher, recommended powdered hog testes, but refined
his recipe by offering the powder in wine [14016].
Since early documentation, these potions continued to be prescribed and consumed up into
the twentieth century. In the 1920s, Testifortan® became a financially successful drug for
treatment of impotence. Its main constituent was testis extracts and yohimbin; after the war
17alpha-methyl testosterone was added without changing the name. Another famous
preparation from the 1920s and marketed until today is Okasa®, which, among other
components, also contains testis sicca and thereby small amounts of testosterone, as could
be determine in the 1970s. However, as the testes synthesize testosterone but do not store
their products in contrast to other endocrine organs such as the thyroid and the pancreas,
the daily production by an adult man of about 6-8 mg is contained in roughly 1 kg of (bull)
testes and even if this amount of testosterone were to be consumed, the testosterone taken
orally would be inactivated by the first-pass effect in the liver. Therefore, all testicular organ
therapy administered orally can only be considered as a placebo medication, which,
however, may not be without its own effects. Ultimately this type of testicular organotherapy
was terminated by the advent of phosphodiesterase inhibitors [14016].
However, organotherapy literally had exploded at the end of the nineteenth century when
Charles E. Brown-Séquard (1847–1894), who until then was a well-reputed scientist and
member of several scientific academies, published the results of his famous self-
experimentation in the Lancet. He gave himself 1 mL injections of a mixture of one part
testicular vein blood, one part semen and one part juice extracted from dog or guinea-pig
testes daily, and after 20 days made astonishing observations on himself: “A radical change
took place in me. I had regained at least all the strength I possessed a good many years ago.
I was able to make experiments for several hours. After dinner I was able to write a paper on
a difficult subject. My limbs, tested with a dynamometer, gained 6 to 7 kg in strength. The jet
of urine and the power of defecation became stronger” [14016].
Certainly all these were placebo effects, but the world had obviously waited for such
quackery, because in no time the ‘extracts of animal organs by the Brown-Séquard method’
were sold all over the (Western) world and factories sprang forth in Europe as well as in
America, for example, next to Central Park in New York. There must have been a real craze
for these products and physicians concerned about the image of the young field of
endocrinology started worrying. The famous neurosurgeon Harvey W Cushing (1869–1939)
and the president of the Association of the Study of Internal Secretions, Edward H.
Rynearson even talked about “endocriminology” in the context of this organotherapy. This
assessment of the medical scene at the time is also reflected in contemporary cartoons and
comic songs from the early twentieth century. Eventually, this type of quackery stimulated
science and decent pharmaceutical companies to search for real hormones [14016].
Testis transplantation
Next to organotherapy there was another sad approach to treat hypogonadism and bring
about rejuvenation and treatment for all sorts of disorders: the transplantation of testes. G
Frank Lydston (1858–1923) in Chicago was one of the first to perform human testicular
transplantation from donors after experimentation in animals. Lespinase published his
experience with transplanting human testes to patients for rejuvenation and Leo Stanley
reported 20 cases of transplantation of testes from executed prisoners to other inmates who
reported signs of revitalization. Later on he turned to animals as sources for his testicular
grafts and reported satisfaction on the part of the patients including 13 physicians [14016].
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In Vienna, Eugen Steinach (1861–1944) performed vasoligation for rejuvenation and one of
his followers, Serge Voronoff (1866–1951) turned to xenotransplantation and used monkey
testes to be transplanted for rejuvenation. He first offered his surgery in Paris, but after some
scandals continued his questionable operations in Algiers, where he was obviously visited by
patients all from over the world. Voronoff had followers in many countries who
xenotransplanted animal testes or pieces thereof to patients in need of rejuvenation, also in
the USA, where this type of treatment caused great interest among the laymen and the
media. As unrest among the medical profession continued to grow, in 1927 the Royal Society
of Medicine (London) sent an international committee to Voronoff in Algiers, which concluded
that Voronoff's claims were all poppycock [14016].
These surgeons had followers in many parts of the world, for example, even in Iceland
where, in 1929, the surgeon Jonas Sveinsson transplanted testis slices from a poor farmer in
need of money to a rich Norwegian businessman who, he then claimed, satisfied his 23-year-
old wife so that he even had three children with her. In the Soviet Union experimentation with
human testicular transplantation continued at least into the 1980s. The only testicular
transplantation resulting in fertility of the recipient was performed by Silber between twin
brothers [14016].
The idea of transplanting tissues and cells continued in a transformed fashion as ‘cellular
therapy’ by injecting suspensions of fresh cells of sheep embryos including testis cells, also
for rejuvenation and revitalization, well into the second half of the twentieth century.
Meanwhile, however, science has progressed and, in the age of cell biology, testicular
transplantation continues with the aim of inducing fertility, but now uses isolated germ cells,
and fertility has indeed been restored by this method in gamma-irradiated cocks. Whether
this may become a method to treat male infertility, for example, in Klinefelter patients
remains to be seen, but at least it is pursued on a rational scientific basis – as far as our
present knowledge goes [14016].
Scientific exploration of the testes and their endocrine function
The declaration of the Netherlands as an independent state during the 30 Years War (1618–
1648), and legalized at the Westphalian Peace Treaty in Münster in 1648, resulted in an
enormous upswing in economy, culture and science in this country. The medical sciences
also boomed, based on proper research, especially in anatomy, as shown in Rembrandt's
contemporary painting “Anatomy of Dr Tulp” (1632). The reproductive sciences benefited
from this boom as well. It was Regnier de Graaf (1641–1673), who not only described the
Graafian follicle (1672), but also published a book about the anatomy of the male
reproductive tract as well as the treatment of its disorders. He produced very detailed
drawings and descriptions of the male genital organs and was the first to discover that the
testes were composed of a “collection of minute vessels or tubules, which confect semen; if
these tubules were disentangled without being broken and tied to one another, they would far
exceed 20 Dutch ells in length” (about 13 m). Having first described this in the edible
dormouse, he then went on to the human: a classical case of translational medicine.
Unfortunately, de Graaf became involved in a quarrel with his contemporary Jan
Swammerdam (1637–1680) about the question of who had first described the ovarian
follicles and during that phase he died under nebulous circumstances at the young age of 32
[14016].
A few years after Regnier de Graaf's early and mysterious death, his friend Antoni A.
Leeuwenhoek (1632–1723), together with the student Johan Hamm, used his newly invented
prototype of a microscope and described the ‘little animals of the sperm’ in a letter to the
Royal Society in London in 1677 (Collected Letters 1948). Considering the primitive
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appearance of his microscope, the details of his morphological descriptions of sperm are
amazing and it is even more amazing that 300 years later we are still quarreling about
normal and abnormal sperm morphology [14016].
But it took another century until Lazzaro Spallanzani (1729–1799), a priest and scientist in
Modena, artificially inseminated frogs and dogs and demonstrated the real function of sperm.
By using sperm that he had preserved on ice he also became the father of cryopreservation
without which modern reproductive medicine and medicine in general would be unthinkable.
He was a very systemic investigator and insisted – quite in contrast to others at the time –
that experiments needed to be repeated before results could be accepted, a principle that
prevails until today [14016].
The anatomist Franz Leydig (1821–1908) in Würzburg described the interstitial cells of the
testes in 1850. Although he did not know their function, they still carry his name. Finally in
Milano, in 1865 Enrico Sertoli (1842–1910) discovered the supporting cells in the
seminiferous tubules, also carrying his name until today. Thus, over roughly two centuries,
the basic morphological elements of the testes had been described, as well as the one major
product of the testes, the sperm. Even the function of sperm and fertilization had been
elucidated so that the time had come to explore the basis of testicular endocrine function
[14016].
Although the endocrine function of the testes was known through their physiological and
clinical effects, their nature remained completely obscure. Although William Harvey (1578–
1657) had discovered the role of the heart and blood circulation in 1628, in some medical
schools Galen's (129–216) concept of the four bodily humors prevailed well into the
nineteenth century. Against this background it is not surprising that the idea of a hormone
working as a signal transduced by circulating blood took so long to be born [14016].
John Hunter (1728–1793) is considered by some to be the father of endocrinology, as he

transplanted testes in chickens. However, his outstanding achievement as a scientist
notwithstanding, he transplanted testes in order to demonstrate the ‘vital principle’ of living
organs. As a surgeon in the Seven Years’ War (1756–1763) he saw the need for
transplantation of organs and limbs, and this is what stimulated his research. He never
described his testis transplantations himself, but we learn about them through a scholar, Dr
W Irvine, in a letter to Prof Th Hamilton in Glasgow in 1771: “…Nay more, he has many hens
just now into whose abdomen when young, he has put the testes of a cock just separated
from his body and his testis has got blood vessels and nerves from the part of the abdomen
or viscera to which it is applied…”. Far from any endocrine thought the goal was to
demonstrate the survival of the transplant due to nerve growth [14016].
Such thoughts were precipitated by Arnold Adolph Berthold's (1803–1861) experiments,

which also concerned transplanting chicken testes. As published in 1849, he castrated four
cocks, two received an ectopic transplantation of one testis, the two others remained
untreated and he observed: “They (the transplanted roosters) crowed quite considerably,
often fought among themselves and with other young roosters and showed a normal
inclination to hens …Since the testes can no longer remain in connection with their original
nerves after being transplanted to a strange place … it follows that the consensus in question
must be affected through the productive relationship of the testes, that is to say, through their
action on the blood, and then through the suitable ensuing action of the blood on the
organism as a whole.” The paper describes only four animals and comprises only four pages
– in contrast to the extensive style of the time, but was epochal. However, Berthold's rival at
the University of Göttingen, Rudolf Wagner (1805-1864) was jealous, tried to repeat the
experiments, but failed and declared them as rubbish. And as he became the full professor of
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physiology, his opinion prevailed. Berthold's personality did not allow him to fight for
recognition of his findings, which fell into oblivion [14016].
The homestretch to arrive at testosterone as a chemical entity
Berthold's unique discovery was superseded by organotherapy as described previously, but

it was not permanently forgotten: Moritz Nussbaum (1850–1915), professor of anatomy in
Bonn, repeated Berthold's experiments at the beginning of the twentieth century and
confirmed the results in frogs, as did Eugen Steinach in rats. Finally, A Pézard confirmed
Berthold's original results in cocks, and the search for the active androgenic substance in the
testes began. From observation of the cock's comb growing under the influence of
transplanted testes first described by Berthold, in 1929 Moore and coworkers established the
standardized capon comb's test measuring androgenic activity in square cm of comb surface.
This first bioassay facilitated determination of androgenic activity in body products as well as
in chemical solutions. Loewe and Voss used the biological effects of androgens on the
accessory sex organs and developed the “cytological regeneration test”, which was based on
regrowth of the seminal vesicle epithelium under androgenic substances (Loewe–Voss-test).
The then still hypothetical male hormone was called “androkinin” [14016].
Simultaneously steroid biochemistry emerged and the great breakthroughs were the
discovery of the ring structure of steroids and bile acids at the National Institute of Medical
Research in London and at the Bavarian Academy of Sciences in Munich. A heated
discussion started about whether there were three of four rings in the steroid structure and, if
four rings, whether the fourth had five or six C-atoms. Under the sponsorship of the Health
Organization of the League of Nations (the predecessor of WHO) famous chemists including
Edmund A Doisy, Adolf Butenandt and Guy Marrian assembled at University College London
in 1932 and reached the consensus that steroids had four rings and the fourth ring had five
C-atoms. Shortly before, these eminent researchers, including Ernest Laqueur, had isolated
pregnandiol and estrone from pregnant mare urine provided by various drug companies
cooperating with scientists in order to replace the miscredited organotherapy and to bring
proper hormone substitution to patients [14016].
In 1931, Butenandt isolated the androgenic steroid androsterone (androstan-3alpha-ol-17-
one) from urine for which he required 15 000 liters provided by young policemen from Berlin,
which was then processed by Schering to obtain 15 mg of this first androgen. In 1935, Ernst
Laqueur (1866–1947) and his group in Amsterdam extracted and isolated 10 mg
testosterone (androsten-17alpha-ol-3-one) from 100 kg of bull testes, which they found more
active than androsterone and named it “testosterone.” In the same year Butenandt and
Hanisch in Göttingen as well as Ruzicka and Wettstein in Basel published the chemical
synthesis of testosterone. This marked the beginning of modern clinical pharmacology and
endocrinology of testosterone and male reproductive physiology [14016].
Development of testosterone preparations
Soon after its synthesis testosterone became clinically available, first in the form of pellets
and then as injectable esters, that is, testosterone propionate with a short half-life and, from
the mid-1950s on, the longer-acting testosterone enanthate appeared, which remained the
major testosterone preparation for half a century. Also in 1935, 17alpha-methyl-testosterone
was synthesized and its oral effectiveness was demonstrated However, due to its 17alpha-
structure it turned out to be liver toxic, a fact that gave testosterone in general a bad name
among physicians, as this toxicity was also suspected for testosterone without reason;
eventually in the 1980s this androgen became obsolete for clinical use in Europe. In the late
1970s the orally effective testosterone undecanoate, absorbed from the gut via the lymph to
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avoid the first-pass effect in the liver, was added to the spectrum of testosterone
preparations used clinically [14016].
In the 1950s and 1960s, the pharmaceutical industry became more interested in new
androgens than in testosterone itself and concentrated its androgen research on the
chemical modification of steroid molecules in order to disentangle the various effects of
testosterone and produce predominantly erythropoietic or anabolic steroids. In 1956,
contemporary textbooks on androgens had already described 256 androgenic steroids and
by 1976 the number had increased to more than 1000 [14016].
However, it proved impossible to produce androgens with only one effect out of the spectrum
of testosterone activities; at best, one of these effects could be emphasized, but the other
effects remained. The steroid with pure anabolic effects on muscles or bones to treat
cachexia, osteoporosis or small stature, or pure erythropoietic effect for the treatment of
anemia without androgenisation could not be found. Nevertheless, anabolic and similar
steroids were clinically used, but disappeared again in the wake of evidence-based medicine.
However, they continued their existence for illegal use and abuse for doping in sports and
bodybuilding potentially causing considerable undesired effects. Regrettably, at that time the
pharmaceutical industry neglected the chance to develop testosterone preparations better
suited for the substitution of hypogonadal patients than the existing testosterone esters. It
remains to be seen whether the current search for SARMs will take a more rewarding course
than did anabolic steroids [14016].
From the 1970s, the newly developed testosterone immunoassays made serial testosterone
determinations in blood possible and, when applied to pharmacokinetic studies, it turned out
that all available testosterone preparations resulted in unphysiologically high or low serum
levels, which were undesirable in substitution therapy. Clinicians assembled at a workshop
on androgen therapy sponsored by WHO, NIH and FDA in 1990 came to the conclusion:
“The consensus view was that the major goal of therapy is to replace testosterone levels at
as close to physiologic concentrations as is possible” and demanded that new testosterone
preparations better suited for clinical use be manufactured [14016].
In the mid-1990s, transdermal testosterone patches applied to the scrotal skin became the
first transdermal testosterone preparation in clinical use. They had been invented by Virgil
Place at ALZA in Palo Alto, a company specializing in new forms of delivery of known drugs.
However, although clinical results with this preparation were excellent and for the first time
physiological serum levels could be achieved under testosterone substitution, physicians
were reluctant to prescribe a medication to be applied to the scrotum and preferred a
subsequently developed nonscrotal system. This, however, caused unpleasant skin
reactions as it required an enhancer to drive testosterone through the skin. For this reason,
the advent of the first transdermal testosterone gel was welcome. This gel became available
in 2000 for the treatment of male hypogonadism, first in the US and later also in other
countries. Since then, several other gels have been developed and brought to the market,
differing slightly in composition and concentrations. The one with the highest testosterone
concentration (2.5% Testotop®) has also been tested for scrotal application and because of
the high absorptive capacity of the scrotal skin only 20 percent of the gel needed for
nonscrotal application is required, making this form of application economically and
ecologically more desirable [14016].
Finally in 2004, the intramuscular testosterone undecaonate preparation entered the market
and soon achieved great popularity as a real testosterone depot preparation. Testosterone
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undecanoate had originally been used in oral capsules, but had been turned into an injectible
preparation by Chinese investigators using tea seed oil as a vehicle. A long half-life could be
confirmed in volunteering hypogonadal men who all showed serum levels in the normal
range. When finally a company could be interested in this fascinating preparation, it was
‘Europeanised’ by using castor oil as vehicle and was developed as Nebido® (or
Reandron®) for clinical use and is licensed today in 97 countries [14016].
The era of anabolic steroids
In 1923 Bob Hoffman formed the famous York Barbell Company in the United States. A
dominant figure in US weightlifting, he published the “Strength and Health magazine” and
sold health and food supplements in his gym. As a weightlifting coach, his success led to him
being named the head coach of the US Olympic weightlifting team. At the 1954 World
Championships in Vienna, he met with a Soviet colleague who told him of a synthetic form of
testosterone developed by the Nazis which produced dramatic improvements in strength and
power. He and his colleagues contacted Ciba Pharmaceuticals in pursuit of synthetic
testosterone. Ciba had conducted a number of studies on the use of synthetic testosterone in
pain patients and the physically disabled. This resulted in the development of danazol, which
rapidly became a doping substance abused by weightlifters [08008].
Anabolic androgenic steroids (substances similar to the hormone testosterone) were used
already directly after World War II by Soviet athletes to increase muscle mass and power in
weightlifting and bodybuilding events. When the Berlin Wall fell, the East German
government's program of performance enhancement by meticulous administration of steroids
and other drugs to young athletes was exposed. These well-documented and controlled
hormonal doping experiments on adolescent athletes by the East German Sports Medical
Service at Kreischa and Leipzig yielded a crop of gold medalists (mostly young females as
they responded more dramatically to male hormones). Some of these athletes later suffered
severe medical abnormalities, including premature death [08009].
The first reports of athletes using anabolic steroids searching for an increase in weight and
power appeared in 1954. After that here has been an increasing use of doping substances
by athletes. Furthermore, it was found that not just stimulants were being used but also
anabolic androgenic steroids (AAS). However, the banned list did not include those
substances. Therefore, the IAAF banned them and developed an immunological method for
their detection. It was used for the first time at the European Athletic Championships in Rome
in 1974. No cases were found as the method was still immature, but the IAAF initiative paved
the way for the IOC who banned steroids in time for the 1976 Games and found eight cases
at the Montreal Games with an improved method. The IAAF experience soon showed the
need for strict procedures to be applied at every stage of a doping control, including the
laboratory analysis. Therefore, the Federation started to work out procedural guidelines for
doping controls as well as specific requirements for laboratories that were used for the
analysis of doping-control samples. Some heads of laboratories were not so happy since
they felt that their competence was questioned, but in 1979 the IAAF decided to only
recognize analytical results from laboratories that met the specific requirements. The
“Accreditation of Doping Control Laboratories” was born. Subsequently, two years later, the
IOC adopted the IAAF system, and for a couple of years laboratories were jointly accredited
by the IAAF and IOC. In 1986 the IOC took over full responsibility for the accreditation
program. Today, doping-control laboratories are accredited by WADA [12005].
The 1960s through 1980s were the golden age of anabolic steroid use in sports. After
learning that the success of the Russian weightlifting team was in part due to their use of
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testosterone, Dr. John B Zeigler began experimenting with Dianabol (methandrostenolone)
on weightlifters at the York Barbell Club in 1958. The weightlifters became strength and
conditioning coaches in a variety of other sports in the United States and spread use of
anabolic steroids to other sports, such as American football. The German Democratic
Republic operated a state-supported anabolic steroid doping program that produced many
medals in the 1970s and 1980s, especially for women in swimming and track and field. The
doping program, and its health effects for the women, was the subject of an excellent review
by Franke and Berendonk and a television special by the Public Broadcasting Service. Ben
Johnson of Canada had anabolic steroids detected in his urine at the 1988 Olympic Games
in Seoul, and he was stripped of his gold medal. While increased muscle mass was the goal
of early doping with steroids, since the late 1990s steroids in Olympic sport have been
primarily used to enhance recovery to allow more frequent and more intense workouts.
Testosterone is also largely responsible for the larger red blood cell (RBC) mass in men as
opposed to women, so it has benefits beyond its effect on muscle. Other anabolic steroids
have a similar effect on RBC production [12006].
Soon thereafter in the 1950s, Russian weightlifters began to outpace American Olympians
through performance-enhancing injections. Attempting to make up lost ground, the then US
Olympic physician teamed with chemists to produce an anabolic steroid for the Americans,
now known as Dianabol. In the decades that followed, steroids and stimulants spread
throughout sports, and in 1959, the first reported case of a high school football player's taking
steroids surfaced. In the 1960s, the International Olympic Committee banned steroid use and
began formal drug testing in the ensuing decade. During the 1980s, the reported positive test
results ranged from 2 to 50 percent, depending on whether the tests were announced or
conducted at random. At the 1988 Seoul Olympics, the first gold metal in track and field was
stripped when the Canadian sprinter Ben Johnson lost his 100-m victory after failing drug
tests. Then, in 1994, an often-referenced survey was conducted by Goldman when aspiring
Olympians were asked 2 simple questions. The first was, “If you were offered a banned
performance-enhancing substance that guaranteed that you would win an Olympic medal
and you could not be caught, would you take it?” Remarkably, 195 of 198 athletes said yes.
The second was, “Would you take a banned performance-enhancing drug with a guarantee
that you will not be caught, you will win every competition for the next 5 years, but will then
die from adverse effects of the substance?” Still, more 50 percent of the athletes said yes.
This survey made it clear that modern athletes often approach their sports with a “win at all
costs” mentality [06003].
In 1954, the first reports appeared of athletes using anabolic steroids searching for an
increase in weight and power. As a result, the misuse of anabolic steroids in sports led to a
ban by the International Olympic Committee of these substances in 1974 and testing was
implemented on a large scale at the 1976 Montreal Olympic Games via radio-
immunoassays. In 1994, the Drugs Supplement Health and Education Act (DSHEA) was
approved in the United States and several new steroids were commercialized as nutritional
supplements. Initially these new steroids were precursors of testosterone, commonly referred
to as “prohormones”. Late 2004, the US Congress approved the Anabolic Steroid Control Act
(ASCA), restricting the sale of anabolic steroids as nutritional supplements. However, by
2004 a range of prohormones derived from other steroids, including 19- nortestosterone,
boldenone and even 17alpha-alkylated steroids were available as over-the-counter
preparations [06004].
The evolution of the history of testosterone therapies is as interesting as the history of its
development. Erectile dysfunction is one of the most researched ailments treated with
testosterone, although any positive effects are questionable. In men with absent to low
circulating levels of testosterone, treatment with testosterone increased libido, improved
233
erectile function, and helped to maintain secondary sexual characteristics. In men with
normal or mild hypotestosteronemia, studies have not shown consistent response to therapy.
Those treated were reported to have increased sexual interest, increased arousal, increased
frequency of intercourse, and nocturnal erections. In the early twentieth century, there was
much interest in the hormonal influence of testosterone on sexuality and sexual preferences.
It even was prescribed to “treat” homosexuals because it was theorized that male
homosexuals had higher estrogen levels [07007].
Testosterone has even played an important role in various ailments affecting women, such
as treatment for some metastatic breast cancers. Approximately one third of breast cancers
are hormone dependent and respond to androgen therapies. Other uses for testosterone are
as postmenopausal hormone replacement therapy, for sexual dysfunction (by increasing
libido), and for increasing bone density. Some clinical case studies showed an increase in
appetite, lean muscle mass, and strength and an improved overall sense of well-being.
Before the use of erythropoietin and bone marrow transplants, testosterone was used to help
treat anemia (i.e. chronic renal failure/hemodialysis). Psychiatrists prescribed anabolic
steroids from the 1930s to the 1980s to treat psychoses, depression, and melancholia.
Testosterone has been used as an adjunct in people with growth hormone deficiency or in
boys with pubertal delay [07007].
Body builders and athletes began using testosterone to increase muscle mass and to
intensify training protocols on the West Coast of the United States in the late 1940s and early
1950s. The US Food and Drug Administration approved methandrostenolone in 1958. In the
1950s, Soviet Union and East German Olympic athletes were using AASs. They later found
their way into the hands of Olympic competitors, including track and field athletes from many
countries. Paul Niehans wrote the 1960 book” Introduction to Cellular Therapy”, in which the
main emphasis was on testicular secretions. He believed that testicle cell injections
increased testosterone derivative excretion. Some of Paul Niehans' famous patients included
Pope Pius XII, Bernard Baruch, and Aristotle Onassis. In 1974, the International Olympic
Committee banned the use of testosterone and its derivatives. AASs were widely abused in a
variety of sports, including volleyball, cycling, swimming, soccer, and bobsledding.
Testosterone was studied using different forms. Scientists quickly learned it was ineffective,
and even toxic (like 17 alpha-methyl testosterone), when taken orally; instead, it was
synthesized into tiny pellets that were inserted subcutaneously. Longer-acting injectable
forms of testosterone were synthesized in the 1950s (i.e. testosterone enanthate). Over the
following decade, the hormone was modified into derivatives that possessed more anabolic
qualities. In the 1970s, oral testosterone undecanoate was synthesized; however, it did not
fare well in the oral form because of hepatic clearance and hepatotoxicity. Transdermal
scrotal patches were derived in the 1990s. These allowed physiologic levels of testosterone
to be acquired. Nonscrotal skin patches were developed, and testosterone gels were
marketed. Today, there are short-acting buccal forms as well as the long-acting injectable
testosterone undecanoate [07007].
By the early 1990s, several pharmaceutical companies had stopped producing AASs. It was
about at this time that the black market sales of AASs and counterfeit products increased
secondary to the ease of Internet shopping and availability. Authentic steroids, as well as
placebos and unpurified forms, were sold and abused. The US Congress placed anabolic
steroids into the schedule III category of the Controlled Substance Act (CSA) in the Anabolic
Steroid Control Act of 1990. This act included testosterone and all related chemical or
pharmacologic substances that promoted muscle growth. Corticosteroids, progestins, and
estrogens were not included in this act. The Anabolic Steroid Act of 1994 was an amendment
to the CSA. It placed anabolic steroids as well as their precursors on the controlled
substance list. Possession of the drugs without a prescription was now a federal crime.
234
Studies of the effects of supplemental testosterone on aging men in the 1990s suggested an
increase in word memory, special cognition, increased libido, decreased bone resorption,
and increased lean body mass and strength. McKinlay reported in the Journal of Urology that
testosterone does not treat impotence. In theory, prostatic tissue, including cancer and
benign prostatic hypertrophy, can be stimulated by testosterone, but no compelling evidence
has been reported that suggests an increased risk [07007].
Androgenic-anabolic steroids (AAS) have been misused by athletes at the Olympic Games,
both before and after they were prohibited in sport in 1974. Systematic doping with AAS
occurred in the German Democratic Republic from 1965 to 1989 which assisted that country
to win many medals at Olympic Games, especially in female events. Currently, androgenic-
anabolic steroids are the most frequent category of prohibited substances detected in the
urine of athletes both globally and at the last two Summer Olympic Games. Scientific
confirmation that AAS are effective in enhancing sports performance was difficult because
ethical approval was difficult for research involving male subjects taking massive doses of
androgens as some athletes and bodybuilders did. Methods to detect androgenic-anabolic
steroids have evolved gradually over the past three decades and currently, despite an
impressive array of sophisticated analytical equipment and methods, anti-doping authorities
and analytical scientists continue to face challenges as have occurred from the use by
athletes of designer AAS during the past few years. The future development and use of
selective androgen receptor modulators can be anticipated to pose problems in the years
ahead. Endocrinologists should be aware that on occasions, replacement testosterone
therapy may be authorized in sport as a therapeutic use exemption (TUE) [08060].
Definitive proof of anabolic steroid abuse in sports was not possible prior to the introduction
of combined gas chromatography/mass spectrometry (GC/MS).It was now given a report of
the early history (1960-1980) of GC/MS and radioimmunoassay, and how these techniques
were utilized in the first years of steroid doping control in athletics. There were several key
individuals and research groups involved in the early technical developments, and their
essential contributions have been acknowledged. The Oakland USA laboratory was the first
IAAF (International Association of Athletic Federations) sanctioned site to do steroid GC/MS
steroid analysis resulting in athletes being disqualified from competition. This gave notable
successes, including the only East German female competitor ever suspended during the
tenure of the DDR (Deutsche Demokratische Republik). By the early 1980s, in anticipation of
the Los Angeles Olympic games, dedicated year-round sports testing facilities had been
established and part-time amateurs could step aside [08125].
High levels of anabolic-androgenic steroids abuse have been attributed to professional

football players, bodybuilders, weight lifters, and track and field throwers since the 1960s.
The exceptional athletic performance of the East German female swimmers in the 1976
Montreal Olympics brought further public attention to AAS athletic use. It was not until the
1980s, however, that the medical community admitted that these substances were effective.
Since that time, the pervasive use of AASs by professional athletes has garnered significant
media attention, culminating most recently in the ongoing investigation of the use of illegal
performance enhancing drugs by some of baseball's top players. “Juiced”, a book by Jose
Canseco, details his steroid use and the widespread use of anabolic steroids in Major
League Baseball [07008].
Detection of abuse
In 1982, Donike and coworkers first reported a method for detecting testosterone abuse.
They based their method on the fact that exogenously administered testosterone is
235
predominantly excreted in the urine as the glucuronide conjugate. By determining the ratio
between testosterone and epitestosterone (T/E), they eliminated the influence of urine
density variations. The mode of the population distribution of T/E ratios is about 1:1 and early
research suggested that ratios above 6:1 were linked to doping. WADA has decreased the
ratio consistent with doping to 4:1. In the early 1990’s, intra-individual biological variability of
the T/E ratio began to be used in combination with population ranges to detect doping.
Sottas reported a “predictive model” that compared the T/E ratio and other steroid
concentrations to previous results from individual athletes [14017].
Background to T/E ratio
It was reported that after oral, rectal, or intramuscular T administration, the excretion
of TG increased more than other T metabolites. Epitestosterone (E) was found not to
be a metabolite of T because deuterated T administration did not result in significant
deuterated EG excretion. The origin of epitestosterone is still discussed. Although
Dehennin showed that half of total E production is of testicular origin, the remaining
50 percent is still debated. Administration of adrenocorticotrophic hormone (ACTH)
results in an increased EG production, indicating an adrenal origin. Also, adrenal
insufficiency as observed in Addison's disease correlates to significantly decreased T
and E excretion rates. Also peripheral production is possible. The mean T/E ratio of
urine samples of Caucasian males and females in the first population study of Donike
et al was 1-2. The values showed a logarithmic normal distribution with an upper limit
value lower than 6. Using these data, the Medical Commission of the International
Olympic Committee (IOC) banned the use ofT in 1982 and stated that a T/E ratio
above 6 was sufficient proof of T abuse. When applying this criterion in research and
routine analyses, cases of naturally occurring T/E ratios above 6 appeared. Dehennin
et al administered testosterone enanthate in several doses intramuscularly to healthy
men over a period of six months. They found via linear interpolation between doses
that the T/E ratio exceeded the cutoff point of 6 when natural production (around 45
mg/week) was doubled by weekly administration of a comparable dose of exogenous
T [00003].
Availability of drugs
Most of the PEDs that athletes and nonathlete weightlifters used prior to the 1990s were
pharmacological agents approved for medicinal or veterinary use. By the 1990s, various
androgen precursors became available over-the-counter as unregulated “nutritional
supplements” [14017].
Androgen receptors
In the 1980s, Dr. Jean D. Wilson citing the singularity of androgen receptor, suggested that
androgenic and anabolic activity of androgens could not be dissociated. Therefore, he and
others have argued that the term androgenic-anabolic steroid is a misnomer and should be
abandoned [14017].
US professionals
Despite years of aggressive anti-doping testing by international sports federations such as

those for cycling, athletics and soccer, steroid abuse scandals involving high profile athletes
236
continue to be front page news across the globe. Professional sports in the United States
were not subject to extensive anti-doping programs, as players' unions and collective
bargaining agreements prevented such extensive testing to be put into place. However, they
did establish limited anti-doping programs, as the professional sports organizations
recognized the potential of doping to harm athletes and their sport. In 1998, when Mark
McGuire, an American baseball player, broke Roger Marris' home run record, it was revealed
that he had been taking a supplement containing a precursor to nandrolone, a steroid. At that
time Major League Baseball did not ban steroids and did not believe that steroids were a
problem within the league. However, subsequent government investigations and former
players revealed that steroid abuse was a problem in the League, which resulted in a limited
steroid testing program [08006].
Anabolic steroid prodrugs in the US
The potential performance-enhancing benefits of testosterone precursors were brought to the

attention of the public and athletic community in the US in 1998 when Major League Baseball
player Mark McGwire set the home run record and openly admitted to using
androstenedione. Sales skyrocketed by 500 percent, and many supplements containing
prohormones became available in the United States market. Questions and concerns of
contamination with other supplements arose but their purity was unknown because these
supplements were not regulated by the FDA. Also, their popularity was fueled by the
misperception that nutritional supplements are natural, and, therefore, safe. In 2004, after
much controversy and debate, the US Department of Health and Human Services (HHS) and
the FDA announced a crackdown on companies that manufacture, market, and distribute
products containing androstenedione. They recognized the potential serious adverse health
risks that were similar to those associated with AASs. As part of their concern about its
safety, the FDA and HHS sent warning letters to 23 companies asking them to stop
distributing dietary supplements that contained androstenedione and warned them that
enforcement actions would be taken if they did not comply. As a result of this action, the
Anabolic Steroid Control Act of 2004 was passed. This act added the steroid precursor
androstenedione to the list of schedule III controlled substances in the United States.
Schedule III substances have limited medicinal use, require a prescription from a licensed
physician, and allegedly can threaten public health without government regulation. DHEA
was not added to the controlled substance list; industry lobbyists contended that it had
proven effective as an antiaging supplement and that its risks were minimal [07009].
Designer drugs
Synthetic organic chemistry can be traced back to 1865 when Friedrich August Kekule
published two theoretical papers on the structure of aromatic organic molecules. Paul Ehrlich
postulated in the early 1870s that differences in chemoreceptors between micro-organisms,
parasites, and cancer cells from those in host cells could be exploited for therapeutic
purposes. In the absence of current ligand-based and receptor-based molecular design
techniques, there were limited approaches to identify minor structural changes in biologically
active compounds that would enhance selectivity and/or potency of therapeutic molecules.
Hamett made the first significant contribution relating structure to activity of small organic
compounds with his study correlating electronic properties of organic acids and bases with
reaction rates and equilibrium constants, focusing on benzoic acid derivatives. Moving
beyond the linear free energy relationships provided by the Hammett equation, the next
major development was the introduction of quantitative structure-activity relationships
(QSAR) by Corwin Hansch et al. in two seminal papers in the early 1960s, providing a new
tool to systematicallyrelate molecular descriptors (electronic, steric, topological, and
237
hydrophobic indices) to biological activity. These early efforts concentrated on naturally
occurring plant hormone mimics, and relied on statistical analysis of published accounts of
the biological activity of phenoxyacetic acid derivatives and other plant growth regulators.
Electronic indices were found insufficient for QSAR of biological systems; rather, a measure
of lipophilicity (classically measured as an octanol-water partition coefficient) was essential to
predict targeting of compounds to specific tissues, cells or organelles, and subsequent
biological activity. John Topliss developed a method to automate QSAR; however, it is of
limited utility in many experimental systems and ignores possible interactions between
multiple substituents. QSAR remains a dynamic tool for drug design and optimization.
Approximately 15 years ago, pharmaceutical companies realized that existing screening
libraries were inadequate for newly developed high-throughput efforts for lead drug design.
The challenge has been to balance size and structural diversity of new libraries against
screening cost, while maintaining affinity and selectivity against a portfolio of targets. Two
major approaches have emerged: fragment-based screening (FBS) and diversity-oriented
synthesis (DOS). Despite major advances in chemical screening and synthesis, discovery of
new drugs is difficult, expensive, and the efficacy of target-based drug discovery has been
questioned [11555].
BALCO
Although a general topic of interest in the 1990s, designer drugs first made international
headlines in 2003 with the Bay Area Laboratory Co-operative (BALCO) scandal involving the
widespread use by athletes of tetrahydrogestrinone (THG). This simple reaction created a
potent agonist for androgen and progesterone receptors whose presence could not be
detected by standard multiple reaction monitoring (MRM) methods used by anti-doping
laboratories for steroid detection. Subsequent characterization of this steroid derivative led to
US federal prosecution of many involved with BALCO, culminating in March 2011, with the
most high profile case so far, involving the former San Francisco Giant (US Major League
Baseball) Barry Bonds [11555].
History of nandrolone
The anabolic androgenic steroid 19-nortestosterone, also called nandrolone, was first
synthesised by Birch in 1950. The use of nandrolone by athletes became popular in the late
1950s. The International Olympic Committee (IOC) prohibited the use of nandrolone in sport
in 1976. A study in 1982 appeared to have found NA, or a similar compound, in the urine of
athletes who had not used nandrolone. In 1996, the IOC stated that a critical concentration
for nandrolone metabolites in the urine had been established. A doping offence for
nandrolone was defined as a concentration of NA in human urine exceeding 2 ng/mL in men
and 5 ng/mL in women [02002].
History of aromatase inhibitors
The use of aromatase inhibitors has been prohibited for male athletes since September 1,
2001 [02003].
History of 5-alpha-reductase inhibitors enzymes
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Scientists in the pharmaceutical industry reasoned that if 5AR could be targeted for inhibition
after the external genitalia were fully formed and mature, then a safe drug to shrink the
prostate, relieve LUTS, and ameliorate baldness and acne might be developed. Eighteen
years after Imperato-McGinley’s first publication,1 the “prostate pill” arrived; the US Food and
Drug Administration (FDA) approved finasteride June 19, 1992 for the treatment of men with
symptomatic BPH. FDA approval for male pattern hair loss (in men only) followed, and in
October 2002, the dual 5ARI dutasteride was approved. Both drugs currently claim to
improve symptoms, reduce the risk of acute urinary retention (AUR), and reduce the risk of
the need for BPH-related surgery. Soon after the guevedoces’ story became known, and the
implications of 5AR deficiency became clear, Merck began an ambitious development
program in the research laboratories at Rahway, New Jersey. Following synthesis of many
potential 4-aza steroid molecules that would inhibit 5AR, a drug known as MK-906 was
selected as the best therapeutic molecule. After successful testing in experimental animals,
where the drug was found to sharply reduce DHT levels and prostate volume, finasteride
went into human trials in 1986. A few years later, reports of phase I testing were reported by
Stoner, Gormley and colleagues, Rittmaster and colleagues, and others. As expected, men
treated with finasteride developed a marked suppression of DHT, no change or slight
elevation in serum testosterone, and no change in all other serum components studied. In
1992, the phase III studies, demonstrating safety and efficacy over 1 year of treatment in
men with symptomatic benign prostate hyperplasia, were published in the New England
Journal of Medicine, concomitant with FDA approval. As finasteride was known to be a pure
Type 2 inhibitor of 5AR, efforts soon began to develop a drug that would inhibit both Type 1
and 2, theoretically a more powerful inhibition. Merck went into phase II testing of such a
molecule in the early 1990s (MK-434), but trials were quickly halted because of potential
toxicity problems, and the drug was never developed. A dual inhibitor from GlaxoSmithKline,
originally known as GG745 (dutasteride), was developed later in the decade, and in 1998,
early-phase clinical trial results were published. The dual inhibitor was found to lower DHT
serum levels significantly more than finasteride (90 % with dutasteride vs 70 % with
finasteride), offering the potential for greater clinical efficacy of the new drug. Although direct,
long-term comparisons of finasteride and dutasteride in a clinical trial are not available; the
phase III dutasteride data published in 2002 showed that dutasteride yielded symptomatic
improvement over placebo as early as 3 months and a prostate shrinkage exceeding 25
percent, both quicker and more profound than what had been seen in the finasteride trials
[04009].
History of blood doping
By the 1930s it was clear that champion endurance athletes had remarkably high maximal O2
uptake (VO2MAX). In the 1950s, 1960s, and 1970s, classic studies were performed on the
physiological determinants of VO2MAX and on its key role in endurance performance. During
this time there was much debate on O2 delivery versus O2 extraction as the “limiting factor”
for VO2MAX. Observations during this era clearly established the role of maximal cardiac
output as a determinant of VO2MAX, and very high maximal cardiac output values were seen
in champion endurance athletes. In addition, the important role of blood volume and total
body haemoglobin as determinants of VO2MAX also emerged. In an effort to better understand
the physiological determinants of VO2MAX, studies were then conducted that attempted to
manipulate O2 delivery using a variety of approaches including altered concentrations of
inspired O2, drugs that speed or slow the heart, and, as will be discussed here, techniques
that altered total body haemoglobin and haemoglobin concentration. In general, by the 1970s
it was clear that manoeuvres that increased total body haemoglobin increased VO 2MAX and
manoeuvres that reduced total body haemoglobin reduced VO2MAX. These changes in VO2MAX
239
appeared to be somewhat independent of total blood volume because volume loading per se
had little impact on VO2MAX, and likewise manoeuvres that cause haemoconcentration did not
increase VO2MAX. Therefore, the importance of total body haemoglobin as a primary
determinant of VO2MAX was emphasised. In parallel with these mechanistic studies on the
determinants of VO2MAX, applied observations on athletic performance and the role of VO2MAX,
lactate threshold, and running economy emerged. As VO2MAX was seen as a key determinant
of performance, the next obvious question was whether or not manoeuvres that increased
total body haemoglobin and VO2MAX would also increase performance. A number of studies
confirming the positive impact of increased total body haemoglobin on performance were
then conducted. In addition, a variety of rumours and innuendo suggested that at least some
endurance athletes were using this technique in an effort to gain a competitive advantage in
international competition. Thus, the term “blood doping” was coined. Although it is clear that
blood doping improves performance, it is unclear how widespread it was in the 1970s and
1980s as detection was difficult because athletes received a reinfusion of their own red blood
cells [03007].
Blood transfusions as a means for improved endurance were researched as early as 1947 by
Pace and coworkers. Transfusion of 500 ml of allogeneic erythrocytes on four consecutive
days reduced the pulse rate during exercise in simulated hypoxia. However, even though the
methods would not meet today’s standards; this was the first of many studies to confirm the
possible performance enhancing effect of blood transfusions. Accordingly blood transfusions
in sports were later banned by the International Olympic Committee (IOC) in 1986 [13005].
Lasse Viren, a Finnish long distance runner who won gold medals at the 1972 and 1976
Olympic Games in the 5,000 m and 10,000 m, is believed to be among the first athletes to
have used blood transfusions to improve performance. It should be noted that this technique
was not banned at the time and although the ethical debate on the topic was in full swing, it
was only in 1986 that the International Olympic Committee banned blood transfusions. Other
than the anecdotal evidence from the Nordic distance runners, there are other reports on
more systematic use of transfusions in the context of major sporting events in the 1980s.
Notably, it is well established that a large part of the US cycling team was involved in a
systematic blood doping program that earned them unprecedented success at the 1984
Olympic Games in Los Angeles. There is also some evidence that blood transfusions were
an integral part of the doping regime used for the enhancement of performance for athletes
from the Eastern bloc (Soviet Union, East Germany) at that time. Nevertheless, it can be
assumed that because of the logistic requirements of blood withdrawal and reinfusion, the
technique was not widespread, as the technical necessities were only available to a small
number of athletes, but nevertheless available to certain elite athletes [13006].
The blood doping situation changed dramatically with the commercial introduction of
recombinant human erythropoietin (rhEPO), the human hormone that regulates the
erythropoietic system in the organism. EPO was first isolated in the 1950s. The gene for
human erythropoietin was successfully cloned in 1983, and the first recombinant
erythropoietin (rhEPO) was approved by the FDA for treatment of anemia in renal failure in
1989. One year later the IOC banned the use of erythropoietin (EPO) [13005].
Studies investigating the effect of rhEPO on performance were soon published and
demonstrated positive effects on maximal oxygen uptake of 6-12 percent. Although the
authorities rapidly banned rhEPO, the easy access to the substance and the huge impact on
performance resulted in widespread abuse of rhEPO during the 1990s/2000s. It is believed
that this substance had a considerable impact on the development of peak performances in
all endurance sports during these years and there are even scientific attempts to prove this
for several sports on the basis of performance analysis. The abuse was facilitated by the fact
240
that no detection method was readily available at that time. From a practical point of view,
the impact of rhEPO on performance in endurance sports is best illustrated by a quote from
Greg Lemond, an American cyclist who won the Tour de France in 1986 and 1989, i.e.,
before rhEPO became available, recalling the 1991 race: “I was the fittest I had ever been,
my split times in spring training rides were the fastest of my career, and I had assembled a
great team around me. But something was different in the 1991 Tour. There were riders from
the previous years who couldn’t stay on my wheel who were now dropping me even on
modest climbs.” These words accurately describe how rhEPO changed the entire world of
endurance sport in the following decades and divided the athletes’ performance primarily
between rhEPO users and non-users [13006].
Logically, following widespread abuse from the 1990s onwards, doping scandals involving
rhEPO or blood transfusions have shaken the world of sport on a regular basis, culminating
recently with the investigation of Lance Armstrong, who subsequently admitted the use of
both rhEPO and blood transfusions throughout his career. Although, it is therefore common
belief that many recent doping cases were not unveiled by conventional anti-doping testing,
but rather by police investigations or admissions from athletes or staff, thus non-analytical
approaches, anti-doping laboratories were able to detect about 400 cases testing positive for
rhEPO between 2003 and 2011 (World Anti-Doping Agency (WADA) statistics). Analytics
have therefore come a long way in the detection of blood manipulation in sports and still
outperform police investigations by 10 to 1 [13006].
Blood transfusions, as a method to enhance endurance performance, first gained attention

after the Olympic games (OGs) in Mexico City in 1968. Before these OGs, evidence was
presented that a lowered atmospheric pressure would decrease performance in all athletic
disciplines dependent on a high level of sustained oxygen uptake. This was indeed
confirmed in Mexico City, where all winning times in running races above 800 m were
significantly worse than the world records at that time. This highlighted the impact of the
oxygen delivery to the working muscles as a limiting factor during whole body endurance
exercise. It also became evident that runners hailing from higher altitudes tended to be
superior to competitors from lowlands because they had “thick blood” with high hemoglobin
content. A relatively straightforward way to increase the hemoglobin concentration (Hb), and,
hence, oxygen delivery to the muscles is, thus, by blood transfusions. This was documented
in the classic study by Ekblom et al from 1972 where a high correlation between Hb and
performance capacity after blood withdrawal and reinfusion was presented. An overnight
increase in Hb by 13 percent caused by the reinfusion of 3 units of stored autologous blood
resulted in an increase in maximal oxygen uptake and physical performance capacity of 9
percent and 23 percent, respectively. The method of transfusing blood in a sport setting was
hereafter dubbed “blood doping” by the media, and its potent effect on athletic performance
was quickly noted in the sports community. Blood doping was used already at the OG in
1972 by a Finnish steeplechaser, and during subsequent OGs, several athletes admitted
having used blood doping. Not until after the OG in Los Angeles in 1984, where the US
cycling team used blood doping and won 9 medals after not having won a medal in cycling
for 72 years, the method (both homologous and autologous transfusions) was prohibited by
the International Olympic Committee, although no method was available to detect its use.
Because of its logistic advantages compared with blood transfusions, human erythropoietin
(rhEPO) became the preferred blood boosting method by athletes after it had been available.
At the OG in Sydney in 2000, two tests for rhEPO were introduced: a “direct test” that was
able to distinguish rhEPO from endogenous molecules by isoelectric focusing and an
“indirect test” based on changes in blood parameters caused by rhEPO administration.
Because of the introduction of these tests, old-fashioned blood doping reentered the scene.
At the Salt Lake City Winter OG in 2002, discarded blood transfusion equipment was found
at the headquarters of the Austrian cross-country skiers. After DNA testing, 2 skiers were
241
disqualified.Although testing for homologous blood transfusions had been performed at the
Lillehammer OG in 1994 by use of antigen testing cards, it was not until 2004 at the OG in
Athens that a test had been validated and implemented. At this event, the gold medal winner
of the men's time trial in cycling was first tested positive, but because the backup sample (B-
sample) was frozen and, thereby, the red blood cells (RBCs) destroyed, no doping offense
could be proven. After he failed further doping tests at the 2004 Vuelta a España, the rider
was suspended for 2 years [12009].
The word “doping” was used in the 1860s to describe a drug used for horse racing that
consisted of opium and narcotics. With human athletes, “blood doping” originally referred to a
process whereby athletes increased their oxygen-carrying capacity by receiving blood
transfusions from previously donated blood to increase their hematocrits a few days before
competition. The first report of blood doping in a controlled experiment was reported in 1947.
Today, “blood doping” is used more synonymously with cheating of any kind, including any of
the various blood-boosting methods or classes of ergogenic aids that are available to
athletes. Advances in genetic medicine have allowed athletes to raise their level of
sophistication significantly by using PESs that are virtually undetectable. In June 1989, the
first rHuEPO product was marketed in the United States. It was isolated and purified from
Chinese hamster ovaries and reproduced using DNA recombinant techniques. The popularity
and effectiveness of rHuEPO in elite endurance athletes is demonstrated by a long list of
anecdotes associated with its misuse during international competition. When the average
speed of the cyclists racing in the Tour de France began to increase suddenly during the
1990s, rumors of rHuEPO use began to circulate. The gene that produces EPO was cloned
in 1985, and rHuEPO was available in Europe by 1987. Between 1987 and 1991, more than
20 Dutch and Belgian cyclists died at rest – some of them while sleeping – as a result of
unexplainable cardiac arrest. Between 1997 and 2000, 18 more cyclists died from pulmonary
embolisms, stroke, and myocardial infraction. Finally, suspicions of rHuEPO use in
professional cyclists competing in Europe were confirmed during the 1998 Tour de France;
boxes of ampules containing rHuEPO were found in team vehicles and the personal rooms
of riders from many of the biggest and most successful teams. It became embarrassingly
clear that rHuEPO use in elite professional cyclists was organized, widespread, and
sophisticated. The International Olympic Committee (IOC) added rHuEPO to its list of
banned substances in 1990, even though all forms of blood doping had been officially
prohibited since 1984. Despite justified suspicions of rHuEPO use in cycling and the inability
of current methods to detect its use, in 1997, the governing body of the International Cycling
Union (UCI) enacted hematocrit cutoffs for male (50 %) and female (4 7%) cyclists while
more reliable methods of detection could be developed. The hematocrit cutoffs were based
on existing normative data on elite athletes, taking into consideration the expected effect of
dehydration, in an attempt not to exclude athletes with normal variations but to protect
athletes from danger. Anyone over that limit would be considered “unfit to race” and could
not compete for 2 weeks, although they were not subjected to official sanctions. To
circumvent this, an athlete could inject rHuEPO every 2 to 3 days over 3 to 4 weeks, along
with some form of iron supplementation, to get a desired effect and then reduce the dose to
match the basal rate of endogenous EPO production to maintain one's hematocrit just below
the “legal limit.” During the 2000 Sydney Olympics, the IOC approved the use of a test
developed by the Australian Institute of Sport to detect rHuEPO users [07010].
Shortly after the discovery of blood circulation by the English physician William Harvey in
1628, the first empiric blood transfusion was attempted. There is therefore a long history of
blood doping, conventionally originating with the anecdote of athletes being encouraged to
drink reindeer blood or something like that to achieve extraordinary performances. Although
the earliest proof of improved sport performances after blood transfusions was provided
already in 1947, the first evidence of blood doping came later, in 1972, when a controlled
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experiment clearly showed a considerable increase in performance of athletes undergoing
autologous transfusion of packed RBCs from an earlier venesection. Since then, there are
consistent records of athletes experimenting with blood transfusions who achieved incredible
success in competitions. Besides the first anecdotal reports, this technique became fairly
popular during the 1980s and was widely used by distance runners, cyclists, and skiers,
particularly during the 1980 and 1984 Olympics. Although no reliable test had been devised
for unequivocal detection, the International Olympic Committee (IOC) officially banned blood
doping after the 1984 Olympics. In the same year, the USA Olympic Committee declared that
seven cyclists, including four medallists, out of 24 athletes of the national team who
participated in the Olympic Games, used transfusions. Years later, following the
implementation of reliable strategies for detecting doping with recombinant erythropoietin and
analogues, blood transfusions, which had fallen out of favor, made a strong comeback. In
March 2002 at the Salt Lake City Olympics, the IOC investigated the discovery of discarded
blood transfusion equipment at the quarters of the Austrian cross-country skiers. Following
DNA testing, two Nordic skiers (who had been placed in the 40s, and not the Austrian team’s
three medallists) were disqualified and had their results cancelled. For the same reason,
some professional cyclists, one of whom nearly died after being injected with poorly stored
blood, were found guilty and suspended in 2004 [06005].
The suspension of several professional road cyclists from the 2006 Tour de France could
represent the tip of the iceberg, with more than 200 athletes in different sports disciplines
implicated in an international doping probe including blood transfusions and exogenous
hormone administration. In an apartment building in Madrid occupied by a doctor, Spanish
police discovered clandestine equipment for international performance enhancement, seizing
more than 200 450 mL blood bags, along with records and several other doping substances,
which allowed investigators to finally match code names of athletes with their highly detailed
doping records. This sophisticated pan-European doping ring either treated athletes locally or
arranged the transport of stored blood through a system of couriers to athletes at race sites.
Hence, based on the riders named in this one investigation, the problem is endemic [06005].
The introduction of recombinant human erythropoietin (rHuEPO) in the early 1990s sparked
a new capacity in performance enhancement primarily for endurance sports such as cycling
and track and field – detection during this time was unlikely. To help athletes avoid detection,
individual physicians in countries such as Italy and Spain ‘masterminded’ drug prescription.
This resulted in high profile cases in the Tour de France and Giro d'Italia such as Marco
Pantani, Tyler Hamilton and, most recently, Lance Armstrong. Arguably, these cases
represent only the “tip of the iceberg” and exposed the insufficiencies of the current
antidoping system. During the 1990s, “blood doping” in the form of rHuEPO use and/or blood
transfusions became widespread. In response, the IFs such as the International Cycling
Union (UCI), FIFA and the IOC introduced the direct urine test and the sampling of blood to
detect blood manipulation as evidenced by direct and indirect parameters. The analysis of
indirect blood parameters conducted during the Tour de France in 1997 revealed significantly
higher levels of haemoglobin, haematocrit and reticulocytes compared to the normal
population, while a similar analysis of blood samples from players participating in the 2002
FIFA World Cup in Korea/Japan was consistent with normative data. In 2004, UCI introduced
a blood test for the detection of homologous blood transfusion to enhance the analysis of
indirect haematological parameters indicating blood manipulation. The two observations from
the UCI and FIFA clearly indicated that the risks of blood doping abuse are different in
individual endurance sports compared with Olympic team sports, for example. Consequently,
appropriate risk management must be based on data from adverse or atypical analytical
findings as well as after considering the estimated risk of doping in different sports. It was
acknowledged that the latter may be difficult to determine accurately [14429].
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During the Olympic Games held at moderate altitude (2250 m at sea level) in Mexico City in
1968, altitude-induced blood adaptations such as an increase in haemoglobin concentration
were considered primarily responsible for athletes living at altitude wining most of the
endurance races and this phenomenon instigated a new research focus on altitude training in
the field of sports physiology. As an example, the pioneering work of Ekblom et al
demonstrated that elevation in haemoglobin concentration increased maximal oxygen uptake
and improved performance. It is notable that the first accounts of blood manipulation in sport
as well as the term ‘blood doping’ emerged soon thereafter in the media. There is evidence
that blood transfusions were being used in the 1970s and 1980s by athletes such as cyclists,
marathoners and cross-country skiers [14430].
Doping in sport seems to adapt rapidly to new available technology. The appearance of
rHumanEPO, which was shown to successfully increase haemoglobin concentration in
patients, radically transformed blood doping practices. Following the “transfusion era”,
rHumanEPO doping took over in the 1990s, as was highlighted by numerous doping cases
during the Tour de France cycle race and especially the Festina affair in 1998. In 2000,
Birkeland et al demonstrated that rHumanEPO administration for 4 weeks in 20 male athletes
increased haematocrit from 43 to 51 percent, VO2max by 7 percent as well as time to
exhaustion by 9 percent. The practice of red cell transfusions has made a strong comeback
in recent years in response to the development of antidoping tests. Based on testimonies
from athletes, it is now known that athletes are using rHumanEPO in combination with blood
transfusions [14430].
In 1990, erythropoietin was included on the list of prohibited substances by the International
Olympic Committee because misuse by athletes was suspected, although no approved test
existed. There were rumours during this time that athletes used large doses of rHumanEPO
to induce very high-blood parameter values. For example, Bjarne Riis, the 1996 Tour de
France winner (who admitted doping in 2007), acquired the nickname of “Mr 60 percent”
which referred to his haematocrit level. Because rHumanEPO was directly undetectable at
the time and as well as to protect the health of athletes, the 50 percent haematocrit rule was
introduced in 1997 by the International Cycling Union. Briefly, any racing cyclist who had a
haematocrit above 50 percent (50 % for male athletes and 47 % for female athletes) was
declared ineligible and was excluded from the race. Despite this rule, the Festina scandal at
the Tour de France in 1998 provided the proof of organised and widespread doping in
professional cycling and highlighted the need for the creation of an independent international
agency, which would set unified standards for antidoping work and coordinate the efforts of
sports organisations and public authorities. The WADA was consequently established in
1999 [14430].
The first alleged use of blood boosting in sport was in the 1960s, when a French four times
winner of the Tour de France (1961-1964) was named as one of the first cyclists to use the
technique. Widespread use among endurance athletes (especially running, cycling, and
cross country skiing) started after the 1968 Olympic Games, in Mexico City which is situated
at an altitude of 2300 m. Here the athletes from higher altitudes performed better in the
endurance events because of various physiological acclimatisation adaptations, including
increased red blood cell (RBC) mass. Blood boosting was the method adopted by many
athletes after Mexico to increase their aerobic performance. It did not come to general public
attention untilmthe early 1970s when it was termed ‘‘blood doping’’ by the media. This
followed a Finnish steeplechaser using the technique before winning two gold medals in
endurance runs at the 1972 Munich Olympics. The technique became more popular during
the 1980s and was used by distance runners (5000 m, 10000 m, marathon runners), cyclists,
and skiers. Specific accusations were made against the Russians, Italians, Finns,
Americans, and East Germans, particularly during the 1980 and 1984 Olympics. Athletes
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who admitted using the technique included the Italian cyclist who beat the one hour world
record in 1984 and a Russian distance runner who specifically admitted to autologous
transfusion with two units by team doctors in 1980. The US Olympic cycling team boost their
RBC mass, and the need for blood boosting diminished. Increased performance after blood
transfusion was first shown as early as 1947, but the first pivotal results came from the
experiments of Ekblom et al on human volunteers in 1972 [04008].
Early testing strategies for blood doping
The IOC's decision to adopt the two EPO tests for the Sydney games was a genuine
milestone in antidoping science. Recombinant EPO, a peptide of 165 amino acids produced
by genetic engineering, is among the world's top selling pharmaceuticals. In approving one of
the EPO tests, the IOC is for the first time requiring athletes to give blood samples for doping
control. Developed by Michael Ashenden and his colleagues at the Australian Institute of
Sport near Canberra, the test measures the EPO concentration in blood as well as four other
factors affected by raised EPO levels. Precursors of red blood cells, known as macrocytes
and reticulocytes, are overproduced in bone marrow when EPO levels are raised, and they
leak out into the circulation. So Ashenden's test measures the levels of red blood cells and
these two precursors. It also measures the serum concentration of a protein called soluble
transferrin receptor, which is involved in iron metabolism – and as such influences the
production of the oxygen-carrying haemoglobin complexes found in red blood cells. The
other test, described earlier in 2000 by Françoise Lasne and Jacques de Ceaurriz of the
French National Anti-Doping Laboratory in Châtenay-Malabry, near Paris, detects directly the
presence of recombinant EPO in urine. The test is based on a subtle difference between
human EPO and that produced in vitro for pharmaceuticals. The recombinant EPO has the
same amino-acid sequence as the natural hormone but, because it is produced from non-
human cells, it has a different number of sugar residues attached to it. As a result, the
electrical charges on the two forms of EPO are different and they can be separated using an
electrophoretic technique called isoelectric focusing. To avoid the possibility of false positive
results in Sydney, sanctions will only be taken against athletes who fail both tests. The blood
changes tested for by Ashenden's method linger for two to three weeks after an athlete stops
taking EPO, but the recombinant EPO itself is flushed out of the body within a few days. After
watching the performance of the EPO tests in Sydney, IOC-accredited labs will decide
whether to include them in their battery of standard tests. In 2000, these cost around 150
euros (USD 131) for competition samples and 100 euros for out-of-competition testing. The
intensity of the war on doping will always depend on money, both for carrying out the tests
and for developing new ones. In 1998, the IOC decided to fast-track the development of the
EPO tests by investing USD1 million. This figure was matched by the Australian government
[00001].
Originally, the only means to test for doping by blood transfusion was the adoption of
arbitrary thresholds for hematocrit and/or hemoglobin. Blood doping practices were
suspected when blood tests showed hemoglobin values exceeding 175 g/L for men and 155
g/L for women (International Ski Federation), and hematocrit values above 0.50 for men and
0.47 for women, with reticulocytes < 2 percent (according to the International Cycling Union).
Athletes with random values exceeding such limits were prevented from racing in official
competitions. Nevertheless, such a questionable strategy involved several drawbacks,
including the difficult interpretation of several hematological parameters because of wide
inter-individual variability, the possible occurrence of false positive results that would have
penalized clean athletes with naturally increased values, and the possibility to arbitrarily
expand or titrate the RBC mass up to the allowable threshold [06005].
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The obvious alternative to homologous blood transfusions is autologous blood transfusions
(ABTs). In 2006, just before the Tour de France, a large Spanish doping scandal evolved,
known as Operación Puerto. A doping ring involving several physicians was uncovered by
the Spanish police, and more than 200 autologous blood units belonging to professional
athletes were found in freezers and refrigerators for subsequent reinfusion. Detailed doping
calendars from individual athletes were published, and the modus operandi of involved
athletes and their physicians was uncovered. From these calendars, it became evident that
besides the massive abuse of a wide range of different performance-enhancing drugs and
masking agents, ABTs were used during important competitions. The procedure of blood
withdrawal and reinfusion was performed numerous times for each individual athlete during
the year by using specialized equipment for phlebotomy and storage. Typically, blood was
withdrawn after competitions and reinfused few days before 1-day races or before and during
multiple-day competitions. Since then, athletes testing positive for other substances have
confirmed the ongoing abuse of ABT today [12009].
Increasing delivery of oxygen to the active muscles and making energy efficiently from the
oxygen is the most effective way to increase performance. Increasing the number of RBCs is
the most effective way to increase aerobic performance. The US pursuit cycling team
unexpectedly won gold at the XXIII Olympic Games in Los Angeles. It was later revealed that
they transfused blood and that some of the cyclists had suffered severe transfusion
reactions. The advent of recombinant protein therapeutics in the late 1980s ushered in a new
era for dopers. The lay press speculated that the deaths of 18 European cyclists were related
to the availability of recombinant human erythropoietin (rhEPO). rhEPO stimulates the
production of red blood cells in the bone marrow, resulting in increased red blood cell mass.
The development of a test for rhEPO caused the athletes to change the route of
administration from subcutaneous to intravenous, decrease the dosage, and increase the
frequency of administration in order to avoid detection. At the Salt Lake City Games in 2002,
three winter endurance athletes had Aranesp (darbepoetin-alpha), a novel erythropoietin
stimulating protein, detected in their urine samples – 7 months after approval in the
European Union and 5 years before the FDA approved its medical use in the United States.
Information gathered from investigations confirms that with the advent of tests for prohibited
peptides and proteins like EPO, some cheating athletes changed to autologous blood
transfusions to increase RBC mass [12006].
History of dectection of recombinant erythropoietin and derivates
Erythropoietin, Epo represents a prototypical success of molecular biology. The presence of

Epo was first suggested in the nineteenth century based on the high blood viscosity of
people living in or returning from high altitude areas. Experimentally, the presence of Epo as
an erythropoietic humoral factor was discovered in the early twentieth century due to the
erythropoietic property of serum from phlebotomized rabbits. Most notably, in 1977, it was.
purified Epo from 2550 liters of urine from patients of aplastic anemia and determined its
amino acid sequence. Based on this finding, the human Epo genes were cloned in 1985.
Then, recombinant human Epo (rHuEpo) was successfully used to treat anemic patients with
end-stage renal diseases (ESRD). Furthermore, studies on Epo gene regulation led to the
identification of hypoxia-inducible transcription factors (HIFs) and hypoxia response elements
(HREs) as the HIF-binding consensus sequence on genome, and to the current
understanding of the molecular mechanisms of cellular adaptation to hypoxia [150005].
The production of red blood cells was linked to blood oxygen pressure more than a century
ago. In 1906 Carnot and DeFlandre described a substance capable of stimulating the
production of red blood cells, later known as erythropoietin (Epo). Recombinant human
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erythropoietin (rHuEpo) was approved for treatment of chronic anemia mainly in patients with
end-stage renal disease in 1989. Soon after in 1990, the American Medical Association and
the International Olympic Committee banned the abuse of rHuEpo in athletes. However,
abuse of rHuEpo and related compounds prevails [150006].
Erythropoietin has been commercially available since 1989 principally for disease states
such as for treating the anaemia seen in chronic renal failure. Athletes were quick to exploit
the drug, especially in professional cycling, where the scandal at the 1998 Tour de France
highlighted this issue when a team employee was caught with a carload of performance
enhancing agents, including EPO [03008].
Direct detection of a forbidden substance in a biological matrix such as urine or blood

obtained from an athlete is the classical forensic approach to prove doping. This approach
has long been the sole strategy, only fine-tuned by improving the sensitivity of the analytical
detection methods and by optimizing the timing of testing. The basic principle of the direct
detection of forbidden substances relies on the fact that these substances are different from
the normal constituents of the human organism. With the introduction of recombinant drugs
such as rhEPO, this principle was not valid anymore, as the recombinant constituent was
virtually identical to the endogenous version of the substance. Thus, at first, doping tests
could not differentiate between the natural, endogenous and the artificial, exogenous
recombinant version of the drug. This has, for a long time, been a major difficulty for the
testing laboratories. In 1995, it was described a method to separate the natural from
exogenous EPO through electrophoresis, but other laboratories could never replicate their
results and the described method never reached the stage of validation. Only in 2000, thus
more than 10 years after the estimated beginning of rhEPO abuse in sports, the first
practicable and validated test to directly detect rhEPO in urine was published. This test relied
on a difference in glycosylation between the endogenous and the exogenous EPO
molecules, which resulted in different migration characteristics during isoelectric focusing
(IEF). The recombinant EPO was industrially harvested from transfected hamster kidney or
ovarian cells and, owing to the difference in cell organelles, a minor posttranslational
difference in glycosylation between the rhEPO (made by the hamster cells) and the
endogenous EPO (made by the human kidney cells) occurred, although the amino acid
sequence is identical. The rhEPO molecules are less negative and will thus move differently
from endogenous EPO in an electric field, which can be demonstrated using the IEF. Further
developing the approach of Wide, IEF is then followed by double blotting, which addresses
the problem of non-specific binding of the EPO molecules. Although relatively cumbersome,
the method soon identified the first athletes testing positive, namely Roland Meier and Bo
Hamburger, both cyclists. Ironically, Hamburger was later acquitted by the court of arbitration
of sports on formal grounds (i.e. a lack of harmonization of the positivity criteria for the EPO
tests between laboratories was identified). This issue has since been addressed and strict
positivity criteria apply, based on acceptance, identification, and stability principles. rhEPO
positive samples, for example, have to show at least three acceptable, consecutive bands in
the basic area and the two most intense bands measured by densitometry must be in the
basic area. When the analysis is performed in blood (serum/plasma) the intensity of those
bands must be approximately twice or more than any band in the endogenous area. Many
laboratories nowadays use computer-based classification algorithms to guarantee objectivity
in this context [13006].
At the 2000 Olympic Games in Sydney, the Australian WADA-certified laboratory first
launched a sophisticated anti-doping test for erythropoietin that required both urine and a
blood sample. Over 300 tests were performed for erythropoietin for the first time in Olympic
history but no positives were reported. This could be due to the fact that the technology for
the test was new and questions still existed about the assay [08006].
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In addition to EPO, there are several EPO analogues that are also effective – for example,
darbypoietin. Fortunately these substances, which have a longer half life, are more easily
detectable, and several athletes were suspended for darbypoietin use at the 2002 Winter
Olympics [03007].
Hypotheses on erythropoietin doping practice

It has been noted that recombinant erythropoietin (rEPO) appeared in Europe in 1987, and
the unusual deaths began soon thereafter. From 1987 to 1991, up to 20 competitive cyclists
died suddenly and unexpectedly. Autopsy results were elusive, but cycling authorities said
the deaths were from ‘‘heart attack,’’ ‘‘cardiac arrest,’’ or ‘‘cardiac failure.’’ However, about 15
of the deaths seemed to fit a profile that suggested another explanation: They were young
and improving fast, ‘‘rising stars’’ who died not during a race but at rest before or after a race.
The rationale for abusing rEPO in competitive cycling is that by raising the hematocrit without
unduly raising blood viscosity, one can enhance aerobic performance by enhancing oxygen
delivery to muscles. By 1990, the first experiment on the effects of rEPO on athletes had
been done, and the lead researcher was quoted to the effect that rEPO might enable an elite
athlete to shave 30 s off a 20-min racing time. The problem is that the higher the hematocrit,
the greater the risk of clots. Blood clots are the proximate cause not only of pulmonary
emboli but also of many strokes and heart attacks. Coagulability hinges partly on blood
viscosity, which is set by the plasma fibrinogen level, the deformability of red cells, and the
hematocrit. Hematocrit also influences platelet adhesion, the first step in arterial thrombosis.
Hematocrit, then, modulates the flow, fluidity, and coagulability of blood. As hematocrit
increases toward 60 percent as in mountaineering, for example, blood clots can become a
menace [13066].
In 1991, a warning appeared in a prestigious medical journal on how fast hematocrit can rise
with large doses of rEPO and how rEPO abuse by athletes could drive hematocrit to
‘‘dangerously high levels.”By 2007, after esteemed studies in patients with renal disease,
cancer, and other major illnesses tied higher dosing of rEPO to greater risk of death from
thrombotic events (heart attack, stroke, or venous thromboembolism) or heart failure, the
Food and Drug Administration issued a black box warning on these risks from rEPO. Even
this did not end the abuse of rEPO by athletes or the deaths. Cyclists continued to dope,
dupe, and die [13066].
Sudden deaths in Swedish orienteers

A spate of deaths in orienteers paralleled the cycling deaths. Suddenly, among young, elite
orienteers (but in no other Swedish sports), the death rate spiked to 1 percent a year for 3
years in a row. From 1989 to 1992, seven elite-level orienteers, all from the same small area
of central Sweden, died during or after competitions or training. They knew one another and
occasionally trained together. All performed very well shortly before they died; some placed
near the top in national competitions. The last, Melker Karlsson, 24, was a rising star who
died after a training run and sauna. His death was the final straw that led to a meeting of
Swedish health experts to probe potential causes and solutions. As in the cyclists, the deaths
were considered ‘‘cardiac,’’ and a popular hypothesis was a transmissible myocarditis,
ascribed first to Chlamydia and then to Bartonella. Their supporting evidence, however, is not
compelling and does not dissuade skeptics, from speculating that a culprit in this spate of
sudden deaths in top Swedish orienteers was abuse of rEPO [13066].
Cyclists
Evidence from many believable anecdotes, from sworn testimony to the United States Anti-
Doping Agency (USADA), and from police raids at the 1998 Tour de France and before the
2006 Tour de France shows that cyclists continue to abuse rEPO. Indeed, top South African
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cyclist David George was caught on rEPO (and admitted it) in August 2012. The culture of
competitive cycling dies hard; despite the deaths and the black box warning, rEPO abuse in
cycling has endured for 25 years. In his affidavit to USADA, Stephen Swart, a teammate of
Lance Armstrong in 1994 to 1995, said that their Motorola Team used rEPO for the 1995
Tour de France, and that most riders, including Lance Armstrong, had a hematocrit over 50
percent. It is widely reported that Marco Pantani abused rEPO and had a hematocrit of 60
percent in a 1995 race. As noted in Tyler Hamilton’s recent tell-all book, Bjarne Riis won the
1996 Tour de France on rEPO, and his peak hematocrit was an astonishing 64 percent. For
cyclists who abuse rEPO, there may be a thin line between winning and dying. Alas, the
deaths continue. From early 2003 to early 2004, eight more European cyclists died, and up
to five fit the profile of a likely rEPO death. Notable were French cyclist Fabrice Salanson, 23,
found dead in his hotel room just hours before he was to start the Tour of Germany, and
Belgian cyclist Johan Sermon, 21, who went to bed early to rest up for a planned 8-h training
ride the next day, but was found by his mother dead in bed at dawn. In early 2009, the
promising Belgian cyclist Frederick Nolf, 21, died at night in his Ritz-Carlton hotel room, after
the fourth stage of the Tour of Qatar. He went to bed laughing and happy and never woke
up. No autopsy was done. How the autopsy results on Salanson were described to the press
may come closest to the truth in this long, sad saga. Dr Jan Dressler of the University of
Dresden Medical Institute said the death was probably caused by the heart enlarging and the
coronary vessels failing to pump enough blood [13066].
Background to the athlete biological passport
In an investigation of samples obtained as part of routine International Ski Federation blood-

testing procedures in participants at the World Ski Championships, abnormal hematological
profiles, defined as those deviating from the 1989 Nordic Ski World Championships and the
IOC Erythropoietin 2000 project data set, were identified in 36 percent of the skiers tested
and finishing within the top 50 places in the competitions. In addition, 50 percent of medal
winners and 33 percent of those finishing from 4th to 10th place had highly abnormal
hematological profiles. In contrast, only 3 percent of skiers finishing from 41st to 50th place
had highly abnormal values. Although these data cannot be immediately associated with
blood doping practices, including blood transfusions, and it is very unlikely that blood doping
would be less common in other endurance sports, the present situation is highly suggestive
of a phenomenon that is not being controlled by the ongoing antidoping testing program. In
fact, it has been hypothesized that a combination of blood transfusion and recombinant
human erythropoietin administration could also be used by such athletes [06005].
History of doping with growth hormone
The increased availability of growth hormone (GH) in the mid-1980s, as a result of advances
in recombinant DNA techniques, has allowed research into the use of this hormone at
physiological dosage, as replacement therapy for adults with GH deficiency (GHD) and at
pharmacological dosages as a possible therapeutic agent, for a number of disease states
[00004].
Growth hormone (GH) was first isolated from the pituitary gland in 1957. By the 1980s, its
anabolic actions had been well described, and GH was established as a drug of abuse. The
performance-enhancing potential of GH for use in sports was first advocated in the
Underground Steroid Handbook in 1983, where it was described as “the most expensive,
most fashionable and least understood of the new athletic drugs.” After Ben Johnson was
stripped his 100-m gold medal from the Seoul Olympic Games, he admitted to having taken
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a cocktail of drugs including GH. A Chinese swimmer, Yuan Yuan, was forced to withdraw
from the 1999 world championship after 13 vials of human GH were discovered in her
suitcase. More recently, during a grand jury testimony, Tim Montgomery (former 100-m world
record holder) admitted receiving an 8-week supply of GH and a steroid compound known as
“the clear” [05001].
One of the more recent drugs to gain popularity among both athletes and nonathletes is
human growth hormone, hGH. Before 1985, patients with growth-hormone deficiency had to
rely on cadaver pituitary extract for their treatment. Supplies were necessarily limited, costs
were high, and use posed the danger of fatal viruses such as Creutzfeldt-Jakob disease.
Fortunately for these patients, recombinant hGH (rhGH) was developed and has been
available in the United States since 1985. This has increased the supply of hGH for GH-
deficient patients, but also increased the possibilities for abuse [14614].
In 1998 at the Tour de France that French customs arrested Willy Voet, a physiotherapist of
the Festina cycling team, for the illegal possession of needles, syringes and over 400 bottles
containing erythropoietin, human growth hormones, steroids, amphetamines, narcotics and
stimulants [08006].
Growth hormone was added to the prohibited list in 1989 [01002].
Ben Johnson also admitted to using human growth hormone along with steroids during
investigations after his disqualification in Seoul. The abuse of growth hormone in sports
seems to be escalating, with large caches of needles and vials of hGH being confiscated at
sporting events worldwide. Six months prior to the 2000 Olympic Games, a pharmacy in
Sydney was broken into and 1,575 multiple dose vials of growth hormone were taken while
nothing else was touched. Also, on their way to Australia, the Chinese swimming team was
detained, as needles, syringes, and vials of human growth hormone were found by customs
officials in their baggage [08006].
hGH is a naturally occurring hormone produced by the anterior pituitary gland and is one of
the major hormones influencing growth and development. Harvey Cushing discovered the
hormone in 1912 and isolated it from human and monkey cadaver brains in 1956. Two years
later it was used to treat dwarfism in children by injection. The unfortunate development of
Creutzfeldt-Jakob disease, a degenerative brain disorder, in boys who were treated with
cadaver growth hormone led to the discontinuation of all products derived from the human
pituitary gland. Because of this ban, the abuse of hGH was rare in sport until the middle to
the end of the 1980s. In 1985 Genentech received approval from the US FDA to market
Protropin® for children with growth hormone deficiency. This was the first recombinant DNA
form of growth hormone (rhGH) that was safer than cadaver extracts used in the past.
Recombinant DNA technology made the production of pharmaceutical grade growth
hormone easier and cheaper. Most human growth hormone used in medicine and diverted to
sports doping is now obtained by recombinant technology, and is simply referred to as hGH
(but it may also appear as rhGH or HGH) [08006].
Growth hormone (GH) is an important and powerful metabolic hormone that is secreted in a
pulsatile pattern from cells in the anterior pituitary, influenced by several normal and
pathophysiological conditions. Human GH was first isolated in the 1950s and human derived
cadaveric GH was initially used to treat patients with GH deficiency. However, synthetic
recombinant GH has been widely available since the mid-1980s and the advent of this
recombinant GH boosted the abuse of GH as a doping agent. Doping with GH is a well-
known problem among elite athletes and among people training at gyms, but is forbidden for
both medical and ethical reasons. It is mainly the anabolic and, to some extent, the lipolytic
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effects of GH that is valued by its users. Even though GH's rumour as an effective ergogenic
drug among athletes, the effectiveness of GH as a single doping agent has been questioned
during the last few years. There is a lack of scientific evidence that GH in supraphysiological
doses has additional effects on muscle exercise performance other than those obtained from
optimised training and diet itself. However, there might be synergistic effects if GH is
combined with, for example, anabolic steroids, and GH seems to have positive effect on
collagen synthesis. Regardless of whether or not GH doping is effective, there is a need for a
reliable test method to detect GH doping. Several issues have made the development of a
method for detecting GH doping complicated but a method has been presented and used in
the Olympics in Athens and Turin. A problem with the method used, is the short time span
(24-36 hours) from the last GH administration during which the test effectively can reveal
doping. Therefore, out-of-competition testing will be crucial [08238].
Today, many medical interventions that begin as treatments for disease often expand into
therapies that reduce disability, lessen disadvantage, or even confer advantage. Forces that
propel profitable drugs, devices, and procedures dominate over considerations of efficient
and equitable distribution of resources. This dominance is fueled by industry-physician
collaborations often biased by prior assumptions, reliant on surrogate outcomes, and
advantageous to marketing. Interventions are justified by "medicalization" of physiologic
variations (e.g. short stature) as defects or disease, and nudged into "standard practice" by
key opinion leaders. The story of recombinant human growth hormone (hGH) treatment of
short stature is one vivid example, but others (e.g. expansion of drug treatment to "optimize"
cholesterol profiles, bone health, psychological well-being) can be found throughout
medicine. In the new obesity era, lessons learned from the hGH era will be needed to keep
the field of pediatric endocrinology empowered to make the key clinical decisions, and free of
unintended consequences for patients and runaway health care inflation for society [11469].
After testing over 1,000 samples, the first adverse analytical finding of growth hormone came
in February 2010 when the British Rugby League player, Terry Newton, tested positive.
Since then, several other positive tests have been reported including the announcement in
September 2010 from the Canadian Center for Ethics in Sport that Matt Socholotiuk, a
University of Waterloo football player, had tested positive for GH use on 31 March 2010. The
following year, Colorado Sky Sox first baseman Mike Jacobs became the first baseball player
to test positive for GH and was subsequently suspended for 50 games by Minor League
Baseball. In 2011, Andrus Veerpalu, an Estonian Olympic gold medal winning skier, tested
positive for GH. However, he pleaded his innocence and challenged the laboratory finding in
the Court of Arbitration for Sport who subsequently acquitted Veerpalu on 25 March 2013 as
the court was not convinced that the threshold for considering an adverse analytical finding
was sufficiently reliable to uphold the doping conviction; nevertheless, the court stated “that
there are many factors in this case which tend to indicate that the Athlete did in fact himself
administer exogenous hGH” [13007].
Testing for growth hormone
In the mid-1990s, the IOC and the European Commission co-funded a three-year
international project called GH2000 to develop tests to detect this substance. The project,
which concluded at the end of 1998, was led by Peter Sonksen, an endocrinologist at St
Thomas' Hospital in London. The GH2000 consortium delivered its report to the IOC in
January 1999. It had developed a series of blood markers that could be used to test for
elevated hGH levels, including insulin-like growth factors and proteins that bind to them.
Working independently of GH2000, researchers led by Christian Strasburger at the Ludwig-
Maximilian University in Munich have developed a direct test for recombinant hGH. This
relies on the fact that hGH exists in different molecular forms, the two major fractions of
251
which have molecular masses of 22 kilodaltons and 20 kDa. Although only half of the body's
own hGH is in the heavier form, for recombinant hGH the figure is 95 percent. The test uses
antibodies to identify the two forms, and so allows any shift in the natural ratio to be spotted.
Sonksen costed validation studies for the GH2000 and Munich tests at around US$5 million,
and requested continued funding. The consortium also responded to a formal call for
proposals for research issued by the IOC in August 1999, but was turned down. The German
Sports Research Institute in Cologne then supported further work to validate the Munich test,
drawing on some of the GH2000 samples. Strasburger hopes that the then newly created
World Anti-Doping Agency (WADA), based in Lausanne, would follow through on statements
that tackling hGH abuse will be its top priority, and provide the money needed to bring the
test into general use [00001].
History of human chorion gonadotropin
hCG was first prohibited in sport during the 1980s. The WADA statistics 2012 reports 93
adverse analytical findings for hCG, however it is not known how many of those are due to
doping or as a result of disease [14014].
History of doping with insulin
It was at the Winter Olympic Games in Nagano in 1998 when a Russian medical officer
enquired as to whether the use of insulin was restricted to insulin-dependent diabetes. This
drew attention to its role as a potential performance-enhancing drug and the IOC were swift
to act and immediately placed it on its list of banned substances [01002].
Insulin physiology
Sir Edward Schafer was Professor of Physiology in Edinburgh when he published in 1916 a
wonderful book called The Endocrine Organs. The book is based on a series of lectures he
delivered at Stanford University in California in 1913. As well as containing a wealth of
interesting insights into the early days of endocrinology, this book is most notable for the fact
that it was the first time that the then hypothetical hormone insulin was named (8 years
before it was discovered). What is even more remarkable, he predicted the formation of
insulin from “pro-insulin” 54 years before it was actually discovered! Schafer was a
contemporary of Baylis and Starling – two eminent academic rivals from University College in
London. Shortly before Schafer delivered his lectures to his American audiences, Baylis and
Starling had isolated, characterised and published about Secretin, the first “hormone” (a term
coined by them to describe a substance produced in one part of the body, carried by the
blood stream and acting elsewhere in the body) to be isolated. Schafer questioned the use of
the word “hormone” and proposed two alternative names:
Autacoids – excitatory substances

Chalones – inhibitory substances
He went on to describe how his new hypothetical hormone ‘insuline’ exhibited properties that
resembled both autacoids and chalones and that the chalonic or “inhibitory” actions were
physiologically the most important. It was, he proposed, lack of this chalonic (inhibitory)
action of insulin that led to a failure to store glucose in the liver with the net result that the
liver overproduced glucose and glucose accumulated in the circulation, and this led to the
hyperglycaemia that is characteristic of diabetes. This was indeed advanced thinking. The
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“black ages” of endocrinology followed early in vitro experiments in the 1950s that showed
insulin to be capable of stimulating glucose uptake into bits of rat muscle and fat. Before long
the biochemists had extrapolated from these experiments to conclude (wrongly) that the
hyperglycaemia of diabetes was due to a “damming back” of glucose in the blood stream as
a result of a failure of glucose to enter cells as a direct consequence of insulin deficiency
[01003].
History of doping with caffeine
Caffeine is a stimulant that is not currently banned by WADA, despite its proven ergogenicity.
In the past it was included on the banned list at urine concentrations above (12 microg/mL),
on the basis that concentrations below this level may be attained from the consumption of
coffee, coca cola and similar sources, whereas above this concentration indicated a
deliberate consumption, probably via tablets, with the intent of performance enhancement. It
was removed from the banned list in 2004 but is still subject to monitoring, although it should
be noted that the ergogenic benefits for a range of sports appear to be attained at modest
doses (3 mg/kg) doses that are easily achieved via intake of everyday dietary sources such
as coffee, cola drinks and energy drinks [150003].
From 1962 to 1972 and again from 1984 to 2003 caffeine was on the WADA banned list, with
a concentration >12 microg/ml in the urine considered as doping. Caffeine has been
demonstrated to be ergogenic at doses lower than those doses that result in a urine
concentration of 12 microg/ml, and higher doses appear to exhibit no additional performance-
enhancing effect. During the second banned period, many athletes tested positive for
caffeine. The sanctions ranged from warnings up to 2 year suspensions (maximum penalty,
usually only 2-6 months). Since 2004, caffeine has been removed from the prohibited list,
however, it is still part of WADAs monitoring program (stimulants but in competition only) in
order to monitor the possible potential of misuse in sport. According to WADA, one of the
reasons caffeine was removed from the Prohibited List was that many experts believe it to be
ubiquitous in beverages and food and that having a threshold might lead to athletes being
sanctioned for social or dietary consumption of caffeine. Furthermore, caffeine is metabolized
at very different rates in individuals and hence urinary concentrations can vary considerably
and do not always correlate to the dose ingested. In addition, caffeine is added to a wide
range of popular food products such as coffee, tea, energy drinks and bars, and chocolate
[13008].
Caffeine was removed from the World Anti-Doping Agency list of restricted or banned
substances in 2004 [14717].
History of doping with ephedrine
Ephedra is a Chinese shrub which has been used in China for medicinal purposes for
several thousand years. The pure alkaloid ephedrine was first isolated and characterised by
Nagai in 1885. It was then forgotten until it was rediscovered by Chen and Schmidt in the
early 1920s. Its actions on the adrenoceptors could be classified into separate alpha and
beta effects – a defining moment in the history of autonomic pharmacology. Ephedrine
became a highly popular and effective treatment for asthma, particularly because, unlike
adrenaline (until then the standard therapy), it can be given by mouth. Ephedrine as a
treatment for asthma reached its zenith in the late 1950s, since when there has been a
gradual and inevitable decline in its therapeutic use. From mainstream medicine, ephedrine
253
moved into the twilight zone of street drugs and nutritional supplements. Ephedra and
ephedrine products are now banned in many countries, as they are a major source for the
production of the addictive compound methamphetamine (crystal meth) [11001].
Ephedrine is not only efficacious in the treatment of numerous ailments, but also has a long
history of misuse. Research was needed to examine ephedrine policy over time in order to
determine potential regulatory flaws that allowed misuse to continue. One review is based on
primary literature derived from systematic searches of historical and scientific archives, as
well as grey literature. Ephedrine managed to pass through numerous regulatory loopholes
within seventy years. Despite warnings of misuse over the latter half of the century,
ephedrine, and its herbal source, ephedra, were regulated in a piecemeal fashion and
remained easily available to the public. Health authorities have struggled to control
ephedrine, as an amphetamine "look-alike," as a methamphetamine precursor, as a dietary
supplement, and as a medication. Despite being a potentially dangerous stimulant, under-
regulation was perhaps more problematic than the substance itself. Tighter control of all
ephedrine products, drugs and dietary supplements alike, might have prevented adverse
outcomes and allowed this substance to remain available in a safer manner. Stringent
regulation of all ephedrine products is necessary to prevent misuse and to protect the
public's health [11002].
Mahuang or ephedra, which companies promoted as a legal alternative to ecstasy, although

a natural product, contains the chemical ephedrine, which stimulates the nervous system and
constricts blood vessels. FDA banned ephedra in 2004, after a 23-year-old Major League
Baseball pitcher collapsed and died during practice and was found to be taking the herb. By
early 1996, it had been linked to at least 15 deaths. Meanwhile, FDA was regularly issuing
warnings about liver, kidney, and other health risks tied to supplements [150003].
History of doping with beta2-agonists
Due to fear of possible doping effects of beta2-agonists, including both improved

performance and possible anabolic effect upon muscle, the Medical Commission of the
International Olympic Committee already in 1993 put certain restrictions upon the use of
inhaled beta2-agonists, allowing only the two short-acting inhaled beta2-agonists salbutamol
and terbutaline for use in sports. However, inhaled salmeterol was allowed by IOC to treat
and prevent exercise induced asthma in relationship to sports from 1 February 1996 [01004].
History of doping with morphine
A quantitative analysis of morphine and codeine in human urine was performed after oral
intake of cakes containing commercially available poppy seeds in order to estimate the
possibility of positive doping results. Therefore, eight products from different manufacturers
(poppy seeds or baking mixtures) and origin were obtained and analyzed by gas
chromatography-mass spectrometry for the presence of the alkaloids. One selected batch of
poppy seeds was used as an ingredient in a typical cake and was the object of an excretion
study with nine volunteers. After application, several urine specimens contained morphine
with concentrations higher than 1 microg/mL, and peak values of approximately 10.0
microg/mL were detected. Because the International Olympic Committee set a cutoff limit for
morphine at 1 microg/mL, high-performance athletes could possibly test positive in doping
control after consumption of products containing poppy seeds [03009].
254
History of doping with cannabis
The medical properties of cannabis have been known for many centuries. The first
documented use of cannabinoids for medical purposes dates back to 2800 BC in the
Chinese herbarium Pen-ts’ao, a herbal pharmacopoeia describing many drugs among which
cannabis, which was referred to as ”ma”, meaning ‘”chaotic”. Pen-ts’ao described the pain-
relieving, stupefying and hallucinogenic properties of cannabis and recommended cannabis
for constipation, malaria, gout, rheumatism, and menstrual anomalies. Cannabis therapeutic
use was introduced in Western medicine during the first half of the nineteenth century by the
Irish physician William Brooke O’Shaughnessy (1809–1889), who studied forensic toxicology
and chemistry at the University of Edinburgh in Scotland. He conducted a number of
experiments in animals and proved that cannabis was safe even at high doses; thus, he
extended the use of cannabis to patients suffering from rheumatism, seizures, and tetanus
[12006].
The International Olympic Committee included cannabis in the banned substance list
beginning in 1989 and since 2004 the World Anti-Doping Agency has prohibited its use for all
sports competition. Cannabinoids are substances prohibited in-competition only [0009].
The class of cannabinoids has been subject of much debate concerning its relevancy for
sports drug testing, fuelled by the increase of the urinary threshold for the main cannabis
metabolite 11-nor-delta9- tetrahydrocannabinol-carboxylic acid (THCCOOH) from 15 ng/mL
to 150 ng/mL (being effective since 11 May 2013) while the MRPL for cannabimimetics
remained at 1 ng/mL as well as prevalence studies demonstrating the widespread availability
and misuse of cannabis and its synthetic analogs. Since the raise of the urinary threshold for
THCCOOH came unexpected, studies from early 2013 concerning improved/accelerated
quantification approaches have become obsolete, even though the principle is certainly still
valid 130009].
History of doping with xenon
On September 1st 2014, a modified Prohibited List as established by the World Anti-Doping
Agency (WADA) became effective featuring xenon as a banned substance categorized as
hypoxia-inducible factor (HIF) activator [150007].
History of doping with alcohol
Alcohol is prohibited in-competition only and it is prohibited in the following sports:

aeronautic, archery, automobile, karate, motorcycling and powerboating. Until 2010, modern
pentathlon was also included in this list. The limit (blood tests) eligible for a doping violation
is 0.10 g/L [13008].
History of doping with gamma-Hydroxybutyric acid (GHB)
GHB was first synthesized in 1960 as an alternative anesthetic to aid in surgery because of
its ability to induce sleep and reversible coma. However, it had little analgesic effect, and
onset of coma was often associated with seizure activity including tonic-clonic jerking
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movements of the limbs or face. In the late 1980s, GHB was marketed and sold in the health
food industry as a “growth hormone stimulator” to help bodybuilders promote muscle mass
and maintain weight, and as an over-the-counter sedative agent. In 1991, the drug was
banned by the FDA after several reports of adverse reactions in individuals using nutritional
and weight loss supplements containing GHB. Despite the FDA ban, GHB continues to be
manufactured and sold clandestinely [00006].
Occurring naturally in many parts of our body from the brain to heart, to most muscles,
kidneys and brown fat, gamma-hydroxybutyric acid or GHB for short was first synthesized in
1960s by Laborit in an attempt to study the effects of GHB and GABA, producing a
compound that would interfere with beta oxidation and cross blood–brain barrier. It was later
discovered that GHB was an endogenous compound and an endogenous metabolite of
GABA. GHB was thus discovered in search for therapeutic GABA analogs. Since its
discovery, GHB has played many roles in the laboratory. It was used to create an absence
seizure model. GHB was also shown to have tissue-protective effects in the setting of
myocardial infarction, stroke, sepsis, small bowel ischemia, hypovolemic shock, ionizing
radiation and oxygen free radicals. Despite promising beneficial effects, GHB has not found
widespread clinical use. In the 1960, GHB was used as a general anesthetic agent but fell
out of favor due to an association with abnormal electroencephalographic (EEG) patterns in
animals. In the year 1980, GHB could be bought in health food stores and the use began to
rise amongst body builders as it was believed that taking this drug could improve muscle
mass or improve exercise performance. While GHB has been present in laboratories and
therapeutic trials for years, it has recently become a public health issue as a drug of abuse.
Hence in the year 1990, FDA imposed ban over the counter sale of the drug throughout the
United States. Simultaneously from 1997 to 1999, several states and countries passed laws
to control the sale and consumption of GHB and finally designated it as a Schedule 1
substance in the United States in the year 2000 [13010].
History of doping with ecstacy
The methylenedioxy-derivatives of amphetamine and methamphetamine represent the

largest group of designer drugs. The most frequently used compounds are 3,4-
methylenedioxy-methamphetamine (MDMA, ecstasy) and 3,4-methylenedioxy-amphetamine
(MDA), first synthesised in 1910 (MDA) and 1914 (MDMA), respectively, to be used as an
appetite suppressant. At the end of the 1960s, non-medical (recreational) use appeared in
the USA, and in the middle of the 1980s in Europe. In Norway, MDMA and related
compounds have been detected in forensic samples since the early 1990s. In order to
bypass the legal regulations and to produce more potent substances, a number of related
compounds have been synthesised, including derivatives with one or more substituents
(methoxy, methyl, halogen or sulphur), attached at different positions to the phenylring of
amphetamine or methamphetamine. A report from 1998 shows that 0.5-3 percent of the adult
European population, mainly young people, has used ecstasy [00005].
History of biomarker approach regarding doping
In the late 1990s, as a first step, “no start” rules were introduced with the official objective to
protect the health of the athletes when certain blood markers exceeded definite limits (e.g.
hematocrit (Hct) above 50 percent or hemoglobin (Hb) above 17 g/dL (International Cycling
Union, UCI) or Hb above 17.5 g/dL in men and 16.0 g/dL in women (International Ski
Federation, FIS). In this time, the widespread use of rhEPO can be assumed on the basis of
256
indirect evidence; e.g. in elite cross-country skiers extreme Hb values up to 20 g/dL were
common between 1994 to 1996 but disappeared after the “no start” rule was introduced in
1997. Yet, mean Hb values continued to rise, suggesting the further use of artificial methods
with fewer extremes. It became obvious that the use of upper limits of definite blood values
may result in athletes who would titrate rhEPO to approach the target Hb or Hct without
exceeding it [13006].
Plasma volume fluctuations resulting from changes in posture, exercise, and training, altitude
exposure, season as well as storage conditions influence concentration-based blood values
such as Hct and Hb and thus represent a major limitation of their use with absolute limits.
Additionally, cheating athletes may manipulate abnormally elevated Hb and Hct values by
intravenous infusions of normal saline leading to hemodilution. On the other hand, even
clean athletes may be declared unfit as Hb and Hct in a normal distribution may exceed the
given limits. The panel of indirect markers was extended and more evidence was gathered
on the effect on blood values of rhEPO administration in training athletes. It was suggested
the use of a combination of indirect markers of altered erythropoiesis (reticulocyte Hct, serum
EPO, soluble transferring receptor, Hct, %macrocytes) in a multivariate statistical model for
detection of rhEPO during a possible administration phase (ON models) and after recent
cessation of rhEPO use (OFF models). The sensitivity of these models was improved with
larger numbers of subjects and resulted in the introduction of the so-called second-
generation blood tests of which the OFF-hr model, a score combining Hb and %retics, is part
of the current ABP according to the WADA ABP operating guidelines. Although OFF-hr was
originally described for the detection of rhEPO use, it is also sensitive to other forms of blood
doping such as blood transfusion [13006].
The application of these models by sports authorities and anti-doping organizations was
problematic despite their scientific impact. The OFF-hr model was used by certain sports
federations as another ‘no start’ criterion. Yet, infringements of the “no start” rule were equal
to failing a “health test” but not considered a violation against WADA’s anti-doping code and
therefore only yielded short mandatory interruptions of competition, e.g. 2 weeks. As even
these improved biomarkers were only compared with a population-based reference range in
a cross-sectional setting (e.g. universal limit of Hct above 50 %, OFF score greater than
122), it already seemed likely in 2000 that a longitudinal, individual hematologic profile, the
so-called hematologic passport, could be advantageous to prevent and perhaps detect blood
doping. Various attempts were made to define the natural within-subject and between-
subject as well as analytical variability to use longitudinal measurements as an instrument
against blood doping [13006].
History of attempts to dope with nutritional supplements
The modern supplement era began in 1994, when Congress passed the Dietary Supplement
and Health Education Act, or DSHEA (pronounced duh-shay-uh). In the decades before, the
supplements industry was overwhelmingly focused on vitamins and minerals. Much of the
regulation centered on recommended daily allowances of products like vitamin C, iron, or
calcium. DSHEA established the first broad framework for regulating supplements. It also
gave supplements a legal definition: as substances intended to “supplement the diet,”
containing “dietary ingredients” such as herbs, botanicals, or vitamins. At the same time, the
law sharply curtailed FDA's power. Companies were not required to notify FDA provided the
dietary ingredient had a history of use before the law was passed. For the first time, DSHEA
allowed them to make claims on the label suggesting supplements affected the structure or
function of the body – for example, by boosting the immune system or protecting prostate
257
health. And DSHEA codified a loose arrangement: Under the law, as FDA notes on its
website, “unlike drug products that must be proven safe and effective for their intended use
before marketing, there are no provisions in the law for FDA to “approve” dietary
supplements … before they reach the consumer.” The agency can act only after a
supplement is on the market and evidence shows it's unsafe. Since 1994, the number of
dietary supplements marketed in the United States has swelled from about 4000 to more
than 75,000. About USD 36 billion worth were sold last year [150008].
In the mid and late 1990s, nutritional supplements gained immense popularity among
professional as well as also recreational athletes, and an alarming number of positive doping
cases for drugs such as nandrolone were reported in different sports including football.
Athletes attributed their positive steroid cases to the intake of nutritional supplements
contaminated with nandrolone or other anabolic steroids. A careful examination of more than
600 nutritional supplements by the WADA accredited laboratory in Cologne supported this
claim as 15 percent of the samples analysed contained anabolic androgenic steroids not
reported on the label. Of great concern was the finding that the majority of contaminated
nutritional supplements were freely available from fitness clubs, health-food stores and the
Internet. Following the publication of these results, the IFs, led by the IOC and FIFA,
launched an educational campaign warning athletes to avoid nutritional supplements that
were not approved by relevant national regulatory bodies. This message was reinforced by a
consensus statement that reaffirmed the view that there was no evidence of ergogenic
effects of dietary supplements (i.e. a positive effect on health or performance) and strongly
discouraged the indiscriminate use of any nutritional supplements. It was recommended that
nutritional supplements should only be taken if advised by qualified sports nutrition
professionals [14430].
History of caffeine-containing nutritional supplements
Tolerability concerns related to caffeine-containing dietary supplements can be traced to

caffeine’s ability to augment the various pharmacologic effects of sympathomimetic amines.
This effect first caught the medical community’s attention in the late 1970s, when synthetic
combinations of caffeine, phenylpropanolamine, and ephedrine – known as amphetamine
look-alikes – gained notoriety as over-the-counter appetite suppressants and legal speed.
Shortly after their appearance, amphetamine look-alikes were linked to an upsurge in serious
adverse drug events, namely, myocardial infarctions, strokes, seizures, and psychoses, most
of which occurred in young adults. In 1982, the FDA deemed look-alikes as unapproved new
drugs that presented a potential health hazard. By 1988, the FDA disallowed the marketing
of look-alikes as nonprescription medications. Outside the United States, however, some
countries allowed combinations of purified caffeine and ephedrine to be sold as prescription
weight loss aids as late as 2002 [14615].
Beginning with Red Bull® in the early 1990s, energy drinks and shots have become the
fastest growing dietary supplement category on the market in terms of number, variety of
products, and sales revenue. The boom in energy drink use, especially among adolescents
and young adults, has also sparked an upsurge in caffeine-related adverse events among
this population. Energy drinks and energy shots differ from conventional caffeinated soft
drinks in that they are not as highly carbonated (making them easier to consume quickly),
have higher caffeine content, and often contain vitamins, amino acids, l-carnitine, taurine,
glucuronolactone, and botanical extracts, such as guarana, ginseng, Ginkgo biloba, and milk
thistle, to name a few. Like Ephedra-free dietary supplements, ambiguity of energy drink
label claims for caffeine content may contribute to the purported health risks linked to these
beverages. For a while certain energy drinks were formulated with alcohol, but tolerability
258
concerns regarding caffeine and alcohol combinations prompted the FDA to preclude sale of
these products in 2011. Nevertheless, the combination of energy drinks and alcoholic
beverages remains a cause for alarm for both health care professionals and law enforcement
officials [14615].
History of doping with creatine
A French scientist named Chevreul is credited with first discovering creatine (Cr) in 1832,
however, it was not until 1926 that the scientists Chanutin quantified Cr storage and retention
in the body. The first reports that phospocreatine content in human muscle can increase up
to 50 percent following daily creatine supplement (5 g Cr monohydrate 4-6 × day for ≥ 2
days) was written by Harris et al in 1992 [03010].
History of carnosine
In describing carnosine among the constituents of muscle tissue in 1900, V. Gulevitsch

opened the question of its real biological role. Investigation of carnosine-related phenomena
occurred simultaneously with the study of its metabolic transformation within the cell. It has
now been demonstrated that carnosine has the ability to protect cells against oxidative stress
as well as to increase their resistance toward functional exhaustion and accumulation of
senile features [00312].
History of gene doping
In 1997, Leiden et al used an adenovirus to deliver the EPO gene in mice and monkeys. This
boosted the haematocrit from 49 to 81 percent in the mice and from 40 to 70 percent in the
monkeys. The effects lasted for over a year in the mice and for approximately 12 weeks in
the monkeys [03011].
The International Olympic Committee (IOC) in 2002 released its new list of banned
substances and methods. The list was effective from 1 January 2003 and replaces the 1
September 2001 list. Amongst the important changes, the category of genetic doping as a
banned method is listed for the first time. At the 1964 Winter Olympics in Innsbruck, a
Finnish competitor Eero Mäntyranta, won two gold medals in cross country skiing. Though
his training programme wasn’t radically different from his rivals, Mäntyranta had a distinct
advantage. He was born with a genetic mutation that increased the oxygen carrying capacity
his red blood cells by 25-50 percent. Mäntyranta had a mutation in the gene coding for the
erythropoeitin (EPO) receptor which prevented the normal feedback control of red blood cell
mass [03008].
Gene therapist Ted Friedmann and multiple Olympic gold medallist Johann-Olav Koss were
the first to describe the possibility of misusing the techniques and experiences of gene
therapy in the athletic arena. In 2006, before the Turin Winter Olympic games, the president
of the World Anti-Doping Agency (WADA), Dick Pound, called gene doping “the new threat
that is now a reality.” Although Pound did not expect gene doping to pose a problem in Turin,
he indicated that it could be a problem at the Summer Games, 2 years hence in Beijing. In
fact, the problem did not materialize in China, in 2008, nor at the London 2012 Olympics, as
far as the then available detection measures could determine [13011].
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History of athlete biological passport
To detect cellular components of blood and those pharmacological agents that are too large
to be excreted in urine, blood collection and analysis was begun in 2008. The Hematological
Module of the WADA Athlete Biological Passport uses the predictive model of Pottgiesser et
al to monitor hematological marker changes within an individual. The fact that intraindividual
variations in a number of blood (and urine) parameters are lower than interindividual
variations has been used in the clinical chemistry laboratory since the 1970s. Blood is also
analyzed for recombinant proteins, such as GH variants and biomarkers [12006].
Serial analysis of biomarkers was already in practice by a number of federations with some
programmes predating the formation of the World Anti-Doping Agency (WADA) and the
implementation of the World Anti-Doping Code. However after the 2006 Torino Winter
Olympic Games, at the request of a number of International Federations (IFs), WADA formed
an ad hoc Haematological Working Group to look at the issue of blood doping and to develop
a harmonised longitudinal profiling programme that was both scientifically and legally robust.
This resulted in the creation of the ABP Guidelines and Related Technical Documents which
were first published in 2009. In 2011, WADA re-established a Haematological Expert Group
to further refine and develop this module [14015].
In 1994, it was shown that between-subject variation can be removed in doping tests by
using a series of measurements obtained from the same individual. Six years later the
concept of the Athlete Biological Passport (ABP) was proposed. The ABP uses a longitudinal
approach where an individual’s previous results are logged and compared to the new results.
In 2006, Sottas et al proposed to use and combine all data contained in a single blood profile
of an athlete. The hematological module of the ABP, used to detect blood doping, has
successfully been in use since 2009, while the module for steroid doping is currently being
finalized for implementation [14432].
The athlete biological passport (ABP) was recently implemented in anti-doping work and is
based on the individual and longitudinal monitoring of haematological or urine markers.
These may be influenced by illicit procedures performed by some athletes with the intent to
improve exercise performance. Hence the ABP is a valuable tool in the fight against doping.
Actually, the passport has been defined as an individual and longitudinal observation of
markers. These markers need to belong to the biological cascade influenced by the
application of forbidden hormones or more generally, affected by biological manipulations
which can improve the performance of the athlete. So far, the haematological and steroid
profile modules of the ABP have been implemented in major sport organisations, and a
further module is under development. The individual and longitudinal monitoring of some
blood and urine markers are of interest, because the intraindividual variability is lower than
the corresponding interindividual variability. Among the key prerequisites for the
implementation of the ABP is its prospect to resist to the legal and scientific challenges. The
ABP should be implemented in the most transparent way and with the necessary
independence between planning, interpretation and result management of the passport. To
ensure this, the Athlete Passport Management Unit (APMU) was developed and the WADA
implemented different technical documents associated to the passport. This was carried out
to ensure the correct implementation of a profile which can also stand the challenge of any
scientific or legal criticism. This goal can be reached only by following strictly important steps
in the chain of production of the results and in the management of the interpretation of the
passport. Various technical documents have been then associated to the guidelines which
correspond to the requirements for passport operation. The ABP has been completed very
recently by the steroid profile module. As for the haematological module, individual and
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longitudinal monitoring have been applied and the interpretation cascade is also managed by
a specific APMU in a similar way as applied in the haematological module. Thus, after
exclusion of any possible pathology, specific variation from the individual norms will be then
considered as a potential misuse of hormones or other modulators to enhance performance
[14433].
For many years, the concept of individual monitoring of biological markers had been studied
to detect the potential abuse of doping substances. Manfred Donike, head of the Cologne
anti-doping laboratory, worked with his group since the beginning of the 80s on the effect of
steroid abuse. In 1989, the Cologne group described the long-term influence of anabolic
misuse on the steroid profile. The idea of individual and longitudinal follow-up in the field of
the fight against doping was then born naturally. The same group made this statement even
clearer by observing that for an individual, the homeostasis of biosynthesis and metabolism
of endogenous steroids was not disturbed by physical workload but of course was influenced
by the use of testosterone or other similar substances [14433].
Regarding blood doping also, the idea of individual and longitudinal monitoring was
established quite soon at the time when recombinant human erythropoietin (rh-EPO) was
introduced in the market. In 1989, blood tests were performed at the World Cross Country ski
championships in Lahti, Finland, in order to constitute a database and to show possible
abnormal individual variation in blood markers due to rh-EPO doping or to blood transfusion.
As the abuse of rh-EPO by endurance athletes increased dramatically in the 90s, several
proposals of indirect detection by markers were suggested. The percentage of circulating
MacroHypo red blood cells (RBCs) as well as the transferrin receptors was proposed. Then,
the percentage of reticulocytes (RETs) was shown to be drastically increased a few days
after the beginning of a treatment showing the potential of the red cell line to be used as a
diagnostic tool to detect blood doping manipulation in the athlete population. All these studies
were carried out at the time when blood doping (including rh-EPO and transfusion) was
certainly the most severe in endurance sports and disciplines. In 1996 and 1997, two major
international sports federations decided to limit blood doping in their population of interest. At
that time International Skiing Federation (FIS) and International Cycling Union (UCI) decided
to introduce a competition rule based on a population-based upper limit of 18 g/dL
haemoglobin (HGB) for FIS and 50 percent haematocrit (HCT) for UCI. This “no-start” rule
was introduced with the double aim to preserve the health of the athlete and also to protect
fairness in the competition, but it became rapidly obvious that a population-based cut-off was
not appropriate to maintain the competition fair due to the large interindividual variability
distribution in a population of several blood markers [14433].
Large database of blood values was then accessible to the main endurance federations and
the concept of individual follow-up slowly entered into the culture. The term of passport was
first introduced by Cazzola and later Malcovati who studied the feasibility of the
haematological passport for athletes competing in endurance sports. This group, as other
authors previously, made at that time a clear statement that the definition of arbitrary limit in
critical haematological markers to evaluate the eligibility to compete was neither a very
specific nor a very sensitive strategy. In fact, the adoption of this kind of limit was risky by
creating false positive cases (e.g. naturally elevated HGB) and many potential false negative
cases with athletes using plasma volume expanders [14433].
Longitudinal monitoring of athlete's hematologic parameters holds considerable promise as a

strategy to detect and thereby deter illicit blood doping. Two cornerstones of this approach
will be Hb concentration and reticulocyte counts, which can both be measured on portable
analyzers relocated to the event venue. Thus samples collected at the time of competition for
routine blood screens could also provide a cost-effective and convenient source of Passport
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data. A crucial element of the Passport approach is to define in advance normal fluctuations
in blood parameters, to enable authorities to discriminate between expected and suspect
changes. It is also necessary to recognize whether biological variability is sport-specific or
whether such changes are universal across different sports. One study documented temporal
changes of hemoglobin concentration (Hb) and reticulocyte counts in elite rowers. Blood
samples were obtained from members of the French National Rowing squad (n=83 males,
n=31 females). Between two and eight (average 5 males and 4 females) EDTA blood
samples were collected from each rower, and were measured on either an ADVIA 120
Hematology Analyzer or a Sysmex Roche XE2100 (instruments were calibrated according to
manufacturers’ specifications). The results were contrasted with previously reported data
comprising longitudinal evaluations from a subset of n=288 male professional football players
measured on average 3 times. Analysis of variance was used to partition the total variation.
These quantitative data illustrate that for Hb the major component of variance is attributable
to between subject variation, which supports the intuitive belief that comparing an athlete’s
current values with their own longitudinal data rather than population-derived thresholds will
provide greater resolution when searching for signs of blood doping. The similarity of within
subject variation apparent across different sports augurs well for the universality of this
approach. Although of secondary importance to the Passport concept (which is contingent
upon consistent within subject variance), there was no apparent explanation for why between
subject variation in the cohort of female rowers was markedly lower than for other groups,
and this deserves closer scrutiny. The results also emphasize the need to quantify, and
adjust for, instrument bias for reticulocyte assays. Instrument bias can be quantified by using
the mean value from a cohort of athletes as a de facto calibration agent, and bias negated by
using a paper adjustment to standardize values. The “within subject” variation in rowers was
comparable to that of athletes from other sports. Reticulocyte results were dependent on the
type of instrument used [04011].
Doping in the Olympics
The ancient Olympic games
The first instance of an athlete doping in competition is unclear, although there are examples
of sportsmen from the Greek era using natural substances to gain an advantage. The
Olympic games, founded in 776 B.C. (date of the earliest recorded Olympic competition) in
Olympia as a tribute to the gods but also to celebrate the virtues of athletic competition,
peaceful coexistence and the magnificence of athletics, constitute Olympia’s perennial
contribution to the world, this symbolized by the eternally burning Olympic flame. We may
still sing today, as did Pindar in his eighth Olympian Victory Ode, "… of no contest greater
than Olympia, Mother of Games, gold-wreathed Olympia…" [12002].
The ancient Olympic games were (almost) men only affairs. Successful athletes were highly
honoured,and, perhaps for this reason, skulduggery was not unknown. For example, it is red
in Wikipedia that “Sotades at the ninety-ninth Festival was victorious in the long race and
proclaimed a Cretan, as in fact he was. But at the next Festival he made himself an
Ephesian, being bribed to do so by the Ephesian people. For this act he was banished by the
Cretans” [12003].
Olympic nationalism
In July 1912, the Boston Medical and Surgical Journal celebrated “American Supremacy,”
noting that the “overwhelming success of the American athletes at the current Olympic
262
games in Stockholm is as interesting physiologically as it is nationally gratifying. Although
Sweden won the most medals, athletes from the United States won the gold medal count by
the thinnest margin, 25 to 24.” The large population of the United States “is more mixed of all
races, and we should therefore be able to select the best strains and breed the best
mixtures.” American athletes were “better nourished and conditioned” than their competition,
“which again should conduce to racial physical superiority.” To top it off, “the intensity of our
national disposition leads our athletes to train much more eagerly and consistently, and with
a keener professional intentness for winning.” Though eugenic undertones have faded,
physicians have maintained their interest in the Olympics. The strobe of the quadrennial
competitions illuminates dramatic changes in medicine and sport [12004].
The first Olympic tests
The first Olympic drug testing took place at the 1968 Games in Grenoble and Mexico City,
but it was the Munich Games in 1972 that marked the introduction of a comprehensive
testing program. Approximately 7000 athletes participated and just over 2000 samples were
collected and analyzed for various types of stimulants. Since then the testing program has
expanded for each (summer) Games, both in number and percentage-wise, and at the 2008
Beijing Games where 10,500 athletes took part, 4770 samples were collected. By then, the
number of banned substances to be analytically identified had increased significantly. The
IOC, however, had limited possibilities to conduct an efficient anti-doping activity as they
governed only two big competitions every 4 years (since 1992, one every 2 years, winter and
summer alternatively). Then, as it is today, the responsibility for the year-round sport
activities rested with the international federations and the national associations, but they
remained remarkably inactive on the doping issue for a long time. The first international sport
organization to pick up the matter in a serious way was the International Association of
Athletics Federations (IAAF), and during the time between the Munich Games and 1999
when the World Anti-Doping Agency (WADA) was created, the IAAF was the leading
international organization in the fight against doping, as will be evident from the following
article. A series of challenges met the anti-doping campaigners when they started in the early
1970s [12005].
Doping during the modern Olympics
One of the most enduring symbols of the Olympics is the torch or flame, an icon of peace
and sportsmanship that has its roots in Ancient Greece. According to the Creed of the
Olympics: "The important thing in the Games is not winning, but taking part. The essential
thing is not conquering. but fighting well." The modern Olympic Games (1896-2000) have
been heavy laden with controversy, as athletes have abused performance enhancing drugs
to thrust themselves into the limelight in search of gold. It was not until 1967 that the
International Olympic Medical Commission began banning drugs. Full-scale drug testing was
instituted in 1972.: Retrospective review of modern summer and winter Olympics Game
sources (1896-2002) was done for documentation of drug abuse, drug-related overdoses,
and positive drug screens. Data were collected for the type of drug documented. the athlete's
name, their country of origin, and Olympic event. Seventy cases were identified. The most
common class of agents were steroids (29), followed by stimulants (22), diuretics (7), beta-2
agonists (2), and beta blockers (1). Alcohol and marijuana, while not historically prohibited,
have been outlawed by several individual sport federations. Toxicities of these 2 agents were
most likely under-reported. Countries of origin of individual athletes included Bulgaria (7),
USA (7), Sweden (4), Spain (4), Japan (2), Poland (2), Greece (2), Canada (2), Hungary (2),
Russia (2), Austria (2), and Great Britain, Norway, Romania, Armenian, and Latvian, each
with 1. The most common Olympic events in which drug abuse was documented were
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weightlifting (25), trackand field (12), skiing (5), wrestling (5), volleyball (3), modern
pentathlon (3), cycling (2), swimming (2), gymnastics (1), and rowing (1). As athletic
pressures and financial gains of the Olympic Games heighten, more toxicities are likely to
occur despite attempts at restricting performance-enhancing drugs [03013].
Athletes have always sought to outperform their competitors and regrettably some have
resorted to misuse of drugs or doping to achieve this. Stimulants were taken by the first
Olympic athletes to be disqualified in 1972. Although undetectable until 1975, from the 1950s
androgenic anabolic steroids were administered for increased strength and power followed in
the 1990s by erythropoietin for enhanced endurance. Both are highly effective doping
agents. As analytical science validated improved techniques to identify these drugs, Olympic
athletes, including many medallists were caught and disqualified. When the International
Olympic Committee (IOC) prohibited beta blockers (beneficial in shooting), diuretics (assist
weight classified athletes) and glucocorticosteroids, some athletes with genuine medical
conditions were denied legitimate medical therapy. To overcome this, in 1992 the IOC
introduced a system known now as Therapeutic Use Exemption (TUE). One paper discussed
Olympic athletes who have been known to dope at past Games and some medical
indications and pitfalls in the TUE process [12007].
The resolut fight during the Olympic games against doping in sports commenced as a result
of the death of a Danish cyclist during the Rome Olympic Games in 1960 - directly seen by
millions of people viewing TV. The International Olympic Committee (IOC) established a
Medical Commission (IOC-MC) which had the task of designing a strategy to combat the
misuse of drugs in Olympic Sport. It's today a far cry from the horror that ensued when drug
testing was first introduced for the 1968 Winter and Summer Olympic Games and an athlete
was busted, for of all things, drinking beer: the Swede modern pentathlete Hans-Gunnar
Liljenwall was stripped of a bronze medal for dipping into the local cerveza at the Mexico City
Summer Games [08012].
At the 2000 Olympics, 10 athletes were caught doping, including 6 medalists, while a record
27 athletes were caught doping at the 2004 Olympics. In total, 84 athletes, including 28
medal winners, have been caught doping at Summer Olympics, 37 of whom were
weightlifters, the most notorious of sports amongst dopers. The Winter Olympics have
generally witnessed fewer tests and fewer doping busts. Since 1968, only 13 athletes,
including 6 medal winners, have been caught doping. Seven of them were cross-country
skiers and 4 were hockey players. According to the International Olympic Committee there
have been 84 infractions for doping since 1984, including such bizarre incidents as one in
2004 in which an Irish equestrian administered an antipsychotic drug to his horse. By
Olympic Games, the failed, missed, refused or falisified test (and medals forfeited) have
been [08012]:
1968 Mexico City 1 (1)

1972 Munich 7 (4)
1976 Montreal 10 (3)
1980 Moscow 0
1984 Los Angeles 12 (2)
1988 Seoul 10 (4)
1992 Barcelona 5 (0)
1996 Atlanta 2 (0)
2000 Sydney 10 (6)
2004 Athens 27 (8)
The Winter Olympics have generally witnessed fewer tests and fewer doping busts. Since
264
1968, only 13 athletes, including 6 medal winners, have been caught doping. Seven of them
were cross-country skiers and 4 were icehockey players.
In total, 84 athletes, including 28 medal winners, have been caught doping at Summer
Olympics, 37 of whom were weightlifters, the most notorious of sports amongst dopers. By
country, the number of medals forfeited as a consequence of drug testing up to 2004:
Bulgaria 7
USA 6 (3 gold and 2 bronze from Marion Jones)
Hungary 3
Germany 2
Sweden 2
Canada, Russia, Poland, Ireland, Romania, Finland, Mongolia, Greece, Spain,
Armenia, the Netherlands & Ukraine 1
By sport 12 medals have been forfeited in weightlifting, 5 in athletics, 2 each in equestrian,

wrestling and cycling, and 1 each in judo, modern pentathlon and rowing.
The increased number of positive tests is in part a function of the increased number of tests
administered at each game. At the 2000 Olympics, about 2000 doping tests were
administered. That number grew to 3700 by the 2004 Olympics. The increasing number is
largely a result of the expansion of the rules governing who gets tested. In the past, the top 4
finalists in an event and 1 other athlete chosen randomly were subjected to tests. However,
in Beijing, the top 5 athletes were tested in addition to 2 chosen at random in each final. As
well, random tests will be conducted throughout earlier stages of competition. Beginning in
2000, Olympic athletes were also subject to pre-Olympic, out-of-competition testing to detect
substances consumed prior to competition that wouldn't later appear on a test. Blood testing
was introduced on a limited basis at the 1994 Winter Olympics and at the 2000 Summer
Olympics. It is the International Olympic Committee itself that administered and monitored
athlete testing in Beijing, not the World Anti-Doping Agency. The latter focuses on policies,
regulations and monitoring the 33 facilities worldwide that have been approved for testing
athletes' samples [08012].
Some International Sport Federations (IF) and National Sports Federations followed suit
when the anti-doping process started, but progress was modest until the world's best male
sprinter (Ben Johnson, Canada) was found doped with anabolic steroids at the Olympic
Games in Seoul in 1988. Further progress was made following the cessation of the cold war
in 1989 and in 1999 public authorities around the world joined the Olympic Movement in a
unique partnership by creating WADA, the World Anti-Doping Agency, which has doubtless
been the start of a new anti-doping era [08013].
The Olympics in medical journals
Even before Pierre de Coubertin revived the Olympics in 1896, lore from the ancient games
circulated in the medical literature. An 1851 essay in the Boston Medical and Surgical
Journal about the power of mind over body described “the old Greek who died on the spot
from excess of joy on seeing his three sons crowned with laurel at the Olympic games”
(1851). Oliver Wendell Holmes invoked this same episode in his valedictory to Harvard
medical graduates (1858). Other authors drew competing lessons from the Olympic legacy.
One warned that excessive athletic training diverted energy from mental development,
leaving adolescents “listless and stupid”: “It was especially remarked by the Greeks that no
one who in boyhood won the prize at the Olympic games ever distinguished himself
afterwards” (1867). An 1891 review, in contrast, expressed the hope that educators would
265
learn from the ancient techniques and improve athletic training in U.S. schools [12004].
When the modern games began in Athens in 1896, physicians only slowly became interested
– and mostly in marathons. Heat and humidity tormented marathoners in St. Louis in 1904:
only 14 of 27 finished. The winner Thomas Hicks, who sustained himself during the race with
strychnine sulfate, five eggs, and brandy, required the care of four physicians in the
aftermath. Heat caused problems again in London and Stockholm. When the games
resumed in Antwerp in 1920, athletes were subjected to physical examinations. The United
States sent its first team physician – one who had fenced in Stockholm – to the Paris games
in 1924. Medical scrutiny has continued ever since [12004].
Physicians have been interested in the Olympics for many reasons. In the 1920s, they
probed the limits of human physiology. One group studied the Yale heavyweight rowers who
won gold in Paris. An ingenious contraption revealed that at their racing speed – 12 mph –
the eight men produced four horsepower, a 20-fold increase over resting metabolism (1925).
A 1937 study published in the Journal showed that athletes at the 1936 Berlin games
consumed 7300 calories each day. Sometimes the venue itself became the issue. The
United States threatened to boycott the Berlin games until Hitler relented and allowed black
and Jewish athletes to compete. Ignoring these tensions, the Journal, which had published a
favorable review of Nazi health insurance in 1935, advertised the exhibits and lectures on
“Medical Theory and Practice in the New Germany” that had been organized for physicians
who visited the Olympics (1936). Boycott politics surfaced again when the Journal's editor,
Arnold Relman, visited the Soviet Union in 1980. Relations had been strained by tensions
over the Soviet invasion of Afghanistan and the United States' threat to boycott the Moscow
games. Other venues created medical concerns. Roger Bannister, who eventually became a
neurologist after being the first person to run a mile in under 4 minutes, “thoroughly
disapproved” of holding the 1968 games at high altitude in Mexico City. And indeed, several
hundred athletes collapsed at those Olympics, from migraine, shock, syncope, or emotional
excitement. Fears of local pathogens emerged before the Olympics in Seoul (Japanese
encephalitis) and Barcelona (multidrug-resistant strep). Each warning met with vehement
rebuttal. Olympic events now attract millions of visitors and require careful medical and public
health planning [12004].
The safety of Olympic sports has remained an enduring concern. The French Academy of
Medicine appointed a committee before the 1924 Paris games “to study the effects of
modern athletics on the human system.”. The resulting tests “revealed an alarming number
of cases of athletic heart.” Subsequent studies from the 1920s (on athletes from the 1928
Amsterdam games) through the 1990s (on 310 Italian Olympians) have produced conflicting
evidence on the question of whether intense physical training can cause cardiac hypertrophy.
Some sports received special scrutiny. On the eve of the Atlanta games in 1996, a scathing
review likened women's gymnastics to child abuse, arguing that although “elite gymnastics
can provide a profoundly meaningful experience for the athletes,” it could also “result in
serious, life-endangering physical and psychological disabilities.” Citing injuries, eating
disorders, and social problems, the authors warned that “talented youngsters at every
competitive level should be supported rather than crippled by their sport as they enter
adulthood” [12004].
A different kind of medical scrutiny emerged in the 1950s. Commenting on a symposium

about “Pheidippidian physiology,” a 1957 editorial highlighted the recent dramatic
improvements in performance at track and field events. What explained “these epidemics of
broken records”? The editorial considered possible contributions from training, diet,
antibiotics, and motivation but focused on “a speculative explanation”: “that amphetamine is
being used by some athletes to help them break otherwise unassailable records.” Such
266
practices, if they were in fact occurring, were both dangerous and “ethically undesirable.”
Each passing decade brought new scandals about performance-enhancing drugs. After the
U.S. Olympic Committee admitted that seven cyclists (including four medalists) had received
blood transfusions at the Los Angeles games, a Sounding Board article in the Journal in
1985 condemned this practice. Not only was the practice dangerous, especially in light of the
emerging AIDS epidemic, but also it “represents an attempt to use a medical therapy to
provide athletes with an unfair competitive advantage.” When Ben Johnson was stripped of
his gold medal at the Seoul Olympics, the Journal reviewed the medical risks and legal
consequences of anabolic steroids in 1989. Erythropoietin came next. A Dutch physiologist
wrote in the Journal that “the next Olympic Games have already been nicknamed the
`Hematocrit Olympics,'” and physicians' obligation seemed clear: “the medical profession has
a responsibility to consider carefully these untoward consequences of scientific progress.”
[12004].
Performance-enhancing drugs have cast a long shadow on the modern Olympics. Whether
the agents are the strychnine, heroin, cocaine, and morphine that athletes used in Athens in
1896 or the amphetamines, steroids, and erythropoietin that some use today, the dilemma
remains the same. As a sports medicine specialist noted in 2004, the “attraction of
performance-enhancing drugs is simply that they permit the fulfillment of the mythical
promise of boundless athletic performance – the hubristic “faster, higher, stronger” motto of
the Olympic Games.” The ensuing systems of medical surveillance have led, inevitably, to “a
new type of competition,” in which some athletes try to stay one step ahead of the authorities
[12004].
There are several historical journal article in the Boston Medical and Surgical Journal (1851-
1925) and in the follower New England Journal of Medicine after that [12004]:
Boston Medical and Surgical Journal

1851. Badeley JC. On the reciprocal agencies of mind and matter. 45:195-201.
1858. Holmes OW. Valedictory address. 58:149-59.
1867. The abuse of physical exercise. 77:425.
1891. Hartwell EM. The principal types of physical training compared. 125:641-4.
1912. American supremacy at the Olympic games. 167:102.
1924. French Academy of Medicine names committee to study effects on the human
system of modern athletics. 190:397.
1925. Stiles PG. Recent progress in physiology. 193:873-6.
New England Journal of Medicine
1930. Bland EF, Sprague HB. Progress in the study of cardiovascular disease in 1929.
203:574-91.
1935. Davis MM, Kroeger G. Recent changes in German health insurance under the
Hitler government. 212:1037-42.
1936. Information for doctors during the Olympiad in Berlin. 215:211.
1937. Burnett FL. Nutrition, health and disease. 217:515-27.
1957. Icarus complex. 257:1194-5.
1968. Lister J. After-dinner speaking — “Hello, there!” 278:953-4.
1969. Jokl E. Altitude diseases. 280:1420-2.
1980. Relman AS. Moscow in January. 302:532-4.
1985. Klein HG. Blood transfusion and athletics: games people play. 312:854-6.
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1988a. Marcus LC. Liability for vaccine-related injuries. 318:191.
1988b. McKinney WP, Barnas GP. Japanese encephalitis vaccine: an orphan product
in need of adoption. 318:255-6.
1988c. Steffen R. Japanese encephalitis vaccine: necessary for the Olympics in Seoul?
319:251.
1989. Hallagan JB, Hallagan LF, Snyder MB. Anabolic–androgenic steroid use by
athletes. 321:1042-5.
1991a. Pelliccia A, Maron BJ, Spataro A, Proschan MA, Spirito P. The upper limit of
physiologic cardiac hypertrophy in highly trained elite athletes. 324:295-301.
1991b. Romijn JA. Erythropoietin. 325:1176-7.
1992a. Barnett ED, Klein JO, Teele DW. Pneumococcal vaccine for Olympic athletes
and visitors to Spain. 326:1572.
1992b. Plasencia A, Segura A, Farrés J, Cuervo JI. Pneumococcal vaccine for Olympic
athletes and visitors to Spain. 327:437.
1996. Tofler IR, Stryer BK, Micheli LJ, Herman LR. Physical and emotional problems of
elite female gymnasts. 335:281-3.
2001. Zarins B. Review of: Endurance in sport. 344:1025-6.
2004. Noakes TD. Tainted glory — doping and athletic performance. 351:847-9.
2006. Kellerman AL. Crisis in the emergency department. 355:1300-3.
Canada
It is now a long time since the Ben Johnson scandal at the Olympics in Seoul, Korea, drew
attention to the issue of doping. In the scandal's aftermath, an independent organization now
known as the Canadian Centre for Ethics in Sport was established to develop a national
antidoping program. It has served as a model for other national antidoping organizations.
Still, 55 Canadian athletes violated antidoping rules in the past 3 years: 27 used
“recreational” drugs such as marijuana, and 28 took performance-enhancing drugs, most
often anabolic steroids or stimulants [08014].
Formation of the IOC Medical Commission
In 1961 the International Olympic Committee (IOC) created a Medical Commission (IOC–
MC) at its 59th Session in Athens, Greece. The decision was triggered by the death of the
Danish cyclist Knud Enemark Jensen during the road race for teams at the Rome Olympic
Games the year before. He was said to have taken some stimulating drug, but was also
reported to have suffered from heat exhaustion and dehydration. Probably a combination of
all this caused his death, but this has never been officially confirmed. At any rate, the IOC
could no longer ignore the use of stimulants that had obviously been in place in certain
sports for quite some time. The creation of the IOC-MC marked the start of the modern era of
the anti-doping campaign. The Commission was requested to propose a strategy for
combating the use of performance-enhancing drugs in Olympic sports. It took quite a while to
analyze the situation and recruit the necessary competence. Not until the 1967 IOC session
were some concrete proposals presented such as a list of forbidden drugs (stimulants) in
Olympic sport and drug testing at the coming Games. Therefore, 1967 is often referred to as
the start of the IOC-MC [12005].
Formation of the World Anti-Doping Agency

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The practice of enhancing athletic performance through foreign substances was known from
the earliest Olympic Games. In 1967, the International Olympic Committee (IOC) established
a Medical Commission responsible for developing a list of prohibited substances and
methods. Drug tests were first introduced at the Olympic winter games in Grenoble and at
the summer games in Mexico City in 1968. In February 1999, the IOC convened the World
Conference on Doping in Sport in Lausanne, Switzerland. The Lausanne Declaration on
Doping in Sport recommended creation of an International Anti-Doping Agency. The World
Anti-Doping Agency (WADA) was formed in Lausanne, Switzerland on the basis of equal
representation from the Olympic movement and public authorities. One of the mandates of
WADA was to harmonize the Olympic antidoping code and develop a single code applicable
and acceptable for all stakeholders. The world antidoping code developed by WADA
included creation of several international standards (IS). The purpose of each IS was
harmonization among antidoping organizations. The ISs were developed for laboratories,
testing, the prohibited list, and for therapeutic use exemptions (TUE). The objective of this
manuscript is to present a brief history of doping in sport and describe creation of WADA in
1999. The components of the World Anti-Doping code (in particular, the Therapeutic Use
Exclusion program or TUE) is described. The WADA code defines a TUE as "permission to
use, for therapeutic purposes, a drug or drugs which are otherwise prohibited in sporting
competition." Experiences of the Canadian Centre for Ethics in Sport Doping Control Review
Board are presented because this national TUE committee has been operational for over 12
years. The challenge of developing a rigorous global antidoping program requires
acceptance of doping as a problem by sport organizations, athletes, and public authorities.
Individual stakeholders must be prepared to preserve the values of sport, which means free
from doping. This will require vigilance by all interested parties for the benefit of elite athletes
and society overall [04010].
Probably the most serious challenge for the anti-doping fight during the 1970s and 1980s
was the unwillingness of most countries and international federations to join the fight. There
were several reasons, namely: the costs; the lack of competence; the negative image should
a top athlete in their own country or sport test positive; and, the Cold War. The East
Germans used success in sport as a political weapon and other countries had followed suit,
although in a less sophisticated way. Officially, sports leaders were against doping, but far
too many only paid lip service and some were even sabotaging the fight. When the Cold War
faded following the political events in 1989 and the years that followed, the anti-doping fight
gained increased support. In 1999 the public authorities actually joined the fight by accepting
the invitation of the IOC to form WADA together [12005].
In 1999, the IOC recognized that an effective fight against doping required cooperation
between sport and government. The First World Conference on Doping in Sport resulted in
the formation of the WADA in 2000, which was charged with harmonizing the international
antidoping efforts. WADA has developed a World Anti-Doping Program, which has been
adopted by all Olympic sports. In order for governments to ratify an equivalent to the World
Anti-Doping Program, it was necessary to develop the International Convention Against
Doping in Sport through the UN Educational, Scientific, and Cultural Organization. To date,
over 160 countries, including the United States, have approved the convention [12006].
When antidoping activities accelerated following the Ben Johnson case at the Seoul games
in 1988 and following worldwide political changes that took place soon afterwards, it became
apparent that the situation and antidoping rules around the world were in chaos. Athletes
received different penalties for the same offence depending on sport and their nationality.
The antidoping campaign could not advance without a harmonised and universally accepted
set of rules. But that could not be carried out by sport alone. Governmental support was
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needed. That was one of the main reasons for the creation of WADA in 1999. Remarkably,
WADA had a draft set of rules – a draft Code – ready by late 2003. It received wide support
mostly from the governments whereas some federations expressed reservations. The IOC
declared, however, that those Olympic federations that did not adopt the Code before the
Athens Olympic Games (2004) would not have their sport included in the games. Thus, when
the Athens Games opened, the world also saw the birth of the “World Anti-Doping Code”.
The governments took their share of the commitment by creating a Convention under
UNESCO in 2005; this to encourage all governments around the world to support the fight
against doping on the basis of the WADA Code [14431].
The World Anti-Doping Code was created on February 20, 2003 and entered into force for
the first time on January 1, 2004 [14425].
The recent development of the World Anti-Doping Agency (WADA) is an endeavor of the
international community to consolidate and coordinate efforts to minimize doping practice. In
this current anti-doping environment and in view of the Athens 2004 Olympic Games, the
organizing committee "Athens 2004" in collaboration with the International Olympic
Committee (IOC), the Medical Committee (MC) and WADA, will develop new anti-doping
policies and will deliver programs and guidelines for doping control [03002].
WADA is the international independent agency that publishes the World Anti-Doping Code,
which is the document harmonizing anti-doping policies in all sports and all countries. The
Code was first adopted in 2003 and became effective in 2004. The Code sets forth specific
anti-doping rules and principles that are to be followed by the anti-doping organizations
responsible for adopting, implementing, or enforcing anti-doping rules within their authority,
including the IOC, International Paralympic Committee, international sport federations (for
example, the International Cycling Union), major event organizations, and national anti-
doping organizations (for example, the US Anti-Doping Agency).WADA revises and
publishes its list of banned substances approximately annually. It specifies those banned
substances and methods that are prohibited at all times (both in-competition and out-of-
competition) because of their potential to enhance performance in future competitions or their
masking potential, and those substances and methods that are prohibited in-competition
only. The list may be expanded by WADA for a particular sport [14612].
Formation of Court of Arbitration (CAS)
During the late 1970s and early 1980s, one athlete after another who tested positive
challenged the IAAF before national courts and the Federation spent a lot of money on court
trials. In an attempt to take care of the problem of escalating legal costs the IAAF included in
its constitution an “Arbitration panel” as the highest authority to settle disputes within
athletics. This could not prevent athletes from bringing their cases before national courts, but
the Arbitration panel served as deterrent. This happened in 1982 and a year later the IOC
created the Court of Arbitration for Sport (CAS), which today is included in the WADA Code
as the final appellate body on doping matters. There are examples of enormous legal costs
following an athlete’s positive sample. The IAAF was ordered by a district court in USA to
pay USD 27.4 million in damage compensation to the 400 m world-record holder Harry
“Butch” Reynolds after their panel had disqualified him for steroid doping in 1990. The
decision was overruled by the Circuit court, and the Supreme Court refused to hear the case.
Thus, after several years of legal battle Reynolds lost the case (and money) but the IAAF
also lost a lot of money. The winners were the lawyers on each side. In recent years, WADA
successfully defended the testosterone analysis before CAS after it had been challenged by
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the Tour de France winner Floyd Landis following his positive test. The defense cost WADA
about USD 2 million. The ever-increasing legal costs are a major challenge for sports
organizations and a true threat to the anti-doping fight [12005].
Out-of-competition testing
The next challenge was to convince the world of sport that doping controls at competition
only is not sufficient. Since AAS are taken during training periods in order to promote the
building of muscles and strength, doping controls will also have to be conducted during
training – so-called out-of-competition controls. Such controls had been in place in Norway
and Sweden since the early 1980s but when the idea was brought to a broader international
sports community it was met with great resistance. A majority saw it as an unacceptable
intrusion into the athlete’s private life. However, the Ben Johnson scandal at the Seoul
Games in 1988 made the skepticism fade and in 1990 the IAAF, as the first international
federation, started testing out-of-competition after the necessary rules had been passed by
the 1989 IAAF Congress. Today, out-of-competition testing is compulsory in the WADA
Code, and any national anti-doping organization or international federation that does not
conduct such testing will be declared noncompliant with the Code and possibly face serious
consequences. The IAAF ambition to clean up its sport resulted in the identification of doped
athletes, some very famous [12005].
History of therapeutic use exemptions (TUE)
The introduction of what today is known as therapeutic use exemption (TUE) was far from
smooth. It started in the mid-1980s when a Swedish athlete asked the nation’s anti-doping
organization for permission to use testosterone as replacement therapy following the removal
of both his testes (unilateral cryptorchism as a newborn followed by cancer in his remaining
testis as a teenager). It was granted with the remark that it was only valid within Sweden.
Soon thereafter a similar case (bilateral testicular torsion) occurred in Australia. At the 1988
Olympic Games two athletes were allowed by the IOC-MC to use banned substances
(corticosteroids for inflammatory bowel disease and diuretics for nephrotic syndrome). The
permissions were granted although no clear rules existed. Some Commission members,
therefore, suggested that such rules be worked out. Other members were against, arguing
that athletes should not be allowed to use prohibited substances. If they were in such need
they should quit sport. In 1992, however, a proposal was presented to the IOC-MC with strict
criteria for “permitted use of prohibited substances” and the formation of a group that should
evaluate applications for such use of IOC-MC. The proposal was accepted, and a
‘”Medications Advisory Committee (MAC)” started its operation at the Barcelona Games in
1992. Although MAC’s authority was limited to the Olympic Games it became an advisory
body for international federations and national anti-doping organizations. MAC worked under
strict anonymity and, actually, in secret. The fear was that if MAC became generally known it
would result in a flood of requests and abuse of the system. When the IOC Juridical
Commission reviewed the 1999 edition of the IOC Medical Code they first refused to include
the concept of “permitted use”. Only after pressure from the IOC-MC was it added as a
brief‘addendum’ When WADA was about to be formed, however, the IOC-MC requested that
the concept of permitted use be accepted and clearly explained in the coming rules. Today
TUE is an important part of the WADA Code [12005].
When the IOC undertook the first extensive doping controls (>2000) at the 1972 Munich
Olympics, the only drugs that were prohibited were stimulants and narcotics. Androgenic-
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anabolic steroids (AAS) were prohibited prior to the 1976 Olympics, although endogenous
steroids such as testosterone could not be identified. During this period, there was no
discussion about athletes needing a Therapeutic use exemptions (TUE). However, after
diuretics, beta-blockers and systemic glucocorticosteroids (GCS) were added to the IOC's
Prohibited List in the 1980s, significant interference occurred in the availability of essential
medications to manage medical conditions in elite athletes. Around 1987-1988, two male
athletes without testes, one in Sweden and the other in Australia, sought to continue their
testosterone replacement therapy and participate in their sport. Both were granted national
approval. At the 1988 Calgary Games, an ice hockey player was approved to take oral GCS
for inflammatory bowel disease (IBD) and later that year in Seoul, a rower with nephrotic
syndrome was approved to continue her diuretic therapy and participate. Both were ad hoc
decisions taken by all members of the IOC-MC, some of whom were not medically qualified.
In February 1991, it was presented a paper “Permission for athletes to use drugs contained
in the IOC List of Banned Classes” to the IOC-MC. This detailed the following criteria that
should be met to approve any such application by an Olympic athlete [13013]:
- The athlete would experience significant impairment of health if the prohibited

medication was withheld
- No enhancement of performance could result from the administration of the prohibited
substance as medically prescribed
- The person would not be denied the prohibited substance if he/she was not a
competing athlete
- No available permitted or practical alternative can be substituted for the prohibited
substance
- Retrospective approval would not be granted
Independently, and at the same IOC-MC meeting, professor Manfred Donike presented a
paper on “Replacement therapy” which focused on testosterone in anorchia. An interim
Medications Advisory Committee (MAC) was appointed (D Catlin, A Ljungqvist and K Fitch,
Convenor) and requested to develop guidelines to implement the proposal. This was
undertaken and included [13013]:
- The complete medical details including the history, clinical findings and investigation
must be submitted
- The necessity to administer the prohibited medication including the dosage, route and
frequency of administration must be certified by a suitably qualified medical specialist
- The medical necessity to administer the prohibited substance cannot be the result,
wholly or partially, of prior use of a drug from the banned classes or banned methods
- Additional investigations requested by the MAC will be undertaken at the athlete's or
his/her National Olympic Committee's (NOC) expense
- Any doctor who provides the MAC with false information will be ineligible to be
accredited as an Olympic team doctor or official
- Under no circumstances will permission be given to use any synthetic anabolic
steroid
However, the concept had its doubters and it would be another year before the IOC-MC
agreed to allow the MAC to start operating prior to the Barcelona Games. However, the IOC-
MC Chairman, supported by some Commission members, would not permit any publicity for
“fear of abuse of the system”. Hence, only two applications were received for Barcelona
1992. Both were for oral GCS for well-documented IBD and were approved. Each athlete
won a medal, although neither was in an individual event [13013].
Anorchia
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During the early years, MAC members were conscious that they were embarking on a new
and potentially controversial undertaking and that their decisions must be able to withstand
the closest scrutiny. In the mid-1990s, the MAC compiled the first document that established
some circumstances in which certain prohibited substances and methods might be approved
to manage specific medical conditions encountered in athletes. A number of conditions and
circumstances for which permission would not be granted were also documented. The
classes of prohibited drugs under consideration were diuretics, corticosteroids, beta-blockers
and AAS (only testosterone). No exemptions were contemplated for stimulants or narcotics.
In 1994, two world-class sailors applied to continue to administer testosterone at the 1996
Olympic Games if selected. Both had anorchia, one a congenital condition and the other
surgical due to bilateral malignancies. Having considered these applications, the MAC
sought the advice of independent expert endocrinologists who were “blinded” as to the
identity of each athlete, his NOC and his medical advisors. Both were approved and although
neither athlete qualified at the 1996 Olympic yachting trials, one did compete in Athens 2004
[13013].
Aplastic anemia
One unusual request was for erythropoietin in a Winter Olympic athlete prior to the 1994
Lillehammer Games because she had recently been a bone marrow donor for her brother
with aplastic anaemia. It was the usual policy of the oncology centre concerned to withdraw
and store a unit of blood prior to harvesting bone marrow and reinfusing this postharvest.
(This would have been a Prohibited Method.) Owing to not wanting to interrupt her Olympic
training programme and the urgency of her brother's need for a bone marrow donation, this
was not undertaken and her haemoglobin fell by 2 g/dl. However, despite the compelling
circumstances, the application was rejected [13013].
Congenital adrenal hyperplasia (CAH) and hypogonadism
Some early requests included an archer with 21-hydroxylase deficiency (salt losing)
congenital adrenal hyperplasia (CAH) seeking oral GCS, which was approved. However,
shortly after this, a shooter with 17-hydroxylase deficiency CAH who had been granted
national approval was denied permission by her International Federation (IF) to administer
GCS and compete internationally. Fortunately, intervention by the MAC reversed this
decision and later, she competed successfully at the 2000 Olympic Games. Prior to Atlanta
1996, a female soccer footballer with C1-esterase deficiency hereditary angioneurotic
oedema was approved to use danazol daily and participated. In contrast, an aging canoeist
who had been prescribed testosterone for alleged hypogonadism and had the backing of his
national antidoping agency sought permission to continue this therapy at Atlanta. The MAC
advised that this athlete did not have a valid justification and rejected the application [13013].
Stimulation medication
A well-publicised case that regrettably reflected that the MAC was compelled to remain
“clandestine” was that of an elite 22-year-old 3 m diver whose severe narcolepsy was
diagnosed in 1996. Immediately, she sought permission from her NOC and then from FINA
to take stimulant medication but was rejected by both bodies. She continued to compete
nationally and internationally and won national titles but seemingly was not required to take a
doping control until a Grand Prix event in 1999 when she admitted the use of
dexamphetamine on the doping control form and tested positive. Despite being advised that
her narcolepsy was so severe that she was at major risk of falling asleep on the 3 m diving
platform and thus of potential injury, FINA's Doping Control Panel imposed a 12 month
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suspension. Intervention by the FINA Medical Commission resulted in the sanction being
reduced to 6 months, but on appeal to CAS, this was further reduced to 2 months. Ironically,
less than a month after her positive test, FINA modified their doping regulations to permit the
use of a prohibited medication in special circumstances. The opinion of the MAC was not
sought until shortly prior to her initial FINA hearing and then at the request of her NOC. The
MAC had no hesitation in advising that she was entitled to a TUE. This may or may not have
influenced both the FINA Medical Commission and the CAS. Had the IOC informed the
stakeholders of the MAC's existence, this diver would have applied and been approved to
take her medication in 1996. Soon after this decision by the MAC, an application was
received from a track and field athlete from the same NOC whose narcolepsy had been
proven conclusively by sleep studies. On this occasion, the opinion of an independent expert
in sleep medicine was sought. This request was granted by the MAC for the recently
released but prohibited modafinil – a wakefulness drug [13013].
Danazol
Between 1992 and 2003, the IOC's MAC (later TUEC) received numerous applications and
its recommendations were believed to have been accepted on all occasions. In addition to
functioning at the Olympic Games without publicity, the MAC provided advice to as many as
15 IFs and 11 NOCs who sought assistance. Few applications were received that could be
deemed to be “opportunistic”, but one from a 33-year-old elite female weightlifter appeared to
be. On the advice of a “Longevity Institute” doctor, she sought permission to administer
transdermal testosterone following her hysterectomy and bilateral oophorectomies performed
for endometriosis. This was in addition to her replacement permitted hormone therapy. The
application was rejected because no woman should ever be granted approval to take any
AAS except danazol for very strict criteria. From its early days, the MAC decided that no
AAS, except testosterone, could be approved for any male athlete and only then with a
conclusive diagnosis and if the necessity was confirmed by an independent expert. This
policy remains today [13013].
Glucocorticoids and diuretics
In late 1998, approval for oral GCS treatment was granted to a young athlete in the sport of
curling who had a successful renal transplant. Earlier that year, he had competed at the 1998
Nagano Olympics with an MAC-approved TUE for furosemide to assist in managing his
chronic renal failure. At that time, a condition of approval was that the specific gravity of urine
collected in doping control must be 1005 or greater by refractometer. However, improved
analytical hardware has made this requirement no longer necessary [13013].
Beta-blocker
A 56-year-old elite international shooter with a history of coronary artery bypass surgery was
denied permission to take a cardioselective beta-blocker and compete. Although his medical
indication was not questioned, beta-blockers had been demonstrated to produce a 13
percent improvement in shooting performance and this was deemed to contravene the
criterion of ‘not enhancing sports performance’. Currently, the status of beta-blockers in
shooting remains unchanged, that is, TUEs should not be approved. Prior to Salt Lake City
2002, a bobsledder with low-serum testosterone after a unilateral radical orchiectomy for
seminoma was denied permission to administer depotestosterone because of the presence
of one intact testicle [13013].
Recognising the concept of TUEs
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Many NOCs and IFs did not accept the policy of therapeutic use, first because it was not in
the Medical Code of the Olympic Movement, and second, because of ignorance on the part
of many sports authorities as to the concepts of therapeutic use. Thus, recognition of the
concept of TUE continued to be difficult. In 1998, the IOC's Juridical (Legal) Committee was
incredulous at being advised by the author that TUEs had been approved for 6 years and
criticised the IOC-MC Chairman for denying the MAC and its operation any publicity. But it
would be another 2 years before the Juridical Committee would finally permit a one sentence
mention of the concept as an addendum to the 2000 edition of the Medical Code of the
Olympic Movement. This permitted a small breakthrough when the Anti-Doping policy for the
Sydney 2000 Olympics made mention of the principle. Australia is believed to be the first
country to have a committee with legislative authority to approve TUEs from August 1999.
During the Sydney 2000 Olympic Games, Australia's TUEC organised a meeting of NOCs,
IFs and interested persons which recommended that templates of established and proposed
criteria for specific TUEs be prepared. This task was undertaken by Australia's TUEC and the
templates circulated to attendees for feedback in 2001 [13013].
It was the World Anti-Doping Agency (WADA), not the IOC, that provided global recognition
and acceptance of TUE. Strenuous attempts to sell the idea of TUE occurred at the 1999
World Conference on Doping in Sport in Lausanne from which WADA had its origins.
Fortunately, two members of the MAC had significant WADA roles from its start and were
able to ensure that the principle of TUE was accepted in the World Anti-Doping Code. An
International Standard of TUE (ISTUE) was prepared between 2001 and 2003 and became
operative when WADA finally assumed global responsibility for doping in January 2004.
Significantly, the TUE criteria and guidelines developed by the MAC in 1991-1992 were
incorporated virtually unaltered in the initial ISTUE. WADA established a TUE Expert Group
in 2004 and members reviewed the Australian TUE templates that had been updated at least
annually. These were offered to WADA in 2005 but the offer was rejected. During the last 6
years, WADA has developed advice termed “Medical Information for TUE Committees”,
which had a rocky start, but by involving experts in each field, it has become a valuable
reference document for TUECs [13013].
Recent Olympic Games
It is difficult to compare TUEs at Olympic Games as changes to the Prohibited List have
necessitated TUEs for different substances and methods. Insulin was prohibited prior to
Sydney 2000 and 5/8 TUEs approved were for insulin-dependent diabetes mellitus (IDDM).
But the number of applications increased significantly after WADA assumed global
responsibility for doping from the IOC. At the Games in Athens 2004, 24 TUEs for athletes
from 19 NOCs and 15 sports were approved with 9/24 (38 %) being for insulin. An appeal
against a rejected application for systemic GCS was heard by WADA and the verdict
confirmed that the IOC's MAC, now termed TUEC, had acted correctly. Intravenous infusions
were prohibited prior to Torino 2006, although no athlete sought a TUE and 2/4 approved
TUEs were for insulin for IDDM. Two applications were considered not to meet the criteria for
approval and were discussed with each athlete's Chief Medical Officer (CMO) who accepted
the TUEC's rationale and both were withdrawn. In Beijing, the TUEC recognised or approved
39 TUEs, of which nine were for IDDM. These 39 athletes were from 19 NOCs and 19
sports. For the first time, five intravenous infusions were approved. Interestingly, there were
two Olympic athletes with well-documented Addison's disease approved for oral GCS. One
application for stimulant medication for adult onset attention deficit hyperactive disorder was
approved and a second withdrawn when a second opinion was sought, which was IOC's
policy and later WADA's [213013].
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Between 2002 and 2008, inhaled beta2-agonists (IBA) were managed by an IOC
Independent Asthma Panel but not considered to be a TUE. However, in 2009, when WADA
included IBAs in the prohibited list, the requirements to approve IBA use were identical to
those of the IOC's Asthma Panel. This decision was partially rescinded just prior to
Vancouver 2010 but two IBAs remained prohibited, formoterol and terbutaline. These
accounted for 94 of the large number of 107 TUEs approved or recognised by the IOC's
TUEC at Vancouver. Of the remaining 13, there were three approvals for insulin for IDDM,
two for ADHD and one for an IF infusion. One IF-approved TUE for a female athlete to take
dehydroepiandrosterone was a fundamental error since no female athlete should ever be
approved to take any AAS except danazol. An appeal to WADA was successful, but WADA
rules permitted the athlete 14 days to cease taking this prohibited medication, which allowed
her to continue until after the Games. This is unsatisfactory and should be changed [13013].
A number of difficulties were encountered at the recently successful London Olympic Games.
From 2004, the IOC's TUEC had agreed to recognise TUEs for participating Olympic athletes
approved by both IF and NADO TUECs after being subjected to close scrutiny. For
Vancouver, the IOC was required to use WADA's Anti-Doping Administration and
Management System (ADAMS). This posed a number of problems which were augmented in
London with almost four times the number of participating athletes and IFs. First, the IOC
TUEC's access to ADAMS was restricted since members were denied the opportunity to
open uploaded medical files to allow them to confirm that an approved TUE had been
granted according to established criteria. Second, more than half of the participating 205
NOCs do not upload TUEs onto ADAMS. Third, few NOCs heeded the request to provide the
IOC's TUEC with a list of valid TUEs for their athletes. Finally, much time was spent in
fruitlessly examining ADAMS to identify athletes believed to be accredited for London 2012
who may have had current a TUE. Almost all TUEs identified on ADAMS for athletes
presumed to be competing in London were either for substances that were currently not
prohibited (mainly IBA) or short-term TUEs that were no longer necessary or valid. Clearly,
the IOC needs WADA to provide superior access and a more efficient way of ascertaining
valid TUEs if ADAMS is to be used at future Olympic Games [13013].
Prior to the opening of the London Olympic Village, CMOs were requested to provide the
IOC's TUEC with details of all valid TUEs for their NOC athletes who would participate at the
Games. Only two complied totally and 21 of the 31 preapproved TUEs that were recognised
were from these two NOCs. During the Games, two athletes tested positive for prohibited
substances and when advised, the NOCs of both athletes confirmed that each had a valid
TUE that had not been disclosed. Hence, one must question how many other athletes had
TUEs and did not advise the IOC. Although at each Games between 2002 and 2010,
between 1/1000 and 1/1500 athletes were known to have IDDM, in London, only three
athletes were reported to have a TUE for insulin (1/3500) which begs the question: were
there more insulin-dependent diabetic athletes about whom the IOC was never advised? In
London, the TUEC approved another 26 TUEs, with as customary most (15) being for
systemic GCS and there were six intravenous infusions. One application was considered
inappropriate and the CMO was contacted and the reasons provided. The NOC agreed to
withdraw the application, institute an alternative permitted treatment and this athlete won a
gold medal [13013].
Legislation
In the early 1900s, endurance events lasted for days without rest. Open-water swimming,
cycling, and long-distance running and walking athletes used stimulants such as strychnine,
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heroin, and amphetamine to alter the perception of fatigue. Only later did governments and
sport recognize the serious health risks associated with the use of stimulants. The
International Amateur Athletics Federation (IAAF; now the International Association of
Athletics Federations) banned the use of stimulants in 1928. The amphetamine-related
deaths of Danish cyclist Knud Enemark Jensen during competition at the 1960 Olympic
Games and British cyclist Tommy Simpson during the 1967 Tour de France illustrated the
seriousness of the problem. In 1966, the cycling, soccer, and track and field international
federations began testing for stimulants. The International Olympic Committee (IOC) formed
its Medical Commission, which included a Subcommission on Biochemistry and Doping in
Sport, in 1967 and tested for stimulants at the 1968 Olympic Games in Mexico City. France
adopted antidoping legislation in 1963; the Council of Europe adopted the first international
Anti-Doping Convention in 1968 [12006].
In 1998, police found a large number of prohibited substances, including ampoules of

erythropoietin, in a raid during the Tour de France. The scandal led to a major reappraisal of
the role of public authorities in anti-doping affairs. As early as 1963, France had been the first
country to enact anti-doping legislation. Other countries followed suit, but international
cooperation in anti-doping affairs was long restricted to the Council of Europe. In the 1980s,
there was a marked increase in cooperation between international sports authorities and
various governmental agencies. Before 1998, debate was still taking place in several discrete
forums (IOC, sports federations, individual governments), resulting in differing definitions,
policies, and sanctions. Athletes who had received doping sanctions were sometimes taking
these sanctions, with their lawyers, to civil courts and sometimes were successful in having
the sanctions overturned. The Tour de France scandal highlighted the need for an
independent, nonjudicial international agency that would set unified standards for anti-doping
work and coordinate the efforts of sports organizations and public authorities. The IOC took
the initiative and convened the First World Conference on Doping in Sport in Lausanne in
February 1999. Following the proposal of the Conference, the World Anti-Doping Agency
(WADA) was established later in 1999. In the 1990s, there was a noticeable correlation
between more effective test methods and a drop in top results in some sports [14612].
Concern regarding the effects of anabolic steroids on athletes resulted in US Congressional

hearings in 1988. Anabolic steroids were scheduled under Class III of the Controlled
Substance Act in 1990. That same year, the Dubin Commission report, commissioned by the
Canadian government because of concerns regarding the use of public money in sport,
documented widespread abuse of performance-enhancing drugs and poor testing by
Canadian sporting authorities. In 1990, two governmental agencies, the Canadian Sport Anti-
Doping Agency and the Australian Sport Drug Agency, were formed to deal with drugs in
sports. The second Council of Europe Anti-Doping Convention was signed. A multilateral
intergovernmental agreement, the International Anti-Doping Arrangement (IADA) was formed
to promote more effective antidoping practices. The IADA group developed an International
Organization for Standardization (ISO) Publicly Available Specification (ISO/PAS 188730) for
collection of urine samples. This document eventually became the basis for WADA's
International Standard for Testing [12006].
Courtroom, economics and anti-doping
Before any new drugs test is introduced, sports governing bodies must be convinced that it
will stand up to legal challenges. If the doping control authorities lose a courtroom battle, the
consequences can be disastrous. In 1997, the British Athletics Federation went bankrupt,
partly as a result of court costs incurred after the middle-distance runner Diane Modahl
challenged its decision to ban her following a positive test for testosterone. Modahl
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convinced the court that bacterial growth caused by a failure to refrigerate her urine sample
properly could have led to a false positive result [00001].
Expelled team doctor in 2000

The International Olympic Committee (IOC) has expelled a Romanian team doctor from the
Sydney games and barred him from the next two Olympic Games because he gave
champion gymnast, Andreea Raducan, a cold remedy that contained a banned substance
while she was competing at games. Raducan tested positive for pseudoephedrine. The IOC
also stripped the 16-year old athlete of her all round gold medal but allowed her to keep a
gold medal from the team competition and a silver from the vault competition. Prince
Alexandre de Merode, IOC drug chief, said she had taken two tablets of an over-the-counter
cold medication. “The medication was prescribed by the team doctor”, said de Merode. “She
is not directly responsible. The fault falls with the medical doctor. But we have rules and we
have to apply the rules.” The Romanian team doctor, named in wire service reports as
Ioachim Oana, was expelled immediately from the Sydney games and is suspended for the
winter games in Salt Lake City, Utah, USA, in 2002 and the summer games in Athens,
Greece, in 2004 [00001].
USA
Although officials have banned PEDs from Olympic competition since 1967, and the
International Olympic Committee has prohibited AAS use since 1975, it was not until 1991
that the US Congress designated AAS as Schedule III controlled substances. In 2004 the
Anabolic Steroid Control Act amended the Controlled Substances Act and expanded its
definition of anabolic steroids. The new definition, which does not require proof of muscle
growth, identified 59 specific substances (including their salts, esters, and ethers) as
anabolic steroids and listed them as Schedule III controlled substances [14017].
The USADA was formed in 2000 by the US Olympic Committee in part to avoid the
perception of the “fox guarding the hen house.” In 2001, Congress designated USADA “the
official antidoping agency for Olympic, Pan American and Paralympic sport in the United
States.” USADA was given authority to develop a comprehensive national antidoping
program including testing, adjudication, education, and research. USADA and WADA have
jointly worked to advance the science (analytical chemistry, biochemistry, endocrinology,
hematology, laboratory medicine, pharmacology, physiology, sports medicine, and
toxicology) of detection of doping [12006].
The Bay Area Laboratory Cooperative (BALCO) scandal was one of the early examples of
information sharing between law-enforcement and antidoping agencies. BALCO was
providing synthetic anabolic steroids not approved by the Food and Drug Administration and
designed to avoid detection to a number of athletes including Kelli White, Marion Jones, and
allegedly Barry Bonds. Sharing of information between the Internal Revenue Service
Criminal Investigations, local law enforcement, and USADA enabled effective prosecution of
the cases in criminal and sport venues, as appropriate. Prior to 2004, detection of a
prohibited substance or its metabolites or markers was required to be prosecuted for a
violation of the antidoping rules. The 2004 edition of the Code recognized other means for
proving a case of doping, including any reliable information. USADA's prosecution of the first
“nonanalytical positive” case that same year resulted in suspension of the athlete [12006].
Industrialized doping
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In 2003, another significant event in the understanding of the institutional nature of doping
occurred. A syringe was anonymously sent to a WADA-accredited laboratory in Los Angeles
that contained tetrahydrogestrinone (THG), a "designer" steroid that was not known and not
on the current WADA prohibited list, made specifically to avoid detection by modern anti-
doping technologies. This led to a series of investigations resulting in the indictment and
subsequent conviction of individuals running a performance-enhancing program for
professional athletes at the BALCO pharmacy in San Francisco [08006].
In May 2006, Spanish police arrested five people and seized a variety of banned
performance-enhancing drugs and blood-doping supplies at a Madrid doping clinic. Here,
professional athletes would receive medically-supervised injections of hormones and other
performance-enhancing drug regimes. The 40-page police report included a clear paper trail
of doping procedures on at least 50 professional cyclists. The report was given to the
International Cycling Union, which led to the disqualification of 23 professional cyclists,
virtually all the top contenders from the 2006 Tour de France. The final of the 2006 Tour was
also tarnished, as the champion, Floyd Landis, was found to have a positive anti-doping test
for steroids. Landis was stripped of the championship and discharged from his team. At this
writing the result is being challenged by Landis and his legal and medical experts, claiming
that the test was invalid since several errors were made in the collection, analysis and
reporting of the results [006].
In a separate investigation in Paris in 2006, 23 individuals were sentenced to 4 years in jail

for trafficking a cocktail of amphetamines and other performance-enhancing drugs known as
"Belgium Pot" to professional cyclists. In October of that same year, the cricket world was
shocked to learn that two Pakistani fast bowlers, Shoaib Akhtar and Mohammad Asif, tested
positive for the steroid nandrolone [08006].
Laboratory testing
Mass spectrometry has played a decisive role in doping analysis and doping control in
human sport for almost 40 years. The standard of qualitative and quantitative determinations
in body fluids has always attracted maximum attention from scientists. With its unique
sensitivity and selectivity properties, mass spectrometry provides state-of-the-art technology
in analytical chemistry. Both anti-doping organizations and the athletes concerned expect the
utmost endeavours to prevent false-positive and false-negative results of the analytical
evidence. The Olympic Games play an important role in international sport today and are
milestones for technical development in doping analysis. Mass spectrometry has playad an
important role in doping control from Munich 1972 to Beijing 2008 Olympics [08015].
One brief note gave also a general overview on the activity of the antidoping laboratories
accredited by the World Anti-Doping Agency outlining the evolution, over the last four
decades, of the analytical methods and techniques in the detection of prohibited substances
and methods. Special emphasis was given to the future trends of the fight against doping in
sports, as seen from the perspective of a laboratory scientist, in the wider context of fair play,
health protection, and perception of the activity of the antidoping laboratories by the general
public [08016].
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There were 35 WADA accredited laboratories in 2009. Approximately 3,300 additional
samples were analyzed in 2009 compared to 2008, with a slight increase in Adverse
Analytical Findings and Atypical Finding, from 1.84 percent (2008) to 2.02 percent (2009).
Anabolic steroids
The initial test for testosterone in urine was developed by Donike and coworkers, who
showed that administered testosterone appeared in the urine as testosterone glucuronide.
They also showed that for a population of athletes, the ratio of testosterone to
epitestosterone (T/E) had a positively skewed distribution, with a modal ratio of about 1:1.
Initially, an athlete sample having a T/E ratio > 6:1 was considered a doping violation. The
concept of intraindividual reference ranges (as opposed to population-based reference
ranges) was introduced into the T/E test in the early 1990s. Computer programs are now
used to compare an athlete's current sample result to their previous sample results. Results
that are inconsistent with previous results are investigated and could result in targeted testing
or an antidoping rule violation. The measurement of 13C/12C ratios in testosterone and its
metabolites has allowed the differentiation of pharmaceutical testosterone from natural
testosterone. Donike's group also began the concept of the urinary “steroid profile,” which
used a combination of other urinary steroids to increase the sensitivity of the test. Other
antidoping research has identified a del/del genotype of UGT2B17 as the cause of a
subpopulation of individuals who have low (<0.5) T/E ratios in urine, the use of 11 steroids in
urine to improve test sensitivity, new metabolites of testosterone (e.g. testosterone
cysteinate) in the urine, and several substances that affect the metabolism and excretion of
testosterone [12006].
To measure testosterone in the urine to detect doping is not adequate because of large
interindividual and intraindividual differences in urinary steroid concentration. However, the
nearly constant ratio of urinary testosterone glucuronide to epitestosterone glucuronide
became the basis of a better test. Epitestosterone is the 17alpha epimer of testosterone and
has no known physiological function. It is not a metabolite of testosterone [08010]. An upper
normal limit of six was calculated for the testosterone/epitestosterone ratio based upon
population studies. In 1983 the Medical Commission of the International Olympic Committee
(IOC) introduced this value as a criterion for testosterone abuse. Ratios above six should be
considered suspicious, and the person concerned should be subjected to further testing. In
2004 the approved upper limit was set at four [08011].
Adverse findings 2005-2011 from the Doping Control Laboratory of Athens
Onearticle concerns the analysis of the adverse analytical findings (AAFs) and the
appropriate alterations made during the period 2005-2011, so that the Doping Control
Laboratory of Athens (DCLA) obeys the updated World Anti-Doping Agency (WADA) List of
Prohibited Substances. The % AAFs of the DCLA was compared with those of WADA-
Accredited Laboratories. In 2008, the term Atypical Finding was introduced by the WADA
representing a reported but inconclusive result. A characteristic example is when a
testosterone-to-epitestosterone ratio is >4 followed by a negative gas chromatography/
combustion/isotope ratio mass spectrometry result. In a total of about 30,000 athlete
samples, 136 athletes were found with an increased testosterone/epitestosterone ratio and
43 with tetrahydrocannabinol metabolite (THCCOOH) of 427 reported AAFs. Twenty-one
athletes in total were found positive with methylhexaneamine, the 11 found after a batch of
1000 samples was reprocessed. Besides, there were AAFs below their Minimum Required
Performance Level (MRPL). The increasing need for higher detectability imposed new
apparatus, e.g., liquid chromatography/quadrupole/time-of-flight mass spectrometry, whereas
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that for lowering the capital costs and reporting times led to the unification of the screening
method which includes stimulants, diuretics, anabolics and other substances [14019].
The 2005 WADA list
The purpose of the World Anti-Doping Code 2003 and the 2004 Prohibited List is to create a
universal international standard to fight doping in competitive sports. The result of this is a
whole series of changes for doctors with regard to their work with competitive athletes. The
revised definition of doping now includes physicians in the group of persons who can fulfil the
elements of a doping offence. Moreover, the mere possession of substances appearing on
the Prohibited List represents a violation of anti-doping regulations. The 2004 Prohibited List
includes several changes to the Olympic Movement List from 2003. Caffeine, for example,
was removed from the list. Cannabinoids, on the other hand, are now prohibited in
competition for all sports. The same is true for all forms of glucocorticosteroids. Therapeutic
use exemptions in an abbreviated process are possible for the administration of
glucocorticosteroids by non-systemic routes, as well as inhalative therapy with the beta-2-
agonists formoterol, salbutamol, salmeterol, and termbutalin. In other cases, a therapeutic
use exemption is possible using a standard application process. Further changes will
become effective in the 2005 Prohibited List. In 2005, it is essential that beta-2-agonists are
prohibited in and out of competition. HCG and LH are prohibited for all athletes.
Dermatological preparations of glucocorticosteroids are no longer prohibited, and
intravenous infusions will be a prohibited method in 2005, except as a legitimate acute
medical treatment. In cases of violations of anti-doping regulations where it is permissible for
the affected person to furnish proof of exoneration, the burden of proof is not higher than that
required to prove the violation. The sanctions provided for in the World Anti-Doping Code
follow a principle of rules and exceptions which at first glance seems difficult to understand.
In the case of doping violations by physicians, the anti-doping code provides--as a general
rule--for exclusion from sports associations for at least four years. Since several of the
changes are questionable under constitutional aspects, it remains to be seen whether the
World Anti-Doping Code 2003 will allow the achievement of a universal standard to combat
doping [05004].
The 2011 WADA list
International anti-doping efforts are harmonized and regulated under the umbrella of the
World Anti-Doping Code and the corresponding Prohibited List, issued annually by the World
Anti-Doping Agency (WADA). The necessity for a frequent and timely update of the
Prohibited List (as the result of a comprehensive consultation process and subsequent
consensual agreement by expert panels regarding substances and methods of performance
manipulation in sports) is due to the constantly growing market of emerging therapeutics and
thus new options for cheating athletes to illicitly enhance performance. In addition, “tailor-
made” substances arguably designed to undermine sports drug testing procedures are
considered and the potential of established drugs to represent a doping substance is
revisited in light of recently generated information. The list that was published and has been
authoritative from 1 January 2011 comprising a total of 10 different classes of banned
substances (S0–S9), three different groups of prohibited methods (M1–M3), and two classes
of drugs (P1 and P2) being banned from selected sports only. In comparison to the 2010
edition of the Prohibited List, few but significant modifications were made. A major novelty
has been the installation of the S0 section, which interdicts the use of any pharmacological
substance that has not (yet) received approval by governmental health authorities (or where
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development has discontinued) as a human therapeutic agent. This addendum is particularly
important in the light of new drug entities that are not covered by any of the established
classes of banned substances, either by their chemical nature or their biological effects.
Ryanodine receptor-calstabin complex stabilizers, which have been proven to enhance
performance in the laboratory setting are currently undergoing advanced clinical trials but do
not represent compounds of S1–S9. These might exemplify such a new category of
substances. The section S2 (peptide hormones, growth factors, and related substances) was
modified concerning the examples of erythropoiesis-stimulating agents (ESAs) by explicitly
listing hypoxia-inducible factor (HIF) stabilizers, which also represent a considerably
heterogeneous emerging class of substances targeted for clinical approval. In contrast to
these additions to S2, the use of platelet-derived preparations has been legitimized and the
paragraph removed in the 2011 Prohibited List accordingly. The category M2 (chemical and
physical manipulation) was extended by a new paragraph (M2.3) that particularly
emphasizes the illicit nature of the “sequential withdrawal, manipulation and reinfusion of
whole blood into the circulatory system”, a strategy that includes, for example, the so-called
UV-activated autohemotherapy (commonly regarded as alternative medicine). M3 (gene
doping) was split into three sub-groups that define (1) the transfer of nucleic acids or
sequences of these; (2) the use of normal or genetically altered cells; (3) the use of drugs
manipulating gene expression with impact on athletic performance as prohibited methods
[12016].
The 2012 WADA list
As of 1 January, the 2012 Prohibited List International Standard has come into effect,
exhibiting minor but relevant alterations from to the previous 2011 version. In agreement with
its predecessor, the List comprises a total of 10 different classes of banned substances (S0–
S9), three different groups of prohibited methods (M1–M3), and two classes of drugs (P1 and
P2). The latter are banned from selected sports only. The major modifications can be
observed in the sections; S3 (beta2-agonists), S4 (hormone and metabolic modulators), and
M3 (gene doping). In the S3 group, quantitative consideration of formoterol has been
considered with the allowance of a maximum daily therapeutic dose of 36 microg of inhaled
formoterol and a urinary threshold of 30 ng/mL. If the determined quantity in urine exceeds
this level, an adverse analytical finding is reported followed by penalty, unless the athlete can
prove (e.g. by means of a pharmacokinetic study) that the concentrations were reached by
the admissible route and daily dosage. The category S4 has been complemented by a new
subsection named “metabolic modulators”. These host peroxisome proliferator activated
receptor (PPAR)delta agonists such as GW1516 and PPARdelta-AMP-activated protein
kinase (AMPK) axis agonists, such as 5-amino-4-imidazolecarboxamide ribonucleoside
(AICAR). These were formerly listed among gene doping (M3.3) in the previous list.
Following a re-evaluation of the impact of the use of alcohol (P1) and beta-receptor blocking
agents (beta-blockers, P2) on the athletes' performance in selected sport disciplines, the
interdiction of alcohol was lifted for Ninepin and Tenpin Bowling (in agreement/on request of
the Federation Internationale des Quilleurs) and so was the ban of beta-blockers for
bobsleigh, skeleton, curling, modern pentathlon, motorcycling, sailing, and wrestling. In order
to probe for potential patterns of abuse concerning selected substances that are currently not
(or not at all times or at any concentration) prohibited, the established WADA monitoring
programme has been expanded. Besides the stimulants bupropion, caffeine, phenylephrine,
phenylpropanolamine, pipradrol, pseudoephedrine (< 150 microg/ml), and synephrine and
the ratio of morphine over codeine, the prevalence of nicotine, hydrocodone, and tramadol
was to be monitored in-competition. Moreover, the (ab)use of corticosteroids in out-of-
competition periods is acquiring concern and appears as a new item on the 2012 monitoring
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programme. Concerning nicotine and its metabolites, a comprehensive compilation of
monitoring data was published outlining an alarmingly high prevalence of nicotine use in
selected sports disciplines. Further to these explicitly stated drugs, alternative medicine has
necessitated greater attention in order to protect both the spirit of sport and the athletes
themselves from inadvertent anti-doping rule violations. In continuation of the endeavor to
keep pace with the changing trends of doping, manipulation, and innovations and
improvements in analytical chemistry, anti-doping laboratories are urged to enhance their
procedures in terms of comprehensiveness, speed, and/or sensitivity. This, in combination
with the fact that the International Standard for Laboratories allows for the long-term storage
and re-analysis of doping control samples, is considered one of the main aspects causing
deterrence to cheating athletes [13012].
The 2013 WADA list
Compared to its predecessor, the 2013 Prohibited List exhibited only few major modifications
such as the re-categorization of insulins from S2 (peptide hormones, growth factors and
related substances) to S4.5 (hormone and metabolic modulators) and the inclusion of M2.3
(chemical and physical manipulation) into M1.1 (manipulation of blood and blood
components) by respective re-wording of the paragraph. In addition, the maximum daily
therapeutic dose of 36 microg of inhaled formoterol (S3) was increased to 54 microg,
resulting in a permissible urinary concentration of 40 ng/mL (formerly 30 ng/mL). In
agreement with prior protocols, an adverse analytical finding is reported (followed by penalty)
if the determined quantity in urine, including the measurement uncertainty, exceeds the
threshold limit. The finding will be processed as an anti-doping rule violation unless the
athlete can prove (e.g. by means of a pharmacokinetic study) that the concentrations were
reached by the admissible route and daily dosage. In agreement with or on request of
international federations, the interdiction of beta-receptor blocking agents (beta-blockers, P2)
was lifted in selected sport disciplines including ninepin and tenpin bowling, aeronautic,
boules, bridge, and powerboating. This is a continuation of the process initiated in 2012,
where another 7 international federations removed the ban of beta-blockers from their sport
[13009].
In continuation of the 2012 Prohibited List, the 2013 version also contained 12 classes of
prohibited substances (S0–S9 plus P1 and P2) and three categories of prohibited methods
(M1–M3). Compared to its predecessor, the 2013 Prohibited List exhibited only few major
modifications such as the re-categorization of insulins from S2 (peptide hormones, growth
factors and related substances) to S4.5 (hormone and metabolic modulators) and the
inclusion of M2.3 (chemical and physical manipulation) into M1.1 (manipulation of blood and
blood components) by respective re-wording of the paragraph. In addition, the maximum
daily therapeutic dose of 36 microg of inhaled formoterol (S3) was increased to 54 microg,
resulting in a permissible urinary concentration of 40 ng/mL (formerly 30 ng/mL). In
agreement with prior protocols, an adverse analytical finding is reported (followed by penalty)
if the determined quantity in urine, including the measurement uncertainty, exceeds the
threshold limit. The finding will be processed as an anti-doping rule violation unless the
athlete can prove (e.g. by means of a pharmacokinetic study) that the concentrations were
reached by the admissible route and daily dosage. In agreement with or on request of
international federations, the interdiction of beta-receptor blocking agents (beta-blockers, P2)
was lifted in selected sport disciplines including ninepin and tenpin bowling, aeronautic,
boules, bridge, and powerboating. This is a continuation of the process initiated in 2012,
where another 7 international federations removed the ban of beta-blockers from their sport.
In addition to the Prohibited List, WADA has established a monitoring program in order to
283
probe for potential patterns of abuse concerning selected substances that are currently not
(or not at all times or at any concentration) interdicted. The “in-competition” monitoring
program, which included the stimulants bupropion, caffeine, phenylephrine, phenylpropanol-
amine, pipradrol, pseudoephedrine (< 150 microg/mL), synephrine, and nicotine as well as
the ratio of morphine over codeine, hydrocodone, and tramadol in 2012, was complemented
by the analgesic tapentadol in 2013. Further, as in 2012, the (mis)use of corticosteroids in
out-of-competition periods has been investigated [14009].
Category 0
The category S0 of the Prohibited List does not explicitly mention any specific substance;
here, any pharmacological compound not covered by the other classes of prohibited
substances and methods and without “current approval by a governmental regulatory health
authority for human therapeutic use” is considered illicit. Potential candidates for this
category are sirtuin-1 (SIRT1) activating drugs such as SRT1720 the characterization,
metabolism. In case of the proposed routine doping control application, the mass
spectrometer was a QqQ instrument with ESI source operated in positive mode and MRM,
while compound characterization was conducted on a quadrupole-time-of-flight (Q-TOF)
system [13009].
Monitoring program
In addition to the Prohibited List, WADA has established a monitoring program in order to
probe for potential patterns of abuse concerning selected substances that are currently not
(or not at all times or at any concentration) interdicted. The “in-competition” monitoring
program, which included the stimulants bupropion, caffeine, phenylephrine,
phenylpropanolamine, pipradrol, pseudoephedrine (< 150 microg/mL), synephrine, and
nicotine as well as the ratio of morphine over codeine, hydrocodone, and tramadol in 2012,
was complemented by the analgesic tapentadol in 2013. Further, as in 2012, the (mis)use of
corticosteroids in out-of-competition periods has been investigated [13009].
The 2014 WADA list
Identical to the 2013 Prohibited List, the 2014 issue also contains 12 classes of prohibited
substances (S0–S9 plus P1 and P2) and three categories of prohibited methods (M1–M3).
Major modifications compared to the preceding 2013 version include the addition of
vasopressin V2 antagonists (commonly referred to as vaptans) to the subclass of diuretics
and the addition of cathinone and its analogues as well as trimetazidine to Section S6
(stimulants). Moreover, as of 1 September 2014, substances acting as hypoxia-inducible
factor (HIF) activators, such as xenon and argon, have been listed as explicitly prohibited,
necessitated by recently surfaced documents on an arguably licit and extensive use of
xenon/oxygen mixtures among selected athletes. WADA further continued the monitoring
programme in order to generate information on potential patterns of abuse concerning
defined substances that are currently not (or not at all times or at any concentration)
prohibited. The 2014 in-competition monitoring programme was complemented by the
narcotic agent mitragynine, covering now collectively the ratio of morphine over codeine,
hydrocodone, tramadol, tapentadol, and mitragynine as well as the stimulants bupropion,
caffeine, phenylephrine, phenylpropanolamine, pipradrol, pseudoephedrine (<
150 microg/mL), synephrine, and nicotine. Further, as in 2013, the potential (mis)use of
corticosteroids in out-of-competition periods has been monitored [14715].
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Erythropoesis-stimulating agents
Besides EPO and its derivatives, hypoxia-inducible factor (HIF) stabilizers/activators are
considered as relevant to doping controls. This class of compounds is particularly
heterogeneous and includes low molecular mass organic compounds such as FG-4592[27]
as well as the recently added noble gas xenon and, as of January 2015 explicitly named, the
inorganic substance cobalt. Ionic cobalt, more specifically Co2+ administered as CoCl2, was
the method of choice for the treatment of renal and non-renal anemia in the pre-EPO era.
Due to serious adverse effects and an undesirable cost-benefit-ratio, CoCl2 was removed
from the therapeutic arsenal [14715].
Amendment to the 2014 Prohibited List
Having been alerted to the substance of Xenon and its potential performance enhancing
characteristics in February, the WADA List Committee discussed the matter during its April
meeting. Following its consideration, the Executive Committee approved the option to modify
Section S.2.1 of the 2014 Prohibited List, which will be effective following the required three-
month notice period:
S2. Peptide hormones, growth factors and related substances

The following substances, and other substances with similar chemical structure or similar
biological effect(s), are prohibited:
Erythropoiesis-Stimulating Agents [e.g. erythropoietin (EPO), darbepoetin (dEPO), hypoxia-

inducible factor (HIF) stabilizers and activators (e.g. xenon, argon), methoxy polyethylene
glycol-epoetin beta (CERA), peginesatide (Hematide)]. The process means that the
amendment to the 2014 Prohibited List will not come into effect until three months after
UNESCO has appropriately communicated the amendment to all States Parties after the
Consensus meeting on antidoping in sport, Zürich, Switzerland, November 29-30 2013
As of 1 January 2015, the revised 2015 World Anti-Doping Code will be operational in the
fight against doping. This will be binding for all stakeholders who unanimously approved the
revised code at the “World Conference on Doping in Sport” in Johannesburg, South Africa on
15 November 2013 [14444].
Implementation of the World Anti-Doping Code 2015
A medical and scientific multidisciplinary consensus meeting was held from 29 to 30

November 2013 on Anti-Doping in Sport at the Home of FIFA in Zurich, Switzerland, to
create a roadmap for the implementation of the 2015 World Anti-Doping Code. The
consensus statement and accompanying papers set out the priorities for the antidoping
community in research, science and medicine. The participants achieved consensus on a
strategy for the implementation of the 2015 World Anti-Doping Code. Key components of this
strategy include:
- sport-specific risk assessment

- prevalence measurement
- sport-specific test distribution plans
- storage and reanalysis
- analytical challenges
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- forensic intelligence
- psychological approach to optimise the most deterrent effect
- the Athlete Biological Passport (ABP) and confounding factors
- data management system (Anti-Doping Administration & Management System
(ADAMS)
- education
- research needs and necessary advances
- inadvertent doping and
- management and ethics: biological data
True implementation of the 2015 World Anti-Doping Code will depend largely on the ability to
align thinking around these core concepts and strategies. FIFA, jointly with all other engaged
International Federations of sports (IFs), the International Olympic Committee (IOC) and
World Anti-Doping Agency (WADA), are ideally placed to lead transformational change with
the unwavering support of the wider antidoping community. The outcome of the consensus
meeting was the creation of the ad hoc Working Group charged with the responsibility of
moving this agenda forward [14018].
The category S0 of the Prohibited List does not explicitly mention any specific substance;
here, any pharmacological compound not covered by the other classes of prohibited
substances and methods and without “current approval by a governmental regulatory health
authority for human therapeutic use” is considered illicit. Potential candidates for this
category are sirtuin-1 (SIRT1) activating drugs such as SRT1720 the characterization,
metabolism, and analysis of which was recently presented. Using a set of five model SIRT1
drug candidates with thiazole-imidazole pharmacophore (as in SRT1720), the mass
spectrometric behaviour under ESI-CID conditions was studied and the detection of the
active substances in human plasma was demonstrated. The analytical system consisted of
an LC equipped with a reversed-phase C-18 column (2 x 50 mm, particle size 3 microm) and
aqueous acetic acid (0.1 %, containing 5 mM ammonium acetate) and acetonitrile were used
as solvents A and B, respectively. At a flow rate of 350 microL/min, the analytes were
separated by gradient elution. In case of the proposed routine doping control application, the
mass spectrometer was a QqQ instrument with ESI source operated in positive mode and
MRM, while compound characterization was conducted on a quadrupole – time-of-flight (Q-
TOF) system. Plasma samples (100 microL) were enriched with eightfold deuterated
SRT1720 as ISTD and 100 microL of water prior to protein precipitation by the addition of
acetonitrile (400 microL). The supernatant was concentrated, reconstituted, and analyzed by
LC-MS/MS. The approach allowed for limits of detection (LODs) between 0.1 and 1 ng/mL at
recoveries of 90-98 percent, demonstrating the fitness-for-purpose of the method [14009].
In order to expand the analytical possibilities for SIRT1 activating drugs to urine samples, in
vitro metabolism studies were conducted to provide insights into metabolic pathways and
potential target analytes in human urine. Mainly hydroxylation and N-oxidation were
observed, and sites of modifications were localized by chromatographic-mass spectrometric
and chemical methodologies. Eventually, an existing routine doping control assay consisting
of enzymatic hydrolysis and LLE with subsequent LC-MS/MS analysis was expanded to
include the new target analytes and LODs of 0.5 ng/mL were accomplished. In the absence
of authentic administration study urine samples, the screening for in vitro/in silico generated
metabolites has proven to be a viable means to identify atypical components in doping
control samples [14009].
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Important persons in anti-doping
Richard W Pound
When talking about performance-enhancing drugs in sport, Richard Pound does not mince
words: “I think it's my job to be everyone's face”, says the 63-year-old Montreal lawyer and
chairman of the World Anti-Doping Agency (WADA), “This is not a diplomatic problem; this is
cheating.” Pound, who swam for Canada in the 1960 Summer Olympics and the 1962
Commonwealth Games, joined the Canadian Olympic Committee when he retired from
competition, eventually becoming its president. He was elected to the Committee in 1978
and put in charge of negotiating television and sponsorship contracts. The lucrative deals he
struck have been credited with freeing the organisation from its dependence on government
largesse. “I was happy as a clam doing that”, Pound recalls. Then, in 1998, in the wake of a
major doping scandal, the then president of the International Olympic Committee Juan
Antonio Samaranch was quoted in the press as saying he thought performance-enhancing
drugs should be allowed as long as they were not harmful, and that the Committee's list of
banned substances was too long. A firestorm of criticism erupted. “It got worse and worse”,
Pound says, “and finally Samaranch had to call an emergency meeting of the executive
board and asks, ‘How are we going to get out of this?’ And we all said, ‘We?’” Pound argued
for setting up an anti-doping agency independent of the International Olympic Committee and
other sports organisations, “because nobody believes the IOC anymore, nobody believes the
international sports federations, like the UCI (Union Cycliste Internationale), and nobody
trusts the national Olympic committees, like the Canadian or US Olympic committees, to
blow the whistle on our own people”. Samaranch eventually asked Pound to lead the effort. “I
said I'd do it for a couple of years to get it up and running and after that I'm out of here. That
was 6 years ago.” When he started the unpaid job, Pound knew nothing about doping: “The
first thing that became clear to me when we started out was that when all is said and done;
far more is said than done. There was an awful lot of lip service being paid and not very
much actual work.” Many coaches and officials had got in to the habit of looking the other
way, Pound says, “We knew our athletes were facing athletes who were doped – East
Germans, athletes from the Soviet Union – and so if our kids leveled the playing field in their
own way, well nobody looked too hard at it. What rules there were, Pound noted, also varied
between organisations and countries. “The rules were all over the ballpark: one sports
organisation had a life ban for the first positive test and another had a 2-week ban that you
could serve between Christmas and New Year's.” So, in February, 1999, the International
Olympic Committee convened a meeting in Lausanne, Switzerland, to draw up the Lausanne
Declaration in Sport, which led to the formation of WADA later that year. The first order of
business was to draft and implement a uniform set of anti-doping rules. These were adopted,
after long negotiations, in 2003 at the second World Conference on Doping in Sport, and
came into effect on Jan 1, 2004. The Code has now been adopted by most major
international and national Olympic organisations and, this October, by UNESCO in the
International Convention Against Doping in Sport. “So now everybody will be playing off the
same sheet; same rules for all athletes, all sports, all countries, and no differences between
domestic laws and sports rules”, Pound says. In addition to drawing up and refining the
Code, WADA maintains and updates a list of banned substances, helps their members to
adopt best practices, and funds scientific research, in particular into the development of drug-
detection technology. But the key to solving the problem of doping in sport will be the
education of athletes and the public, Pound says: “Part of my job is to make it as visceral as I
can: I ask ‘How would you like it if your kid, who had trained for 10 years to go to the
Olympics, lost by a tenth of a second to somebody who was all doped up?’” Now that the
Code has been adopted, Pound notes that the next challenge for WADA is to make sure the
Code is applied. This challenge is a difficult one, but he is hopeful. “If you and I had had this
conversation in 1999 when WADA was being founded, and I was to say to you within 5 years
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we're going to have an international code that will apply to all countries, all sports, in place,
adopted by 202 national Olympic committees, 75 international sports federations, the IOC,
and we'd have an international convention under the umbrella of UNESCO unanimously
approved, you'd look at me and say, ‘You're out of your mind” [05006].
Arne Ljungqvist
Professor emeritus Arne Ljungqvist from Karolinska Institutet, Stockholm, who has served in
various high positions in the IOC, International Association of Athletics Federations (IAAF),
Swedish Sports Confederation, to mention some of them, has dedicated a great deal of his
life to service in sports and sports medicine. Arne Ljungqvist has especially dedicated his
career to the fight against doping and to the protection of the health of the athletes. It all
started over 60 years ago when Arne became a Swedish senior champion in high jump in
1951, jumping as high as 201 cm. Arne was multi-talented and won also the Swedish junior
championships in pole vault and javelin. He was one of the favourites for the Gold medal in
the high jump competition in the Olympic Games in Helsinki 1952, but unfortunately could not
compete because of an injury that he sustained during a medical student carnival in the
autumn of 1951 when a group of us were jumping for the general public in the streets with
numerous hard landings on asphalt. Arne had to end his jumping career as his injury could
not be cured, and it was not until the 1960s that the diagnosis of patellar tendinopathy could
be made. Arne, however, continued his medical studies and soon became very successful.
Arne was appointed professor at the Karolinska Institutet in 1972 because of his excellent
medical research in the fields of renal and cardiovascular diseases and, later, oncology. He
held several high professional positions such as Vice Dean of Medical Faculty, Karolinska
Institutet, 1972–1977; Pro-Rector, Karolinska Institutet, 1977–1983; Chairperson,
Department of Pathology and Cytology, Karolinska Hospital, 1983–1992; President, Swedish
Council of Sports Research, 1980–1992; Dean Swedish School of Sport and Physical
Education, 1992–1996; and President of the Swedish Cancer Society, 1992–2001.
Interestingly enough, he took time to serve Chamberlain to His Majesty the King of Sweden,
1977-1986 and has since then been Lord in Waiting to His Majesty the King of Sweden. In
1971, Arne returned to sport as he was elected to the Board of the Swedish Athletics
Association, where in 1973 he became chairperson. In this time period, athletes used all
kinds of medicines to enhance their bodies to achieve success. An anonymous survey
among Sweden’s best athletes indicated that nearly half of them were using anabolic
steroids, which indeed was legal until 1975, when reliable tests had finally been developed to
identify users. Arne realised that something had to be done to create a healthy and ethical
environment in sports. In 1975, he became a member of the Swedish Sports Confederation
and was part of the initiation of the Swedish Commission against doping in 1977 with its own
doping rules by 1979. During 1989–2001, Arne became the President of the Swedish Sports
Confederation and in 1989 a member of the Swedish Olympic Committee. Arne has had an
outstanding international career, which started when, in 1976 he was elected to become a
Council Member in IAAF and elected Vice President in IAAF in 1981. He served in this
position until 1999 and became thereafter the Senior Vice President of IAAF until 2007 and
Chairperson of the Medical Committee and Anti-Doping Commission. Since 1987 he has
been a Member of the IOC Medical Commission and in 2003 he was appointed Chairperson
of the Medical Commission of IOC. In 1994, Arne was elected Member of the IOC. When
WADA was founded in 1999, Arne became the Member of WADA’s Foundation Board and
Chairperson of WADA’s Health, Medical and Research Committee. Since 2003, Arne has
been a Member of WADA’s Executive Committee and since 2008 has been WADA’s Vice
President. Arne has been the front-line fighter against doping and his name is today one of
the world's most respected within international sports. Arne’s contributions in the fight against
doping are second to none. He has been an athlete himself, which makes him understand
the special language that is present in the athletic situation and also in the locker rooms. In
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his autobiography “Doping's Nemesis”, Arne gives an unrivalled insider’s view of the biggest
dope scandals over the years, including the Ben Johnson and Balco affairs and the history of
the Greek sprinters at the Athens Olympics in 2004. Arne’s actions together with the Italian
police during the Torino Olympics 2006 against the Austrian team are classic. Doping seems
to be steadily on the decline, and there is no doubt that Arne has played a key role in this
successful work. Arne’s legacy in the fight against doping is lasting [13014].
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ORGANISATION OF ANTI-DOPING
The fight against the use of performance-enhancing drugs in sports has been in effect for
nearly 90 years. The formation of the World Anti-Doping Agency in 1999 was a major event
because an independent agency was entrusted with harmonization of the antidoping
program. In addition to sports governing bodies, governments have endorsed WADA and its
programs by signing a United Nations Education, Science, and Cultural Organization
Convention on Doping. The first step in the harmonization process was the development of
the World Anti-Doping Program. This program consisted of five documents – the Code, the
International Standard for Testing, the International Standard for Laboratories, the Prohibited
List, and the International Standard for Therapeutic Use Exemptions – which unified the
approach of the international federations and national antidoping agencies in applying
antidoping rules. For laboratory testing, the International Standard for Laboratories
establishes the performance expectations for and competence of laboratories recognized by
WADA, including accreditation under ISO/IEC 17025. The antidoping rules are adjudicated
by arbitration using the internationally recognized Court of Arbitration for Sport [10006].
In the current World Anti-Doping Code, there are already eight means to an Anti-Doping Rule
Violation (ADRV) and it is not simply the presence of a prohibited substance in a biological
matrix, that is, a positive analytical test that can lead to sanctions. Article 2.2 states that the
use of a prohibited substance may be established by any reliable means including witness
statements, documentary evidence or evaluations of longitudinal profiling. Other sections of
Article 2 refer to tampering, possession and trafficking as well as administering or attempting
to administer prohibited substances. It is clear that the athletes’ entourage is the target of
some of these sections [14015].
After 1.5 years of widespread stakeholder consultation, a revised World Anti-Doping Code
and International Standards were adopted at the 4th World Conference on Doping in Sport in
November 2013 and will come into effect in January 2015. The importance of investigations
has been strengthened and the International Standard on Testing (IST) has been renamed
the International Standard on Testing and Investigations (ISTI) to reflect that change. As
examples of new elements, there are requirements for antidoping organisations to
automatically investigate Athlete Support Personnel in the case of any ADRV involving a
minor or when those personnel have provided support to more than one athlete found to
have committed an ADRV. There are further sections that address the intimidation of
potential witnesses as well as a new section on prohibited association with those athlete
personnel who have been sanctioned and serving a period of ineligibility. Athletes were
particularly adamant that athlete support personnel, including doctors and coaches, should
suffer more significant consequences as result of encouraging or aiding doping behaviour
[14015].
An international background to the anti-doping movement
Besides blatant over-commercialization, there is no more ominous threat to sports than

doping. Drug-use methods are steadily becoming more sophisticated and ever harder to
detect, increasingly demanding the use of complex analytical procedures of biotechnology
and molecular medicine. It should also be mentioned in passing that doping is not limited to
human sport competitions but is also practiced in animal sports, e.g. equestrian sports.
Underlying this distressing phenomenon is regrettably a worldwide pervasive attitude that
represents the exact contrary of the original and declared aims of the event as promulgated
by de Coubertin in 1903, namely, the aspiration ‘‘not to have conquered but to have fought
290
well’’. It is important to note that there is frequently differentiation between the official
methods employed for anti-doping detection in WADA/IOC laboratories and strategies
applied experimentally in various other laboratories. However, laboratories that are not
accredited by WADA may apply methods and develop strategies, such as WADA
laboratories, in support of the Athlete Biological Passport Program, where all data produced
by laboratories can be collated. Over the past few decades, doping has become ever more
complex and widespread, increasingly involving exploitation of the fields of endocrine-
pharmacology and molecular biology. It thus currently represents not only an individual
health hazard but also a menace to society itself, undermining the principles and significance
of all the great sports events, and in particular the Olympic games. Faced with this problem,
the scientific world is today striving to confront the challenge, in particular in regard to the
recent development of hormones abuse, ever more complex methods and new technology
being deployed for the task. It is evident that anti-doping policy should proceed to
implementation of newly developed analytical methodology and advanced instrumentation as
part of a strategy to clearly distinguish between the use of legitimate medication and the use
of illicit substances. Furthermore, a multidimensional strategy to counter the scourge needs
to be developed combining strict ‘‘prohibitionist’’ measures with preventive-educational
programs. Meanwhile, the effort needs also to entail an individualized approach,
incorporating counselling on a personal basis, which takes strongly into account the social
and psychological background of each athlete. Meanwhile, with regard to athletics, there
must be full support of the effort exerted by the WADA to detect banned substances and
compounds so as to eradicate doping, accompanied by legislative changes and longer
disqualifications. By continuing to upgrade collaboration with national Anti-Doping Agencies,
WADA will be enabled to considerably improve efficacy, thereby gaining ever better control
of doping, consequently reducing fraud while, vitally, lessening the health risks incurred by
athletes. The prevention of harm to the athlete and the guarantee of fair play should be the
target [12011].
The international sports community has recognized for many years the dangers of all forms
of doping, but it has been only in the recent past that serious and increasingly effective
regulatory mechanisms have been put into place for the detection and control of drug-based
doping in sports. It has been clear that, given the opportunity, athletes and their trainers and
handlers will resort to many illicit techniques and substances to provide a competitive
advantage in sports. One should only remember the pervasive and officially sanctioned and
operated doping programs established in East Germany between 1970s and 1980s; how
effective these were in the short term, and how harmful these were to the athletes in the long
term. It seems very likely that the world of sports will continue to seek out new drugs and
stealthy drug delivery methods and even gene-based enhancement to ensure victory in
competition. Athletics represents one of the provinces of human activity, most susceptible to
the application of existing and future advances in the field of human gene therapy, for the
enhancement of nondisease human traits. Modern athletics is as much an entertainment as it
is a sport and is sodden with huge amounts of money to assure the victories and records that
the public demands. Athletic events are also some of the most powerful instruments for
international politics. The prestige, nationalism, and jingoism compel our political institutions
to demand victory. Finally, athletes are by nature risk-takers who are driven to compete,
excel, and win, even at the cost of injury and other harm to themselves. But even worse,
athletes are highly vulnerable to potentially harmful manipulation by dishonest and venal
rogue trainers, sports technicians, and sports associations and federations who disregard the
ideals of sports and the welfare of the athletes in the interests of victory at all cost. The fact
that a sport is already filled with many pervasive drug-based forms of doping should convince
even the most skeptical that all current and future advances in pharmacology, sports
physiology, and sports medicine, whether based on ever more sophisticated drugs, gene
transfer technology, or other still unrecognized technologies, will be applied to the world of
291
sports and will almost certainly occur before the underlying technology is known to be
effective and truly safe [06006].
Taking as an example the Tour de France, it can be posited that in the last century, in
virtually each year the winner and/or runner-ups were either known to have used doping or
strongly suspected of having done so. Aspiring to organize a Tour without doping can
therefore be seen as trying to invent a Tour that has never existed before. The difference
between earlier and more recent Tours, admittedly important, is the kind of performance
enhancing technology that has been made available by the biomedical revolution over the
last decades. If in the early days of sports the arsenal of performance enhancing compounds
was quite limited, today’s advances of biomedical science have indeed opened up Pandora’s
box with unlimited possibilities but also increased health risks. Since a doping culture has
always been part of cycling it is quite understandable that these new possibilities from bio-
medical research were, and still are being exploited for performance enhancement practices
by cyclists and their entourage. WADA’s claim that a culture of doping-free sport will develop
and help attain the eradication of doping in sports remains to be proven; for competitive road-
cycling, recent publications suggest that although doping practices certainly have changed, a
culture of doping in professional cycling still prevails. A provocative editorial in the journal
Nature in 2007 proposed that perhaps the Tour de France should be the first competition to
accept pharmacological performance enhancement [12012].
World Anti-Doping Agency (WADA)
The World Anti-Doping Agency (WADA) was established in 1999 as an independent,

international agency with the aim of creating an environment in world sport that is free of
doping. WADA and associated anti-doping organisations such as the Australian Sports Anti-
Doping Authority (ASADA) strive to ensure that there is a “level playing field” in high-
performance sport and to optimise the safety and welfare of athletes. The World Anti-Doping
Code (the Code) is the document that provides consistency of anti-doping policies across
sports and across international boundaries. It is based on five international standards aimed
at bringing consistency among anti-doping organisations. It covers:
 testing and investigations

 laboratories
 therapeutic use exemptions
 the list of prohibited substances and methods
 protection of privacy and personal information.
The world of sports doping is constantly changing. One of the key functions of WADA is to
support high-quality research in order to stay abreast and ahead of individuals and
organisations who seek to illegally enhance sporting performance. The Code also requires
frequent updating to adapt to changing knowledge and the changing doping environment. A
new Code was introduced in 2015 with ramifications for athletes, sporting organisations and
medical practitioners who deal with high-level athletes. Athletes bear strict liability for any
substances found within their bodies. As some commonly prescribed drugs are prohibited in
sport, it is crucial that medical practitioners and others advising athletes have access to up-
to-date anti-doping information. Exemptions may need to be obtained if the athlete requires
the therapeutic use of a drug [150057].
To improve the fight against this new potential kind of abuse, the International Olympic
Committee (IOC) and national sports federations collaborated in 1998 to establish the World
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Anti-Doping Agency (WADA), an agency jointly funded by the IOC and cooperating nations
and committed to develop programs for detection and control of athletic doping. It carries out
its tasks by compiling and constantly updating a list of substances and methods that are
inconsistent with the ideals of sports and that should be banned from athletic competition. It
is also responsible for developing and validating new, scientifically sound detection assays
and implementing effective international programs for incompetition and out-of-competition
screening of athletes. The WADA has implemented its program on drug control in sports by
issuing and continually updating the world Anti- Doping Code, including a list of banned
substances and methods, the latest of which is presented as an appendix to this volume
[06006].
One article provided a review of the leading role of the World Anti-Doping Agency (WADA) in
the context of the global fight against doping in sport and the harmonization of anti-doping
rules worldwide through the implementation of the World Anti-Doping Program. Particular
emphasis is given to the WADA-laboratory accreditation program, which is coordinated by
the Science Department of WADA in conjunction with the Laboratory Expert Group, and the
cooperation with the international accreditation community through International Laboratory
Accreditation Cooperation and other organizations, all of which contribute to constant
improvement of laboratory performance in the global fight against doping in sport. A
perspective is provided of the means to refine the existing anti-doping rules and programs to
ensure continuous improvement in order to face growing sophisticated challenges. A
viewpoint on WADA's desire to embrace cooperation with other international organizations
whose knowledge can contribute to the fight against doping in sport is acknowledged
[12010].
Background
Athletes have a long history of using substances in an attempt to gain an advantage in

sporting competitions. The ancient Greeks and Romans used herbs, fungi, poppy seeds and
stimulants such as strychnine in order to boost performance. In the modern era, this practice
continued mostly with the use of stimulants and narcotics. Sports federations took notice and
in 1928 the International Association of Athletics Federations (IAAF) became the first
federation to prohibit the use of performance-enhancing drugs (PEDs), although there would
be no testing in sport for another 40 years. Amphetamine use was involved in the deaths of
cyclists Knud Jensen and Tommy Simpson in the 1960 Olympic Games and the 1967 Tour
de France respectively: this spurred the development of the International Olympic
Commissions (IOC) Medical Commission, which published the first IOC Prohibited List in
1967. This became the de facto Prohibited List for Olympic Sport Federations. The “Festina
affair” (1998 Tour de France), where a team trainer's car was found to contain a panoply of
PEDs, was the catalyst to create a new organisation to harmonise, coordinate and promote
the fight against doping in sport in all its forms. The IOC convened the first World Conference
in Doping in Sport in 1999, which resulted in the formation of the World Anti-Doping Agency
(WADA) [13015].
National antidoping organizations (NADOs)
Doping control for national- and international-level athletes has undergone major changes in
the past few years, and will continue to change at an accelerated rate. National antidoping
organizations (NADOs) such as the United States Anti-Doping Agency (USADA) are being
established by major nations to work with national governing bodies of sport. The World Anti-
Doping Agency has been established to coordinate worldwide antidoping efforts with the
NADOs and international federations of sport, and to implement a recently drafted World
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Anti-Doping code, which clarifies the definition of doping and establishes procedures to
harmonize international efforts in sample collection process, testing laboratory accreditation,
result reporting, and result adjudication. A number of substances and methods currently used
in doping present serious challenges to the scientific community, and are described briefly. In
addition, brief descriptions of other issues of significance to doping control, including the role
of physicians in doping and the operation of the USADA, are presented [03026].
Pre-WADA history
In the modern era, doping practice continued mostly with the use of stimulants and narcotics.
Sports federations took notice and in 1928 the International Association of Athletics
Federations (IAAF) became the first federation to prohibit the use of performance-enhancing
drugs (PEDs), although there would be no testing in sport for another 40 years.
Amphetamine use was involved in the deaths of cyclists Knud Jensen and Tommy Simpson
in the 1960 Olympic Games and the 1967 Tour de France respectively: this spurred the
development of the International Olympic Commissions (IOC) Medical Commission, which
published the first IOC Prohibited List in 1967. This became the de facto Prohibited List for
Olympic Sport Federations. The “Festina affair” (1998 Tour de France), where a team
trainer’s car was found to contain a panoply of PEDs, was the catalyst to create a new
organisation to harmonise, coordinate and promote the fight against doping in sport in all its
forms.3 The IOC convened the first World Conference in Doping in Sport in 1999, which
resulted in the formation of the World Anti-Doping Agency (WADA) [13015].
One study investigated the anti-doping policy promoted by the IOC historical sociologically
focusing on the period from 1968 to 1999. Public opinion surrounding doping control has
emerged as a large amount of drug possession by athletes who had participated in the 1952
Olympics was caught, as well as following the acident where an athlete had died during the
competition as a result of doping. From 1960, as many doping cases in sports games were
exposed, several international organizations proclaimed fight against doping in order to seek
a preventive measure. In 1961, the IOC newly established a medical commission within the
organization. It was decided to implement doping control and female sex testing at the same
time for all athletes who participated in the 1967 Olympics, and they were implemented from
1968 winter and summer Olympic Games. In 1971, the provisions for the tests were
prescribed as mandatory on the IOC charter. From 1989, the OCT system was introduced as
a measure to overcome limitations of the detection during competition period. As political
problems and limitations emerged, WADA (World Anti-Doping Agency) was established in
1999 to professionally manage and push for doping control. Female sex testing policy
contributed to preventing males from participating in female competition by deceiving their
gender to some extent. However, it was abolished due to strong public condemnation such
as women's rights issues, social stigma and pain, and gender discrimination debate. In 1984,
a doping control center was established in Korea, which enabled drug use or doping in the
sports world to emerge to the surface in our society. Korea Sports Council and KOC articles
of association that supervise doping related matters of Korean athletes were revised in 1990.
The action of inserting doping related issue in the articles of association was taken 20 years
after the start of IOC doping policy. Beginning with two international competitions in the
1980s, Korean athletes experienced doping test directly, yet education about doping was
limited. However, some national team level athletes tested positive on the doping test and
underwent disciplinary action. In addition, athletic federation or leaders acquiesced athletes
doping made secretly; this indicated that South Korea was also not free from doping. It was
found that Korea world of sports showed very passive countermeasures and development
process [14622].
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The code
WADA is a unique, independent body representing equally sport and the governments of the
world. The World Anti-Doping Code is the core document on which anti-doping programmes
are modelled. The first version of the Code came into effect in January 2004. There are
presently over 600 signatories, including almost all the world's sport federations. The Code
applies to Athletes, as defined by their national anti-doping organisations (NADOs) or
international federations. Who is considered an athlete for anti-doping purposes may vary
widely and a NADO may still test recreational athletes but not apply all elements of the Code,
for example, the requirement for whereabouts or advanced therapeutic use exemptions.
Athletes may be subjected to sanctions based on possession or trafficking of prohibited
substances and not simply due to a positive doping test. However, it is important to be aware
that criminal legislation exists in certain countries (e.g. for narcotics) which may be in addition
to, or completely separate from, anti-doping sanctions [13015].
Doping in sport presents an ongoing challenge to fair competition and the health of athletes.
The paucity of drug testing in a number of countries, varying sanctions for the same offence
and the way in which some athletes have been able to avoid out-of-competition testing are
causes of concern. World Anti-Doping Code (WADC) globally addresses doping in sport and
represents a worldwide code for adoption by all nations and all sports. Its purpose is to
protect the right of the athlete to participate in doping-free sport, thereby promoting health,
fairness and equality for athletes world wide, and to ensure there are harmonised,
coordinated and effective anti-doping programs. The code provides international standards
for laboratories engaged in drug testing, the prohibited list of drugs and the granting of
therapeutic use exemption. Education of athletes and ongoing research are aspects
encouraged in the code. At the Second World Conference on Doping in Sport held in
Copenhagen in March 2003, 80 countries were represented [03027].
Definitions
It has never been possible to have a simple one sentence definition of doping. In the code,
the definition of doping is the occurrence of any one or more of the following eight anti-
doping rule violations [03027]:
1. The presence of a Prohibited Substance or its Metabolites or markers in an

athlete's bodily specimen
2. Use or attempted use of a prohibited substance or a prohibited method
3. Refusing or failing to submit to sample collection or otherwise evading sample
collection
4. Violation of requirements regarding athlete availability for out of competition
testing including failure to provide required whereabouts information and
missing tests
5. Transferring or attempting to tamper with any part of doping control
6. Possession of prohil~ited substances and methods
7. Trafficking in any prohibited substance or prohibited method
8. Administration or attempted administration of a prohibited substance, or
prohibited method to an athlete assisting, covering up or any complicity
involving an anti-doping rule violation or attempted violation
Fundamental of the doping tests
A new version of the Code came into force on January 1st 2015, introducing, among
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other improvements, longer periods of sanctioning for athletes (up to four years) and
measures to strengthen the role of antidoping investigations and intelligence. To
ensure optimal harmonization, five International Standards covering different
technical aspects of the Code are also currently in force: the List of Prohibited
Substances and Methods (List), Testing and Investigations, Laboratories,
Therapeutic Use Exemptions (TUE), and Protection of Privacy and Personal
Information. Adherence to these standards is mandatory for all antidoping
stakeholders to be compliant with the Code. Among these documents, the eighth
version of International Standard for Laboratories (ISL), which also came into effect
on January 1st 2015, includes regulations for WADA and ISO/IEC 17025
accreditations and their application for urine and blood sample analysis by antidoping
laboratories. Specific requirements are also described in several Technical
Documents or Guidelines in which various topics are highlighted such as the
identification criteria for gas chromatography (GC) and liquid chromatography (LC)
coupled to mass spectrometry (MS) techniques, measurements and reporting of
endogenous androgenic anabolic agents (EAAS), and analytical requirements for the
Athlete Biological Passport (ABP). Compounds are divided into nonthreshold
substances, for which their simple identification could be considered an adverse
analytical finding (AAF), and threshold substances (e.g., ephedrine and derivatives,
salbutamol, and carboxy-THC), banned above a fixed level and for which quantitative
determination in the biological sample is needed. To obtain homogeneous results
between laboratories, Minimum Required Performance Levels (MRPL) for analytical
methods have been established by WADA, indicating the minimum capabilities for
the detection of nonthreshold substances; these values do not apply to threshold
compounds, which are covered by other dedicated documents [150058].
Urine and blood (whole blood, serum, and plasma) are considered the matrices of
choice for routine antidoping analysis. The advantages of urine samples include its
noninvasive collection and accessibility to large volumes of matrix, whereas blood
collection is still considered invasive and with a limited volume. For these reasons,
the majority of antidoping controls is still carried out on urine, even if the percentage
of blood testing is continuously increasing. Therefore, determination of the presence
and/or absence of a doping agent in urine is routinely carried out through a common
workflow including an initial testing procedure (screening) followed by a confirmation
procedure, if applicable. The screening step must be fast, selective, and sensitive to
limit the risk of false-negative and false-positive results. In the case of a suspicious
result, the latter should be established with a confirmation procedure targeting the
potentially incriminating substance(s), including possible metabolite(s). To achieve
this, and considering the important chemical diversity and wide range of
physicochemical properties of forbidden substances (approximately 250
compounds), antidoping laboratories should use multiple analytical techniques,
including immunological, biochemical, and chromatography–mass spectrometry
methods [150058].
Overview of prohibited drugs
Medical practitioners need to be aware that, when treating athletes who are subject to drug
testing, certain medicines that are not illegal to prescribe to the general community could
result in the athlete breaching anti-doping rules. Some of these prohibited medicines are
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likely to stand out as being of concern for athletes, for example anabolic steroids, growth
hormone and stimulants. Other medicines may not be so obvious, for example insulin,
probenecid, diuretics, beta blockers and terbutaline. Some medicines such as insulin are
banned for their direct anabolic effects while other medicines such as diuretics and
probenecid are banned because they can be used to mask banned substances in the urine.
Beta blockers can reduce tremor in particular sports such as golf and shooting.
Methylphenidate, a phenethylamine derivative, is banned in sport because of its stimulant
effects. There are some drugs that are banned during competition, but are not banned out of
competition, for example oral corticosteroids. Other drugs, such as salbutamol and
pseudoephedrine, are permitted, but are prohibited above a threshold serum concentration.
Salbutamol can be taken by inhaler without incurring an anti-doping rule violation, but
nebulised salbutamol could put the serum concentration beyond the prohibited level. An
athlete taking more than 1600 microgram of salbutamol by inhaler, within a 24-hour period,
may potentially exceed the threshold serum concentration. Athletes who have a therapeutic
use exemption for a diuretic and are also using inhaled salbutamol may require another
therapeutic use exemption for their salbutamol. This is because the diuretic could increase
their salbutamol concentration above the prohibited threshold. Most medical practitioners
working with high-performance athletes refrain from prescribing pseudoephedrine on the day
of competition. While an athlete could feasibly take a moderate dose of pseudoephedrine on
the day of competition and remain below the threshold, there is high inter-individual variability
in the urinary concentration of pseudoephedrine. WADA advises athletes to refrain from
taking pseudoephedrine 24 hours before competing. Of particular note for medical
practitioners should be the rules about the use of intravenous fluids in athletes. As a result of
the abuse and inappropriate use of intravenous fluids in sporting environments, the Code
lists as a prohibited method intravenous infusions and/or injections of more than 50 mL per 6
hour period except for those legitimately received in the course of hospital admissions,
surgical procedures or clinical investigations. This effectively means that high-performance
athletes should not be administered intravenous fluids except for medical indications
[150057].
The prohibited list
WADA also took over the role of publishing the Prohibited List (List), revised annually since
2004. The List has expanded considerably from the original IOC Prohibited List of the 1960s
and contains numerous classes of substances as well as prohibited methods such as blood
manipulation. A substance (or method) is considered for inclusion if it meets any two of the
following criteria:
- potential for performance enhancement

- detrimental to the athlete's health
- contrary to the spirit of sport
The deliberations on whether to include substances in the List are a highly interactive and
consultative process which includes stakeholders and experts. It is impractical to list all
known and possible compounds; thus, most of the prohibited classes contain an important
clause stating: “…and other substances with similar chemical structure or similar biological
effect(s)” [13015].
Some substances have permitted routes of administration, (e.g. glucocorticosteroids are

allowed by inhalation or topically). A few substances are permitted but only to a certain
threshold level (e.g. pseudoephedrine). The List is divided into substances prohibited in
competition only (eg, stimulants), and those prohibited at all times (eg, anabolic steroids and
erythropoietin). It is irrelevant whether the prohibited substance is synthetic or from botanical
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sources or whether it is considered a pharmaceutical product or a dietary supplement. “Strict
Liability” means that every athlete is responsible for the substances found in their bodily
specimen during a doping control sample analysis. The first line of defence for many
cheating athletes has been to claim that the positive test resulted from a tainted dietary
supplement. Many of these same athletes later confessed to deliberate ingestion of a
prohibited substance. The athlete's responsibility to explain how a prohibited substance
entered his/her body (Strict Liability) has existed for many years, being initially implemented
by the IOC. It has withstood the scrutiny of the Court of Arbitration in Sport and civil courts,
and is a balance between protecting all athletes by ensuring fair, clean sport and the rights of
individual athletes [13010].
Codes since 2004

WADA is a unique, independent body representing equally sport and the governments of the
world. The World Anti-Doping Code is the core document on which anti-doping programmes
are modelled. The first version of the Code came into effect in January 2004. There are
presently over 600 signatories, including almost all the world’s sport federations. The Code
applies to Athletes, as defined by their national anti-doping organisations (NADOs) or
international federations. Who is considered an athlete for anti-doping purposes may vary
widely and a NADO may still test recreational athletes but not apply all elements of the Code,
for example, the requirement for whereabouts or advanced therapeutic use exemptions
[13015].
Not only doping testing

Athletes may be subjected to sanctions based on possession or trafficking of prohibited
substances and not simply due to a positive doping test. However, it is important to be aware
that criminal legislation exists in certain countries (e.g. for narcotics) which may be in addition
to, or completely separate from, anti-doping sanctions. WADA also took over the role of
publishing the Prohibited List, revised annually since 2004. The List has expanded
considerably from the original IOC Prohibited List of the 1960s and contains numerous
classes of substances as well as prohibited methods such as blood manipulation. A
substance (or method) is considered for inclusion if it meets any two of the following criteria:
- potential for performance enhancement

- detrimental to the athlete’s health
- contrary to the spirit of sport
The deliberations on whether to include substances in the iist are a highly interactive and
consultative process which includes stakeholders and experts. It is impractical to list all
known and possible compounds; thus, most of the prohibited classes contain an important
clause stating: “…and other substances with similar chemical structure or similar biological
effect(s).” Some substances have permitted routes of administration, (e.g. glucocortico-
steroids are allowed by inhalation or topically). A few substances are permitted but only to a
certain threshold level (e.g. pseudoephedrine). The list is divided into substances prohibited
in competition only (e.g. stimulants), and those prohibited at all times (e.g. anabolic steroids
and erythropoietin) [13015].
The 2015 Prohibited list
Each year WADA specifies substances and doping methods that are not permitted in sport.
The Prohibited List is the international standard that outlines the substances and methods
that are prohibited in sport. For a substance or method to be prohibited, it must meet at least
two of the following conditions:
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 The substance or method has the potential to enhance, or does enhance,
performance in sport.
 The substance or method has the potential to risk the athlete’s health.
 WADA has determined that the substance or method violates the spirit of sport.

The Prohibited List is complex and detailed. Even experienced sports medicine practitioners
refer to the list carefully when dealing with potential doping matters [150057].
Substances and methods prohibited at all times (in and out of competition)
Prohibited substances
 S0. Non-approved substances
 This includes veterinary drugs and those which have not been approved by regulatory
bodies such as the Therapeutic Goods Administration.
 S1. Anabolic drugs
 S1.1 Anabolic androgenic steroids
a. exogenous e.g. danazol
b. endogenous e.g. testosterone and its metabolites
 S1.2 Other anabolic agents e.g. tibolone
 S2. Peptide hormones, growth factors, related substances and mimetics
 S2.1 Erythropoietin-receptor agonists
i. erythropoiesis-stimulating agents e.g. erythropoietin (EPO)
ii. non-erythropoietic EPO-receptor agonists
 S2.2 Hypoxia-inducible factor stabilisers, and activators e.g. argon
 S2.3 Chorionic gonadotrophin and luteinising hormone and their releasing factors in
males
 S2.4 Corticotropins and their releasing factors
 S2.5 Growth hormone and its releasing factors
 S3. Beta2 agonists
 Inhaled drugs, such as salbutamol, can be used within specified limits.
 S4. Hormone and metabolic modulators
 S4.1 Aromatase inhibitors e.g. anastrozole
 S4.2 Selective oestrogen receptor modulators e.g. tamoxifen
 S4.3 Other anti-oestrogenic substances e.g. clomiphene
 S4.4 Drugs modifying myostatin function
 S4.5 Metabolic modulators e.g. insulin
 S5. Diuretics and masking agents
 The masking agents include drugs such as probenecid.
Prohibited methods
 M1. Manipulation of blood and blood components
 This includes retransfusion of the athlete’s own blood.
 M2. Chemical and physical manipulation
 This includes tampering with samples.
 M3. Gene doping
 This includes normal as well as genetically modified cells.
Substances prohibited in competition
 S6. Stimulants e.g. amphetamines, pseudoephedrine
 S7. Narcotics e.g. methadone
 S8. Cannabinoids
 S9. Glucocorticosteroids
Substances prohibited in particular sports
 P1. Alcohol (banned in air sports, archery, motor sport, motorcycling and
powerboating)
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 P2. Beta blockers (banned in archery, motor sport, billiards, darts, golf, shooting,
some skiing and snowboarding events, and some underwater events)
The 2015 Prohibited List came into effect on 1 January. There are some important changes
from the previous list:
 Mimetics have been included in the section on peptide hormones and growth factors
(S2) to reflect the fact that synthetic analogues are also prohibited substances.
 Non-erythropoietic EPO-receptor agonists have been added.
 Hypoxia-inducible factor stabilisers have been included because of their growing
importance in doping, particularly in relation to the use of inhaled xenon and argon.
 Examples of chorionic gonadotrophin and luteinising hormone-releasing factors such
as buserelin have been added.
 Corticorelin has been included as an example of corticotropin-releasing factor.
 Growth hormone-releasing factors have been divided in a more precise
categorisation to illustrate the varying biological properties.
 The wording in relation to diuretics has been altered to clarify that diuretics are not
only masking agents but can be abused for other purposes such as rapid weight loss.
 The whole family of phenethylamine derivatives has been identified to address the
growing number of illegal, designer stimulants derived from phenethylamine.
Certain drugs, while not prohibited, are monitored to assess their use and to guide future
changes to the list. The following changes have been made to the monitoring program for
2015 [150057]:
 Monitoring of pseudoephedrine will cease, but urinary concentrations above 150

microgram/mL are prohibited during competition.
 Telmisartan (angiotensin II receptor antagonist) has been added to the monitoring
program as it may enhance endurance by inducing metabolic changes such as
mitochondrial biogenesis and changes in skeletal muscle fibre type.
 Meldonium (Mildronate) has been added as it has potential cardiac stimulant effects.
Sanctions of violations
For an athlete confronted with an anti-doping rule violation, section 10.5 of the Code allows
for no sanction, or reduced sanctions, if the athlete can demonstrate no fault or no significant
fault. As far as supplements are concerned, simply stating the unknowing ingestion of a
tainted dietary supplement is not sufficient – an athlete would have to demonstrate clearly
that every reasonable precaution was taken to avoid ingestion of a prohibited substance
[13015].
The World Anti-Doping Agency (WADA) is introducing enhancements to doping

investigations in its 2015 Code, which include improved sharing of information between
antidoping organisations (including sporting bodies) and enhanced accountability of athlete
support staff. These additions will improve the control of links between sports doping and
organised crime. In February 2013 the Australian Crime Commission released a report that
linked several professional sporting codes, professional athletes with links to organised
crime, performance enhancing drugs and illicit substances. Following this report the
Australian Football League (AFL) partnered the Australian national antidoping organisation to
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investigate peptide use in Australian football. One review compared the model proposed by
Marclay, a hypothetical model for anti-doping investigations that proposed a forensic
intelligence and analysis approach, to use the forensic capabilities of the AFL investigation to
test the model's relevance to an actual case. The investigation uncovered the use of peptides
used to enhance athlete performance. The AFL investigation found a high risk of doping
where athlete support staff existed in teams with weak corporate governance controls. A
further finding included the need for the investigation to provide a timely response in
professional team sports that were sensitive to the competition timing. In the case of the AFL
the team was sanctioned prior to the finals as an interim outcome for allowing the risk of use
of performance-enhancing substances. Doping violation charges are still being considered.
Antidoping strategies should include the investigation of corporate officers in team doping
circumstances, the mandatory recording of all athlete substance use during competition and
training phases, the wider sharing of forensic intelligence with non-sporting bodies
particularly law enforcement and collaboration between antidoping and sporting
organisations in doping investigations. Thus, the AFL investigation illustrated the importance
of the 2015 WADA Code changes and highlighted the need for a systematic use of broad
forensic intelligence activities in the investigation of doping violations [14415].
Australian football is an indigenous football code that is a physical, body contact sport, with
physiological demands that involve aerobic capacity, strength, power and ball handling skills.
The AFL competition is conducted as a professional league of 18 teams with over 810 active
players. The age of players range from 18 to 38 years and all players are bound by a
standard contract with obligations to their team and the AFL. The revenue of the AFL in 2012
was AUD425 million. The operating budgets of the 18 teams varies from AUD35 million per
annum to AUD75 million pa and the club supporter bases, effectively the shareholders of
each team, varies from 35 000 to 80 000 members. The income of players varies from
AUD80 000 per annum to AUD1.2 million per annum. In February 2013 the AFL and
Australian Sports Anti-Doping Agency (ASADA), the Australian National Anti-Doping
Organisation, started an investigation into peptide use at three AFL clubs by using modern
investigative techniques. This investigation took 7 months and involved nine investigators
and several technical experts. The Australian Crime Commission released a report that
linked several professional sporting codes, professional athletes with links to organised
crime, performance enhancing drugs and illicit substances. Following this report the
Australian Football League (AFL) partnered the Australian national antidoping organisation to
investigate peptide use in Australian football. One review compared the model proposed by
Marclay, a hypothetical model for anti-doping investigations that proposed a forensic
intelligence and analysis approach, to use the forensic capabilities of the AFL investigation to
test the model's relevance to an actual case. The investigation uncovered the use of peptides
used to enhance athlete performance. The AFL investigation found a high risk of doping
where athlete support staff existed in teams with weak corporate governance controls. A
further finding included the need for the investigation to provide a timely response in
professional team sports that were sensitive to the competition timing. As reported by the
Australian Crime Commission and partly confirmed by the investigation, the substances
supplied to athletes by team support staff were loosely labelled “peptides” and “amino acids”
by the perpetrators. The substances named in the ACC report included CJC 1295, GHRP-6,
GHRP-2, AOD 9604, hexarelin, ipamorelin, semorelin and other substances, some of
unknown nature. The mix of these peptides varied from team to team and athlete to athlete.
Generally the substances were given in injection form by non-medical trained team support
personnel. In the AFL investigation, due to the lack of individual athlete and team
documentation, the precise nature and doses of substances that were given to each athlete
was uncertain or unknown. Under the WADA Prohibited List many of these substances are
prohibited, being human growth hormone (hGH) releasing substances or sections of the hGH
molecule. They are listed on the WADA Prohibited List under categories S2 (peptide
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hormones, growth factors and related substances) and/or S0 (non-approved substances). In
the case of the AFL the team was sanctioned prior to the finals as an interim outcome for
allowing the risk of use of performance-enhancing substances. Doping violation charges are
still being considered. Antidoping strategies should include the investigation of corporate
officers in team doping circumstances, the mandatory recording of all athlete substance use
during competition and training phases, the wider sharing of forensic intelligence with non-
sporting bodies particularly law enforcement and collaboration between antidoping and
sporting organisations in doping investigations. The AFL investigation illustrated the
importance of the 2015 WADA Code changes and highlighted the need for a systematic use
of broad forensic intelligence activities in the investigation of doping violations. Immediately
following the AFL investigation one team was charged with “bringing the game into disrepute”
under AFL Rules. The team was fined AUS 2 million, sanctioned with loss of player draft
picks and not permitted to compete in the 2013 championship finals. By accepting the
sanctions, the team acknowledged there had been a risk its players had been administered
WADA-prohibited substances, the basis of the initial charges. The impending 2013 AFL
championship finals highlighted the need for the League to promptly respond to potential
team-based doping, in contrast to the longer legal process for determining whether team or
individual doping violations had actually occurred. The team's elimination from the
championship finals avoided the possibility of athletes who had potentially used banned
substances contributing to a team's success in finals. The determination of doping violations
for individuals and/or the teams is a complex legal exercise and, at the time of writing this
paper, is under consideration by ASADA. The findings of the AFL investigation demonstrated
that poor management of athlete support staff combined with weak corporate governance
controls, posed a doping risk. The investigation further verified the emergence of peptides in
doping activities and the role of antiageing clinics and compound pharmacies as potential
conduits of doping activities. In professional team sports there is the need for timely
management of investigation outcomes that are not easily aligned with normal legal
antidoping practices. Antidoping strategy changes that could be considered include the
mandatory recording of athlete substance use during competition and training phases, the
wider sharing of forensic intelligence with non-sporting bodies particularly law enforcement,
the potential role of an examination of corporate governance practices within a sporting
organisation and further improved collaboration between antidoping and sporting
organisations in doping investigations [14445].
Indirect evidentc of doping effects: world records
Progression of world records in athletics is a reliable mean to assess the potentiality of the
human body, which also reflects how society has evolved over time and will continue to
evolve. It was conducted a quantitative analysis of world records in measurable Olympic
events from nine representative disciplines (100, 400, 1500, 10,000 m, marathon, long jump,
high jump, shot put and javelin throw) in order to identify progression and trends for the years
1900-2007 from the database of the International Olympic Committee. Overall, the relative
improvement of athletic performance was higher in women than in men, being nearly
doubled across the different specialities. The biggest increases were observed for javelin
throw and shot put, in both men and women, respectively. Conversely, the improvement in
race time was directly related to the race distance. It was also observed a consistent
significant linear model of world record progression in time, although the improvement has
substantially stopped or reached a plateau in several specialities. The observed trend might
be explained by a variety of factors, including social and environmental changes, natural
selection, advances in training and sport physiology, ergogenic aids and, possibly, doping
[08017].
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The performance-enhancing effects of doping and the introduction of intensive doping
surveillance are especially reflected in the decline in performance in several women's track
and field events [08018].
physical performance in metric sports. It was used time series analysis to investigate the
factor-1 (IGF-1) being the primary candidate explaining the observed effects [12013].
Doping is a very serious issue bedevilling the sporting arena. It has consequences for
athletes' careers, perception of sports in the society and funding of sports events and
sporting organisations. There is a widespread perception that doping unfairly improves
results of athletes. A statistical study of information on best lifetime results of top 100 m
sprinters (males better than 9.98 s, females 11.00 s), over the period of 1980-2011 was
conducted. Athletes were divided into categories of “doped” (n=17 males and 14 females),
based on self admission, the confirmed detection of known doping agents in their bodies or
doping conviction, and “non-doped” (n=46 males and 55 females). No significant differences
(unpaired t-test) between dopers and non-dopers were found in their average results:
male”'dopers” 9.89 s identical with “non-dopers” 9.89 s, females 10.84 s and 10.88 s
respectively. Slopes of regressions of best results on dates for both “dopers” and “non
dopers” were not significantly different from zero. This indicates that no general improvement
as a group in 100 m sprint results over a quarter of a century occurred irrespective of doping
being or not being used. It was concluded that since there are no statistical differences
between athletes found "doping" and the others, one of the following must be true: (1)
"doping" as used by athletes so detected does not improve results, or (2) "doping" is
widespread and only sometimes detected. Since there was no improvement in overall results
during the last quarter of the century, the first conclusion is more likely. Objectively, various
"doping" agents have obvious physiological or anatomical effects. These may not translate
into better results due to the clandestine use of doping that prevents its scientific structuring.
Perception of the effectiveness of doping should be reconsidered. Policy changes may be
required to ensure the continued fairness and equity in testing, legislation and sports in
general [12014].
Anti-doping rules
The ramifications of doping are not limited to topclass athletes who may feel compelled to
risk their health for fame and money, but also extend to amateur athletes eager to exhibit
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superiority in the athletic field. Owing to difficulties in actually proving the intent to cheat, the
World Anti-Doping Agency (WADA) enforces a principle of strict liability for positive test
results for banned substances. Antidoping laws encompass a broad, continuously updated
panel of laboratory tests for the most recent list of banned substances, which includes
traditional as well as promising new drugs and techniques that may find actual applications in
doping athletes [06005].
The rules
FIFA introduced an anti‐doping programme in 1966 at the World Championship, being one of
the first international sports federations to do so.The fundamental aims stipulated in the FIFA
doping control regulations in 2006 are quite similar to the purpose of the World Anti‐Doping
Code programme. According to the definition of doping in the World Anti‐Doping Code,
doping is defined as the occurrence of one or more of the following violations [06002]:
- the presence of a Prohibited Substance or its Metabolites or Markers in an Athlete's

bodily Specimen (strict liability rule).
- possession by an athlete at any time or place of a Substance that is prohibited in
out‐of‐competition testing or a Prohibited Method, unless the athlete establishes that
possession is pursuant to a therapeutic use exemption granted in accordance with
the FIFA Doping Control Regulations regarding the therapeutical use of forbidden
substances or other acceptable justification.
- possession of a Substance that is prohibited in out‐of‐competition testing or
Prohibited Method by athlete Support personnel in connection with an athlete,
competition or training, unless the athlete support personnel establishes that the
possession is pursuant to a therapeutic use exemption as described previously
- trafficking in any Prohibited Substance or Prohibited Method is still a violation of the
anti‐doping regulations and in most of the law systems an illegal act against the
medical preparations law
- administration or the attempted administration of a Prohibited Method to any athlete,
or assisting, encouraging, aiding, abetting or covering up as well as any other type of
complicity involving an anti‐doping rule violation or any attempted violation
As set forth in the preamble of the World Anti‐Doping Code, the purposes of the World
Anti‐Doping Program are:
- to protect the athletes' fundamental right to participate in doping‐free sport and thus
promote health, fairness, and equality for Athletes worldwide
- to ensure harmonized, coordinated, and effective anti‐doping programs at the
international and national level with regard to detection, deterrence, and prevention of
doping
Prohibited substances in the context of these regulations are regularly published in the
WADA list of prohibited substances (www.wada‐ama.org) [06002].
Strict liability rule
The reason for the strict liability rule has been comprehensively stated by the Court of
Arbitration for Sport, Lausanne in some cases. “It is true that a strict liability test is likely in
some sense to be unfair in an individual case, where the athlete may have taken medication
as the result of mislabelling or faulty advice for which he or she is not responsible –
particularly in the circumstances of sudden illness in a foreign country. But it is also in some
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sense unfair for an athlete to get food poisoned on the eve of an important competition be
altered to undo unfairness. Just as the competition will not be postponed to await the
athlete's recovery, so the prohibition of banned substances will not be lifted in recognition of
its accidental absorption. The vicissitudes of competition, like those of life generally, may
create many types of unfairness, whether by accident or the negligence of unaccountable
persons, which the law cannot repair. Furthermore, it appears to be a laudable policy
objective not to repair an accidental unfairness to an individual by creating an intentional
unfairness to the whole body of other competitors. This is what would happen if banned
performance‐enhancing substances were tolerated when absorbed inadvertently. Moreover,
it is likely that even intentional abuse would in many cases escape sanction for lack of proof
of guilty intent. And it is certain that a requirement if intent would invite costly litigation that
may well cripple federations – particularly those run on modest budgets in their fight against
doping” [06002].
It is irrelevant whether the prohibited substance is synthetic or from botanical sources or

whether it is considered a pharmaceutical product or a dietary supplement. Strict Liability
means that every athlete is responsible for the substances found in their bodily specimen
during a doping control sample analysis. The first line of defence for many cheating athletes
has been to claim that the positive test resulted from a tainted dietary supplement. Many of
these same athletes later confessed to deliberate ingestion of a prohibited substance. The
athlete’s responsibility to explain how a prohibited substance entered his/her body (“Strict
Liability”) has existed for many years, being initially implemented by the IOC. It has withstood
the scrutiny of the Court of Arbitration in Sport and civil courts, and is a balance between
protecting all athletes by ensuring fair, clean sport and the rights of individual athletes
[13015].
For an athlete confronted with an anti-doping rule violation, section 10.5 of the Code allows
for no sanction, or reduced sanctions, if the athlete can demonstrate no fault or no significant
fault. As far as supplements are concerned, simply stating the unknowing ingestion of a
tainted dietary supplement is not sufficient – an athlete would have to demonstrate clearly
that every reasonable precaution was taken to avoid ingestion of a prohibited substance. An
athlete taking a spiked supplement with intent to dope may claim that he/she did not realise
the product contained a prohibited substance. It is difficult to know the intent: nevertheless,
the athlete will benefit from the ergogenic effect of the prohibited substance and have an
unfair advantage over their competitor. There are many cautionary tales of athletes taking
energy boosting supplements before or during the games and subsequently being
sanctioned. Many dietary supplements that promise to enhance performance either contain a
prohibited substance or are an example of false advertising [13015].
The reality is that a significant percentage (5-20 %) of supplements contains prohibited

substances, either by inadvertent contamination or deliberate adulteration, during the
production process. This phenomenon has been demonstrated repeatedly, and sporting
federations as well as anti-doping organisations continue to impress this warning upon
athletes. For example, several athletes have been recently sanctioned over the stimulant
methylhexaneamine (MHA), explicitly prohibited since 2009. This was considered to be a
dietary supplement from geranium oil, despite the fact that several studies demonstrated that
its presence in supplements was not from geranium oil but due to the addition of synthetic
MHA. Whether natural or synthetic, athletes need to avoid these types of products [13015].
The whereabouts rule
Apart from such special cases, effective doping controls are bonded to out‐of‐competition
tests. Without accurate athlete location information such controls may be inefficient and
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sometimes impossible. This so called “whereabouts rule” requires athletes and/or teams that
have been identified for out‐of‐competition control to be responsible for providing and
updating information on their whereabouts so that they can be located for No Advance Notice
out‐of‐competition control. The applicable requirements are set by the responsible sport
federation or national anti‐doping organisation to allow some flexibility based upon varying
circumstances encountered in different sports and countries. A violation of this rule may be
based on either intentional or negligent conduct by the athlete, but it is known that the
whereabouts rule may not be realistic in international team sports, in which players are
normally playing for a club far from their home nation [06002].
Separation of power
An important legal principle is the separation of power between the anti‐doping executive
authorities and the disciplinary committee responsible for the administration of anti‐doping
sanctions. This is to minimise any accusations of bias or conflict of interest in the application
of the Code. This principle is applied in a practical sense by having the Doping Control
Sub‐Committee (representing medical, pharmacological, and medicolegal expertise) dealing
with the medical and biochemical aspects of the alleged doping event and, once this issue
has been determined, a separate Disciplinary Committee which awards the appropriate
sanction in view of the individual circumstances of the athlete concerned [06002].
Since 2011, this category (S0) of banned substances has been a part of WADA’s prohibited
list and encompasses a virtually infinite number of compounds currently not covered by any
of the other sections (e.g. anabolic agents, peptide hormones, growth factors and related
substances). New representatives of this class of compounds are low molecular weight
luteinizing hormone (LMWLH) receptor agonists. Focusing on two series of drug candidates
based on either pyrazole or thienopyrimidine core structures, two model substances were
synthesized and used to establish a targeted/non-targeted screening method employing both
diagnostic precursor-product ion pair detection and precursor ion scanning. In the absence of
metabolism study data, the presence of the intact drug or at least a conserved nucleus must
be present to allow the detection using the proposed strategy [12017].
This newly established category of banned substances encompasses a virtually infinite

number of compounds with corresponding physicochemical and pharmacological properties;
however, only those agents currently not covered by any of the other sections (e.g. anabolic
agents, peptide hormones, growth factors, and related substances) are considered relevant
for S0. These compounds [016]
- have not received approval for human therapeutic use

- comprise structures not related to any other listed group of banned substances
- exhibit biological effects that are different from all other drugs included in the
Prohibited List.
Privacy
A concern for athletes when required to provide confidential and personal details, both as to
whereabouts and personal medical conditions, is that strict privacy will be preserved. Such
information must be tightly held with access only available to a small number of people who
require it and they are morally, ethically and legally bound to observe such confidentiality.
WADA will similarly be required to provide such tight privacy provisions in dealing with
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information which is essential to assure drug free sport and, in some cases, the capacity for
the athlete to be able to compete [03027].
Random testing
It was assessed the deterrent effect of mandatory, random drug testing among high school
(HS) athletes in a controlled setting. Two high schools, one with mandatory drug testing (DT)
consent before sports participation, and a control school (C), without DT, were assessed
during the 1999-2000 school year. Athletes (A) and nonathletes (NA) in each school
completed confidential (A) or anonymous (NA) questionnaires developed for this study,
respectively, at the beginning and end of the school year. Positive alcohol or drug tests
required parent notification and mandatory counseling without team or school suspension.
Thirty percent of the DT athletes were tested. Data were analyzed using the end of the
school year measure, adjusted for the initial questionnaire results. Demographics of the
athlete sample revealed that mean age was 16 years with 82 percent white, 10 percent
Hispanic, 5 percent Asian, 3 percent American Indian/Native Alaskan, 1 percent African-
American, and 1 percent Native Hawaiian/Pacific Islander. A (n=276) and NA (n=507) were
assessed at the beginning (baseline) and at the end of the school year (A, n=159; NA,
n=338). The past 30-day index of illicit drugs (4-fold difference) and athletic enhancing
substances (3-fold difference) were lower among DT athletes at follow-up without difference
in alcohol use. However, most drug use risk factors, including norms of use, belief in lower
risk of drugs, and poorer attitudes toward the school, increased among DT athletes. Although
a reduction in the illicit drug use index was present among nonathletes at the DT school, at
the end of the school year, it did not achieve statistical significance. It was concluded that
random DT may have reduced substance use among athletes. However, worsening of risk
factors and small sample size suggests caution to this drug prevention approach. A larger
long-term study to confirm these findings is necessary [03028].
Education and research
Education and research are aspects of the Code, the former addressing:
- Substances and methods on the Prohibited List

- Health consequences of doping
- Doping control procedures
- Athletes' rights and responsibilities
Anti-doping research can include sociological, behavioural, judicial and clinical studies in
addition to medical, analytical and physiological investigation [03027].
Laboratory testing
Out-of-competition anti-doping laboratory's analytical menu
All out-of-competition samples were analysed at the laboratory of the Department of Anti-
Doping Research at the Institute of Sport in Warsaw to ensure consistency of analysis and
results across samples. All procedures were supervised by experienced experts from other
WADA accredited laboratories during the pre-and in-competition phase. All samples were
received by the laboratory within 24 hours of collection and were analysed within 48 hours of
receipt (with the exception of IRMS analysis, which was conducted within 96 hours). All
samples collected in the out-of-competition programme came back negative. Blood
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parameters were only analysed in the out-of-competition programme. A full blood count was
obtained for all blood samples, and the stimulation index (OFF score) and abnormal blood
profile score (ABPS) were calculated. The conditions required for athlete blood passport
(ABP) tests were not fulfilled, as players were not required to rest for two hours post-match.
Serum was tested for the presence of human growth hormone in both the out-of-competition
and in-competition programmes. All HGH tests were prepared using a special
chemiluminescence immunoassay kit for HGH. Only negative samples were observed.
NSAIDs were detected in a large number of samples, both in-competition and out-of-
competition. A relationship was found between the duration of the tournament and the
number of samples containing NSAIDs. The most frequent compound was diclofenac, which
was used more towards the end of the tournament [144416].
In-competition testing programme
Doping controls were conducted after every match at the tournament, with two players from
each team drawn to undergo testing, plus two reserves. Reserves were available to be
tested in the event that a selected player sustained an injury that rendered him incapable of
completing the test (which did not occur at any tournament match). Any of the 23 squad
players could be drawn for testing, regardless of whether they actually participated in the
match. Thirty-two doping control chaperones (four per stadium) were appointed to assist with
the in-competition programme from the pool of tournament volunteers. Where possible,
chaperones came from the national anti-doping organisations of the host countries. All
chaperones were trained by UEFA's anti-doping unit in advance of the tournament via a
specially designed distance learning programme. In addition, a support service was made
available within the anti-doping unit to ensure that any questions regarding the role could be
answered by experienced staff. Finally, all chaperones were given a full pre-match briefing
by a DCO before their first match at the tournament [14446].
Doping control stations (DCSs) at tournament stadiums were of extremely high quality, as
most of the stadiums were newly built, with specific consideration given in the planning stage
to the design and location of the facility. All DCSs used at the tournament were inspected by
members of the UEFA Medical Committee and anti-doping unit staff at site visits conducted
in 2011 and 2012. In-competition samples were analysed for the full WADA analytical menu
plus additional screening for EPO, HGH, blood transfusion and SARMS, with IRMS analysis
conducted as required. All samples were received by the laboratory within 24 hours of
collection and were analysed within 24 hours of receipt (with the exception of EPO and IRMS
analysis, which was conducted within 48 hours). All results were reported before the team's
next match. All samples were analysed by the WADA-accredited laboratory in Warsaw under
the supervision of experienced experts from other WADA accredited laboratories. All 124
players’ blood and urine samples collected in the in-competition programme were returned
as negative. On three occasions, blood could not be successfully collected from a player
(with two of these incidents involving the same player). Following the conclusion of the
tournament, all samples were placed in storage (at -20°C) and will be retained for re-analysis
by UEFA as required. All samples with a T/E ratio in excess of 4.0 were analysed using
IRMS in accordance with the requirements laid down in the WADA International Standard for
Laboratories. No indication of the use of synthetic testosterone or testosterone precursors
was identified. The Warsaw laboratory also sent all steroid profile parameters to UEFA in
order to compare those results with the results of previous analysis. The presence of
recombinant erythropoietin was tested for using ultrafiltration, isoelectric focusing (IEF),
double blotting and chemiluminescence detection. All results were negative. Each of the
OOC samples was analysed for the presence of plasticiser metabolites. Although the
presence of elevated levels of these indicators is not unambiguous proof of the use of blood
308
transfusion, this represents important additional evidence alongside the blood passport
programme. No suspicious results were detected [14446].
Therapeutic use exemption (TUE)
Sporting authorities and medical practitioners working in high-performance sport are

cognisant of the need to ensure that anti-doping rules do not impact negatively upon the
health of the athlete. To ensure that athletes can be treated for a legitimate medical
condition, WADA can provide a therapeutic use exemption for an otherwise banned
substance. International and national athletes should apply for a therapeutic use exemption
prospectively. In cases where a medical emergency necessitates the use of an otherwise
prohibited substance, an athlete may apply for a retrospective therapeutic use exemption.
Athletes should check with their sporting organisation in the first instance. Most sporting
organisations will have a chief medical officer who can assist with the therapeutic use
exemption process, or the sport’s administrators should be able to direct the athlete
appropriately [150057].
Therapeutic use exemption is available to all athletes who require it and who satisfy specific
conditions. This allows an athlete who might be otherwise denied the opportunity, because of
a severe medical condition which can only be managed by the use of a drug prohibited in
sport, to compete at the highest level. Now, via the code, athletes who require to use a
prohibited drug in sport can obtain therapeutic exemption where [03027]:
1. The prohibited substance is essential for the health of the athlete and impairment
would result were it to be withheld
2. Therapeutic use of the prohibited substance would produce no additional
enhancement of performance other than a return to a normal state of health after
treating a legitimate medical condition
3. There is no reasonable therapeutic alternative to the otherwise prohibited substance
4. The necessity for use of the prohibited substance is not a consequence of the
previous use of a prohibited substance
The need for therapeutic use exemptions (TUEs) or the permitted use of Prohibited
Substances and Prohibited Methods by athletes to treat significant medical conditions arose
when several classes of drugs used commonly in medicine were prohibited in sport by the
International Olympic Committee (IOC) during the 1980s. However, although the IOC
Medical Commission (IOC-MC) gave qualified support for the concept to formally start at the
1992 Barcelona Olympics, the Commission's fears that athletes might abuse the mechanism
resulted in minimal publicity and its non-inclusion in the Medical Code of the Olympic
Movement for 8 years. TUEs would not be widely publicised until the advent of the World
Anti-Doping Agency which not only approved the principles of TUEs as developed by the
IOC's Medications Advisory Committee (MAC) in 1991, but also introduced the name of TUE.
Several changes to the Prohibited List have resulted in TUEs being necessary for
substances that were permitted 20 years ago as disclosed in a review of TUEs approved at
the 11 Olympic Games that the IOC's MAC, later the TUE Committee (TUEC), has operated.
The IOC and its TUEC played a pivotal role in developing the concept of TUE which is now
globally accepted [13013].
Athletes can be allowed to use substances from the prohibited list (the doping list) if they
have a medical condition. If so, a Therapeutic Use Exemption (TUE) is required. The
boundaries between the use of pharmacological substances due to a medical need and
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doping are sometimes blurred. Although manipulating the system of TUE granting potentially
represents an entry stage for doping, few studies examine how athletes perceive TUE
management and relate this to current anti-doping policy. 645 Danish elite athletes (mean
age 22) representing 40 sports completed a web-based questionnaire about their experience
and perception of TUE (response rate: 43 %). Nineteen percent of the respondents had been
granted a TUE. Eightyfive percent of athletes granted a TUE regarded their use of the TUE
system as necessary to compete on equal terms with other athletes. Administrative hurdles
for TUE prevented 7 percent of athletes from applying. Fifty-three percent of the athletes
considered that being "allowed" to dope by means of a TUE was of importance for their
(hypothetical) wish to try out doping. Fifty-one percent believed that athletes in their sport
received TUEs without a medical need. Athletes granted TUEs had more than twice as high
odds to distrust the efficacy of the system than athletes never granted a TUE. The belief that
TUEs were misused was especially common among endurance athletes, regardless of them
having experience with TUEs or not. Four percent believed it would be okay to receive a TUE
without a medical need. The results confirm that TUE is a problem in anti-doping policy. The
fact that distrust in TUE administration increases once an athlete has experience of TUEs
represents a challenge for anti-doping policy. We suggest more critical research on TUEs be
carried out in order to improve harmonization and increase transparency in the regulations
[13020].
Athletes who have either physical symptoms or disease after injury may need to be treated
with specific medicines that are on the list of prohibited substances. Therapeutic use
exemption may be granted to such players, in accordance with strictly defined criteria-these
are presented in this article. Procedures of how to request for an abbreviated or a standard
therapeutic use exemption are explained, and data on therapeutic use exemptions are also
presented. A therapeutic use exemption (TUE) permitting the use of such a substance or
method that is on the prohibited list, may be granted to the player, depending to the clinical
situation. An exemption will be granted only in strict accordance with the following criteria
[06007]:
- the athlete shall submit an application for TUE no less than 21 days before
participating in an event
- the athlete would experience a significant impairment to health if the prohibited
substance or method were to be withheld in the course of treating an acute or
chronic medical condition
- the therapeutic use of the prohibited substance or method would produce no
additional enhancement of performance other than that which might be
anticipated by a return to a state of normal health following the treatment of a
legitimate medical condition. The use of any prohibited substance or method to
increase “low‐normal” levels of any endogenous hormone is not considered an
acceptable therapeutic intervention
- there is no reasonable therapeutic alternative to the use of the otherwise
prohibited substance or method
- the necessity for the otherwise prohibited substance or method cannot be a
consequence, wholly or in part, or prior non‐therapeutic use of any substance
from the prohibited list
- an application for a TUE will not be considered for retroactive approval except
in cases where:
o emergency treatment or treatment of an acute medical condition was
necessary
o due to exceptional circumstances, there was insufficient time or
opportunity for an applicant to submit, or the granting body to consider,
an application prior to doping control
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- confidentiality of information: The applicant shall provide written consent for the
transmission of all information pertaining to the application to members of the
granting body and, as required, other independent medical or relevant scientific
experts. If the assistance of external, independent experts is required, all
details of the application will be circulated without identifying the player
involved in the doctor's care. The applicant shall also give written consent to
the decisions of the granting body to be distributed to the involved medical
personnel of other relevant anti‐doping organisations under the provisions of
the Doping Control Regulations. The members of the granting body involved
will conduct all of their activities in strict confidence according to the
Hippocratic Oath and the medico‐legal and ethical rules of confidentiality.
Abbreviated TUE
Abbreviated TUE requests are valid as soon as an approved anti-doping organisationhas

received the request. Treatment may start immediately after the receipt is confirmed.
However, the anti-doping organisation has the right to ask for additional information should
the applied indication for glucocorticosteroids or beta2-agonists appear doubtful. WADA has
also decided that for a beta2-agonist TUE, following a clinical diagnosis of exercise induced
or allergic asthma the results of lung function tests have to be submitted to substantiate the
clinical diagnosis. This decision of the WADA Medical Committee clearly stresses the
importance of sound clinical diagnosis following state of the art assessment to avoid misuse
of β2 agonists in the absence of clear clinical diagnosis [06007].
Standard TUE
In the case of a standard TUE application for which specialist expertise is required, the TUE
committee appoints external independent experts for a second opinion to justify the decision.
Standard TUE requests are valid as soon as an appropriate anti-doping organisation has
sent the player a certificate of approval, except in rare cases of an acute life threatening
condition, for which retroactive approval may be considered. TUEs are indispensable for
improving medical cover of athletes with health impairments while avoiding anti‐doping
violations [06007].
Therapeutic use exemptions (TUEs) at the Olympic Games
That Olympic athletes, indeed all athletes with documented and significant medical
conditions, may seek and be approved to administer a Prohibited Substance or a Prohibited
Method and train for and compete in sports is now widely acknowledged and accepted. This
is termed a Therapeutic Use Exemption (TUE), although some were granted to Olympic and
non-Olympic athletes before this name was introduced in 2001. The International Olympic
Committee (IOC), through its Medical Commission (IOC-MC), had a pivotal role in the
development of TUE and its TUE Committee (TUEC) has functioned since prior to the 1992
Olympic Games in Barcelona. London 2012 was the 11th Olympic Games at which it had
operated. But in its early years, acceptance of the concept of TUEs had many difficulties.
However, although the IOC Medical Commission (IOC-MC) gave qualified support for the
concept to formally start at the 1992 Barcelona Olympics, the Commission's fears that
athletes might abuse the mechanism resulted in minimal publicity and its non-inclusion in the
Medical Code of the Olympic Movement for 8 years. TUEs would not be widely publicised
until the advent of the World Anti-Doping Agency which not only approved the principles of
TUEs as developed by the IOC's Medications Advisory Committee (MAC) in 1991, but also
introduced the name of TUE. Several changes to the Prohibited List have resulted in TUEs
311
being necessary for substances that were permitted 20 years ago as disclosed in a review of
TUEs approved at the 11 Olympic Games that the IOC's MAC, later the TUE Committee
(TUEC), has operated. The IOC and its TUEC played a pivotal role in developing the concept
of TUE which is now globally accepted [13013].
Two decades of experience of TUEs at the Olympic Games have demonstrated that from a
semiclandestine procedure in 1992, it has evolved so that currently there are established
principles, a robust mechanism to apply and approve or reject applications, globally
recognised medical indications and a mutual recognition of appropriately approved TUEs, at
least at the Olympic Games. It is gratifying that the IOC's 1991–1992 pioneering criteria and
guidelines have undergone only minor modifications to those of WADA that operated at
London 2012. However, despite widespread understanding of the TUE principles and
processes, it is essential to continue to strive for superior knowledge of the topic, better
qualified and more experienced TUECs and hopefully a mutual recognition of correctly
approved TUEs from experienced TUECs [13013].
Prevalence of use of TUEs in asthmatics
The prevalence of asthma and the use of anti-asthmatic medication is high among elite
athletes. Elite athletes require a TUE certificate (Therapeutic Use Exemption) if they require
anti-asthmatic medication which is on the prohibited list. The aim of one study was to
determine the distribution of Danish TUE certificates and to examine the use of anti-
asthmatic medication among Danish elite athletes. A cross-sectional study of all applications
for TUE certificates in 2005 was carried out. It was focused on applications including anti-
asthmatic medication. All applications resulted in certificates being issued. A total of 694 TUE
certificates were issued. Of these, 445 (64 %) concerned anti-asthmatic medication. Short-
acting beta-2-agonists (SABA) were the most frequent medication (79 %). Only 2 percent
received long-acting beta-2-agonists (LABA) as single therapy. Inhaled steroids were used
by 69 percent. Swimmers received significantly higher doses of inhaled steroids compared to
all others athletes (1031 microg/day; n=148). The applications for TUE certificates were
generally handled by general practitioners (78 %) [08039].
Prioritation in anti-doping
There is debate concerning whether the guiding paradigm for anti-doping policy should be
the current legalistic approach or a "harm minimisation" approach prioritising athlete health.
This study sought to determine whether a representative sample of Australians prioritises
health above other concerns using the World Anti-Doping Code's Spirit of Sport statement
which lists the 11 attributes that define the moral basis for anti-doping. A Best-Worst Scaling
(BWS) Balanced Incomplete Block Design experiment using 11 choice sets of five Spirit
attributes from the set of 11, with the attributes within each choice set in a random order. A
representative sample of 168 Australians responded to an on-line survey. The BWS scores
defined the relative ranking of each attribute to define an aggregate model and
demographically defined models (gender, education, sports participation and sports
following). Health was ranked as 7/11 in the aggregate model. Only those who did not follow
sport prioritised health (2/11), with other demographic models failing to show a meaningful
departure from the aggregate model. It was concluded that Australians ranked health below
other attributes in the Spirit of Sport, appearing to prioritise "rule following" consistent with
the legalistic approach. This challenges the harm minimisation approach to managing the
role of drugs in sport and suggests that rule-following and legalistic approaches to drug use
should take precedence over health messages [12031].
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Strategy to reduce illicit drug
Australian football
The World Anti-Doping Agency (WADA) prescribes that drug testing is conducted in sports
competitions to detect drug use in athletes. This testing includes performance-enhancing
drugs as well as illicit substances such as marijuana, amphetamines and cocaine. Illicit drugs
are tested for on match days but not on non-match days. Some athletes are known to use
illicit substances for recreational purposes, away from competition times and this poses a
serious health and welfare issue not addressed by the usual sport drug testing regimes. This
paper reports the results of the first 7 years of an illicit drug-testing programme that included
non-match day testing in the elite Australian Football competition, the Australian Football
League (AFL). Players in the AFL were tested for illicit drugs both in-competition and out-of-
competition. Players were selected for illicit substance tests either randomly or targeted
based on previous test history or time since previous test. The number of tests conducted
was increased each year from 2005 to 2011 and testing was focused on high-risk times
during non-competition periods. There were no positive match day tests. There was a
significant reduction in positive tests (19-6) for illicit drugs during non-competition periods
over the 7 years. The reduction in positive tests may be related to player education, the
greater number of tests conducted and the harm minimisation approach of the illicit drug
policy [12032].
Sports supplements from Australian point of view
The sports supplements industry is largely unregulated. The vast majority of the many
ingredients found in sports supplements have not been subject to scientific scrutiny to
support their use. Efficacy and safety data are lacking for many ingredients. Many sports
supplements have been found to contain little or none of the active ingredients claimed by
the manufacturer. Even more concerning is that several studies have found a substantial
proportion of sports supplements contain ingredients which are not mentioned on the label
but which could result in an anti-doping rule violation. In 2013 the Australian Crime
Commission reported that performance-enhancing and image-enhancing drugs, including
peptides and hormones, were being used in some sections of professional sport. Section S2
of the Prohibited List addresses this issue by including growth hormone, erythropoietin and
“other substances with similar chemical structure or similar biological effects”. During 2013-
14, ASADA conducted an assessment of sanctioned athletes and found that 54 percent of
publicly disclosed anti-doping rule violations involved a prohibited stimulant found in a
supplement. Athletes and the professionals supporting them need to be vigilant about the
dangers of an inadvertent violation of anti-doping rules occurring as a result of taking sports
supplements [150057].
Agreements with the pharmaceutical industry
With a steady stream of new therapeutic agents – from stimulants to steroids to protein
hormones – with potential for abuse in sport entering the marketplace, antidoping scientists
and collaborators are continually developing new approaches for detection of prohibited
substances and methods. WADA has signed a declaration with the International Federation
of Pharmaceutical Manufacturers and Associations, whose members will voluntarily assist in
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identifying products with doping potential in advance of their introduction into the marketplace
[12006].
The misuse of medicines for performance enhancement in sport (doping) is not approved by
regulatory agencies, and is illegal in many countries. In addition to the 'traditional' doping
agents such as steroids, beta-blockers and blood transfusions, the list of agents and
techniques used in doping is increasing and now includes newer medicines such as
erythropoiesis-stimulating agents and growth hormones. Innovative new medicines are of
particular interest as would-be dopers may believe them to be undetectable by current
methods. Close collaboration between the biopharmaceutical industry and anti-doping
agencies such as the World Anti-Doping Agency is critical to a successful anti-doping
strategy. Industry is ideally placed to identify the doping potential of new medicines at early
stages and to support early development of detection assays. A strong, united front between
the biopharmaceutical industry and anti-doping agencies is essential to counter the misuse of
medicines for performance enhancement, as well as to promote fair play and clean sport
[12033].
WADA is making significant progress in building relationships within the pharmaceutical and
biotech industries. Cooperation, similar to that which now exists with Roche,
GlaxoSmithKline and the International Federation of Pharmaceutical Manufacturers
Association (IFPMA), will give WADA a head start by working on pipeline compounds that
have the potential for abuse in sport [12034].
David A Cowan and Campbell P Barker spoke to Ryan De Vooght-Johnson at Bioanalysis in

May 2012 about the partnership between industry and academia for the setup and running of
the doping-control laboratory for the London 2012 Olympic Games. David A Cowan is
Director of the London 2012 anti-doping laboratory as well as Director of the King's College
London Drug Control Centre, the UK's only WADA-accredited anti-doping laboratory, and
Head of the Department of Forensic Science and Drug Monitoring. Cowan co-founded the
Drug Control Centre in 1978 and became its Director in 1990. He has published extensively
in the field of pharmaceutical analysis, especially as it relates to detecting drug administration
in sport, and was awarded a personal chair in pharmaceutical toxicology in 1996. Cowan
became Head of the Department of Forensic Science and Drug Monitoring at King's College
London in 2002. He has served on a number of national and international committees,
including the Council of Europe Working Party Investigating Drug Abuse in Sport that led to
the first World Anti-Doping Convention, the Laboratory Representative on the International
Olympic Committee's Medical Commission, and WADA's Laboratory Accreditation
Subcommittee. He is a member of the Crippen Club for Distinguished Toxicologists. In 1998
he was awarded the IOC Trophy for Sport Ethics by the BOA. He was a founding member of
the World Association of Anti-Doping Scientists and became its first President serving on its
Executive Board between 2001 and 2004. He was a Visiting Laboratory Director at the Salt
Lake City Winter Olympic Games 2002, where the first novel erythropoiesis-stimulating
protein (NESP) positive was discovered. He was also a senior advisory scientist at both the
Turin Winter Olympic Games in 2006 and the Beijing Olympic Games in 2008. He was also a
member of the IOC Medical Commission for the Sydney Olympic Games in 2000 and the
Vancouver Winter Olympic Games in 2010. The Drug Control Centre undertook the sample
analysis during the 2002 Manchester Commonwealth Games and Cowan was Co-Director of
the laboratory for the Commonwealth Games in Kuala Lumpur in 1998. Cowan, who has
directed the laboratory at King's College London for many years, was a member of the bid
team making the presentation to the International Olympic Committee in February 2005.
Campbell P Barker has been leading GlaxoSmithKline's delivery of the London 2012
laboratory services since September 2009. Prior to that time he was Director of Global
Strategic Projects in GlaxoSmithKline's Consumer Healthcare business from 2006, and from
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1994 to 2006 he worked in R&D for Procter & Gamble. Barker holds a PhD in chemistry from
the University of Durham [12035].
Doping controls in practice
Drug testing is now ubiquitous in sport, and it often falls to the team physician to perform a
variety of roles including interpreting test results, designing drug-testing programs, acting as
medical review officer, and providing therapeutic use exemptions, education, and counseling.
Proper understanding of current testing methods for drugs such as anabolic-androgenic
steroids, erythropoietin, and growth hormone is essential if the team physician is going to
assume these positions. One article outlined the basics of athletic drug testing from the
collection process through the interpretation of results to assist the team physician in this
field [06008].
International Sport Federations in the protection of the athlete's health
To determine the priorities and activities of International Sport Federations (IFs) with respect
to the promotion of health in their sport and for the general population all 35 IFs participating
in Olympic Games in 2014 or in 2016 were asked to rate the importance of 10 indicated
topics, and to report their programmes, guidelines or research activities on 16 health-related
topics using an online questionnaire (response rate 97 %). On average, the “fight against
doping” had the highest priority followed by “health of their elite athlete” and “image as a safe
sport”. The topics with the lowest importance ratings were “health of their recreational
athlete”,”'increasing the number of recreational athletes” and “health of the general
population”. All except one IF reported to have health-related programmes/guidelines/
research activities; most IFs had 7 or 8 of the listed activities. Eight IFs (24 %) stated to have
activities for “prevention of chronic diseases in the general population” but only FIFA and
FINA reported related projects. It was concluded that IFs aimed to protect the health of their
elite athletes through a variety of activities, however the health and number of their
recreational athletes was of low importance for them. Thus, IFs are missing an important
opportunity to increase the popularity of their sport, and to contribute to the health of the
general population by encouraging physical activity through their sport. FIFA's “Football for
Health” and FINA's “Swim for All” projects could serve as role models [13025].
During the Olympics
One article outlines the process of preparation of an anti-doping laboratory in view of the
activities to be performed on the occasion of the Olympic Games, focusing in particular on
the accreditation requirements of the World Anti-Doping Agency (WADA) and ISO/IEC
17025, as well as on the additional obligations required by the International Olympic
Committee, which is the testing authority responsible for the anti-doping activities at the
Olympics. Due to the elevated workload expected on the occasion of the Olympic Games,
the designated anti-doping laboratory needs to increase its analytical capacity (samples
processed/time) and capability by increasing the laboratory's resources in terms of space,
instrumentation and personnel. Two representative cases, one related to the Winter Olympic
Games (Torino 2006) and one related to the Summer Olympic Games (Beijing 2008), are
presented in detail, in order to discuss the main aspects of compliance with both the WADA
and ISO/IEC 17025 accreditation requirements [12054].
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Medication use in athletes selected for doping control at the Sydney Olympics
During the Olympic Games held in Sydney in September, 2000 Doping Control was
undertaken as specified in the International Olympic Code. During this process information
about the medications taken by athletes was collected as a routine and formed part of the
paperwork associated with a urine test. In their Post Games Report the World Anti-Doping
Agency (WADA) recommended that the information about medications be collated with a
view to assessing their use by athletes. Mandatory doping control for winners of events as
well as random selection of athletes both during competition and out of competition allowed
data to be collected about medications and supplements used by athletes. At the Doping
Control Stations all competitors selected for a test, after providing a urine sample for
analysis, were asked the same question: "what medications have you taken in the past three
days?" The answer was to include all prescription drugs, over-the-counter medications, any
other substances taken by mouth, injection, inhalation, ointment or by suppository, as well as
vitamins, minerals, and all other supplements. This paper reviews the data from the 2758
Declaration Forms obtained at doping control. The prevalence of use of medications, the
number used by an individual, and the pattern of use by these elite sports people were
examined. The trends seen in this survey point to a dangerous overuse of nonsteroidal anti-
inflammatory agents and an unnecessary overuse of vitamins in this population, while
pointing out the increased prevalence of asthma and the dangers of drug interactions. The
main objective here was to review some of the medications used by athletes in the Olympic
Games in Sydney 2000. During these Games Doping Control was undertaken as specified
by the International Olympic Committee. As well as a urine test, information about
medications routinely taken was collected. Mandatory doping control for winners of events as
well as random selection of athletes both during competition and out of competition required
data to be collected about medications and supplements used by athletes as part of the
sample collection protocol. At the Doping Control Stations all competitors selected for a test,
after providing a urine sample for analysis, were asked the same question: "what
medications have you taken in the past three days?" The answer was to include all
prescription drugs, over-the-counter medications, any other substances taken by mouth,
injection, inhalation, ointment or by suppository, as well as vitamins, minerals, and all other
supplements. In the article it was reviewed the data from the laboratory copy of the 2758
Declaration Forms obtained at doping control. The cut down version of the Declaration Form
submitted to the laboratory had all information identifying the athlete removed. Thus all
information used in this article is completely anonymous. The prevalence of use of
medications, the number used by an individual, and the pattern of use by these elite sports
people were examined at the request of the IOC. In their Post-Games Report, the World Anti-
Doping Agency (WADA) acting as independent observers of the anti-doping process
recommended to the IOC that the information obtained in the Athlete Declaration Forms
concerning medications be collated with a view to assessing their use by athletes. The trends
in their use seen in this survey point to an overuse of supplements as well as a dangerous
overuse of drugs such as nonsteroidal anti-inflammatory agents together with multiple drug
use emphasising the dangers of drug interactions and points out the increased prevalence of
asthma in this population [03021].
Illicit drug-related harm during the Sydney 2000 Olympic Games

It was described presentations to emergency departments during the Sydney 2000 Olympic
Games for conditions related to the use of illicit drugs; to discuss the implications of such
presentations for surveillance and public health action at similar events in the future.
Identification of target presentations in sentinel emergency departments; entry of data into a
purpose-built database; and daily electronic transfer of data for central collation and analysis.
Fifteen sentinel emergency departments in the greater Sydney metropolitan area for a 38-
day period spanning the Sydney 2000 Olympic Games met 424 presentations to sentinel
emergency departments with conditions related to illicit drug use. The mean daily number of
316
presentations for adverse events due to illicit drug use was significantly higher (13.3 versus
8.8 presentations) in the 2-week Olympic Games period than in the lead-up to the Games,
culminating in a large peak following the closing ceremony. There was also a significant
increase (5.1 vs 1.7 presentations) in the mean daily number of presentations related to use
of ecstasy or amphetamines, whereas no change was noted in presentations related to
heroin use. Over half (52 %) of presentations occurred at two emergency departments in
areas known as being “hot-spots” for illicit drug use. It was concluded that enhanced
surveillance of adverse events following illicit drug use, possibly targeting known 'hot-spots',
should be considered for future mass events. Advance preparation of preventive strategies,
such as “party-safe” messages, will enable rapid response to unusual patterns of illicit drug-
related harm during future mass events [03022].
London 2012
A brand new 2012 London Olympics anti-doping lab made the headlines worldwide,
accompanied by strong anti-doping messages for prospective Olympians and illustrated by
an iconic photograph depicting the laboratory’s head showing a blood sample to the Minister
for Sports and the Olympics. The 4’400 m2 laboratory, sponsored by a multinational
pharmaceutical company, was operating 24 h/day during the Games, analysing urine and
blood samples of one out of two participating athletes while a part of the samples will be
stored for eight years, using the threat of future testing technology as a further deterrent
[12012].
Biomarkers
Biomarker monitoring can be considered a new era in the effort against doping. Opposed to
the old concept in doping control of direct detection of a prohibited substance in a biological
sample such as urine or blood, the new paradigm allows a personalized longitudinal
monitoring of biomarkers that indicate non-physiological responses independently of the
used doping technique or substance, and may cause sanctioning of illicit practices. This
review presents the development of biomarker monitoring in sports doping control and
focuses on the implementation of the Athlete Biological Passport as the current concept of
the World Anti Doping Agency for the detection of blood doping (hematological module). The
scope of one article extends to the description of novel biomarkers and future concepts of
application [12055].
"Exercisenomics"
Laboratory medicine is complex and contributes to the diagnosis, therapeutic monitoring and
follow-up of acquired and inherited human disorders. The regular practice of physical
exercise provides important benefits in heath and disease and sports medicine is thereby
receiving growing focus from almost each and every clinical discipline, including laboratory
medicine. Sport-laboratory medicine is a relatively innovative branch of laboratory science,
which can provide valuable contributions to the diagnosis and follow-up of athletic injuries,
and which is acquiring a growing clinical significance to support biomechanics and identify
novel genomics and "exercisenomics" patterns that can help identify specific athlete's
tendency towards certain types of sport traumas and injuries. Laboratory medicine can also
provide sport physicians and coaches with valuable clues about personal inclination towards
a certain sport, health status, fitness and nutritional deficiencies of professional, elite and
recreational athletes in order to enable a better and earlier prediction of sport injuries,
overreaching and overtraining. Finally, the wide armamentarium of laboratory tests
represents the milestone for identifying cheating athletes in the strenuous fight against
doping in sports [12056].
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Multi-class and multi-analyte test methods
Traditionally, doping control analytical assays have been drug-class dedicated and tailored to
address requirements concerning sample preparation and chromatography/mass
spectrometry resulting from specific physicochemical properties of target compounds.
Improved analytical instrumentation (particularly based on liquid chromatography-(tandem)
mass spectrometry have enabled the development of numerous cost-effective and rapid
alternatives, allowing for multi-class/multi-analyte test methods. The trend towards
comprehensive and preferably combined targeted/non-targeted screening procedures has
been motivated in part in the requirement for analytical approaches to meet the minimum
required performance levels (MRPLs) stipulated by WADA. Within the last year, several LC-
MS(/MS)-based approaches were published representing options to complement or expand
the currently employed methodologies of doping control laboratories. For example,
employing targeted multiple-reaction monitoring (MRM), the detection of a total of 61
analytes (plus two internal standards) from urine covering seven classes of prohibited
substances (S1–S7) and one agent categorized under M1 was reported. While these assays
are all designed to specificallymeasure amultitude of target compounds with dedicated
precursor-/product-ion pairs and thus gate out all other information (for the advantage of
sensitivity and speed), a trend towards combined targeted/nontargeted analytical methods
has been recognized over the last few years. Here, particularly LC-MS(/MS) approaches with
high resolution/high accuracy mass analyzers such as time-of-flight (TOF) and orbitrap as
well as hybrids consisting of quadrupole or ion trap mass selective devices and TOF or
orbitraps have been used. The benefit of analytical information being recorded in utmost
extent (limited essentially only by sample preparation and/or ionization capability) has been
especially recognized and appreciated [12017].
Statistics
London Olympics
More than 1000 samples were analyzed within a few days after each event for stimulants,
steroids, masking agents, recombinant proteins like erythropoietin and growth hormone
(GH), and other substances on the World Anti-Doping Agency (WADA) Prohibited List in the
London Olympics in 2012 [12006].
Negative tests despite later confessed doping
The Armstrong case, where more than 250 negative doping tests are confronted with the
athlete's confession of erythropoietin use, blood doping, steroid, and growth hormone abuse,
illustrates the limitations of current laboratory tests in detecting doping in sport. Despite
numerous doping controls and simultaneous indications of common doping abuse among
professional athletes in the last two decades, the number of positive urine tests for
recombinant human erythropoietin (rHuEPO) remains remarkably low. Athletes are using
various masking strategies, among them protease inhibitors, intravenous injections of
rHuEPO and alternative erythropoiesis stimulating agents. As one of the countermeasures,
the Athlete's Biological Passport has been introduced. The sensitivity of the Athlete's
Biological Passport is limited if the effect of a low-dose doping remains within the intra-
individual reference range. A possible solution could be the use of a novel Epo test (MAIIA
Diagnostics). Another performance-enhancing strategy is the return to 'old' doping
techniques, such as autologous blood transfusions. Several indirect methods to detect
autologous blood transfusions have been proposed with the majority relying on changes in
erythropoiesis-sensitive blood markers. Currently, an algorithm based on the haemoglobin
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(Hb) level concentration and the percentage of reticulocytes (OFF-hr model; Hb(g/L)-
60·√%ret) is approved by the World Anti-Doping Agency. Genetic factors have been
identified which may interfere with test interpretation. A large inter- and intra-ethnic variation
in testosterone glucuronidation and excretion has been described. Consideration of genetic
variation should improve performance of the testosterone doping test. Taking into account
the pre-analytical care and better tailoring of the threshold values could increase test
sensitivity. Anti-doping laboratories should routinely adjust for multiple testing as failure of
doping control to detect cheaters could lead to more frequent controls. Finally, despite the
huge technological progress, there is a need for increased collaboration between
physiologists, analytical chemists, biostatisticians, and ethicists to reduce doping in sport.
[14005].
Legislations
Brazil
Aiming to suppress the constant importation of substances and drugs, a regulation was
published in the Federal Official Gazette on February 1, 1999 (Ordinance 344, May 12,
1998), with the Brazilian Ministry of Health approving a Technical Regulation on Substances
and Prescription Drugs. This Ordinance is designed to “control and supervise the production,
trade, handling, or use of anabolic substances” that should be decided and implemented in
conjunction with the Ministry of Health, the Ministry of Finance, the Ministry of Justice and its
counterparts in the states, the municipalities, and the Federal District. Doping in Brazil,
according to Article 1 of Resolution No. 02 (May 05, 2004), published in the Official Gazette
(Federal Official Gazette, May 12, 2004) in Section I, the Ministry of Sports and National
Council of Sports, is conceptualized as being a substance, method, or agent capable of
altering an athlete's performance, his/her health, or spirit of the game, during sports
competition – inside or outside of Brasil. Law No. 9965 of April 27, 2000, according to Article
1 thereof, restricts the sale of anabolic steroids, forcing the user to present a prescription
issued by a physician or a dentist, duly recorded in the respective board's professional
registry, and the prescription or script will be retained by the drugstore or pharmacy. With the
implementation of Ordinance No. 101, dated July 29, 2003, the Ministry of Sports created a
Commission to Combat Use of Doping. It was expected that the Commission would
comprise13 members from various institutions involved with sport and a representative of the
National Agency for Sanitary Vigilance, a representative of the Federal Council of Pharmacy,
and a representative of the Brazilian Society of Sports Medicine. Among the various powers
of the Commission to Combat the Use of Doping, it is noteworthy that Item II of ART. 5,
reads: “enforce the World Anti-Doping Code.” The Resolution No. 2, was created on May 5,
2004 by the Sports Ministry and established Basic Standards for Control of Doping in Sports.
This Resolution comprised nine chapters. It begins by discussing the definition of doping and
act of doping, after describing the doping control procedures (identification and selection of
athletes, the control sample; reports and results; and control), the Resolution describes the
punishment for athletes who dope. At the end of Section II, Chapter IX, it provides for the
repeal of Ordinance No. 531 of the MEC (Ministry of Education). This resolution also serves
as the theoretical basis for the construction of the National Sports Policy, approved by
Resolution No. 5, dated June 14, 2005. One important law concerning the issue of doping is
the Brazilian Code of Sports Justice, published in 2010. This law is based on the following
documents: Legislative Decree No. 306 of 2007, which “approves the text of the International
Convention against Doping in Sport, held in Paris on October 19, 2005”; Decree No. 6653 of
November 18, 2008, that “promulgates the International Convention against Doping in Sport,
held in Paris on October 19, 2005,” and the Resolution of the National Education Council No.
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27 of December 21, 2009, that “Approves the list of prohibited substances and methods in
sport.” On May 15, 2012, published in the Federal Official Gazette, was the creation of Law
No. 12638 of May 14, 2012, where the President of the Republic instituted January 15 as the
National Day of Fair Play and to Fight Doping in Sports. Besides these laws, resolutions, and
ordinances that regulate and supervise the use of doping substances and methods, Brazil
has published annually, since 2004, a list of substances and methods prohibited in sports.
Unfortunately, there remains some inconsistency regarding the issue of combating doping in
Brazil because, although there are laws and regulatory agencies, evidence proves that Brazil
is still below expectations when it comes to doping prevention. On January 6, 2005, the
Ministry of Sport made public a report titled “Brazil as an example to combat doping,”
describing that so far no Brazilian had tested positive in doping control tests. On November
24, 2009, the Federal Council of Physical Education, provided on its website, a story from the
newspaper Correio Brasiliense with the title, “Brazil does not fight doping.” The author of this
story revealed that on October 30, 2009, athlete Daiane dos Santos was considered “doped”
in a surprise test done by the International Gymnastics Federation. In addition, the author
also described that in August of the same year, 10 days before the World Championships,
five Brazilians returned home after testing positive in doping tests. The author began his
attack by writing that “Brazil does not have a policy to combat doping” and continued by
describing that while the Ministry of Sports has a Commission to Combat Doping, it only
functions to publicize the WADA list of banned substances created in 2004”. Additionally, the
journalistic site “Estadão” published, on July 31, 2012, the following news: “Brazil is about 30
years too late ‘in the fight against doping’,” according to the information in a publication by
Swedes’ Gunnar Gunnarsson and Arne Ljungqvist, head of the IOC Medical Commission
and vice president of WADA. The international agency to combat doping says, “It is not just
late for 2016. Brazil is about 30 years late. Long ago this structure should have already
existed.” The author states that, with the same speed that laboratories specialize in doping
analysis, the pharmaceutical market provides substances that cannot be immediately
detected. Fortunately, the author draws attention to the importance of doping controls that,
through the discussions generated in the process, help to combat the practice.
Consequently, Brazil has, through covenants (IOC, WADA, and IMETRO), increasingly
combated doping. Since the founding of the Laboratory of Technological Development
Support Institute of Chemistry, Federal University of Rio de Janeiro (Ladetec-IQ/UFRJ) in
1989, the country has intensified campaigns to combat and control doping through the
training of staff for testing and analysis, with antidoping campaigns outside of competition.
Furthermore, aiming at greater efficiency in the analysis of performance-enhancing drugs
during international events (World Cup, Olympics, among others), Brazil has held regular
conferences with international institutions (IOC and WADA), and has adopted technologies
that enable greater security during analysis. Unfortunately, owing to problems related to lack
of infrastructure and proper preparation of staff, Ladetec, the only Brazilian laboratory
certified to conduct doping tests, was disaccredited by WADA in August 2013. According to
the agreements reached during the meeting in Canada, it is expected that Ladetec will once
again be accredited during the second half of 2015, therefore being able to exercise their
laboratory testing functions during the 2016 Rio Olympics. This is an indication and an
example of the known, unknown at a given time, and unknowable complexities associated
with doping and its intervention options and activities [14425].
Whenever it is talked about Dopagem (the Portuguese term adopted by the Autoridade
Brasileira de Controle de Dopagem – ABCD – for the act of doping) people usually think only
about the consumption of performance-enhancing drugs. Sometimes this is true. However,
the larger truth is that doping is not restricted to drugs of any type, it is about ethics; it is
about values. When an athlete uses prohibited substances and methods in a sport which she
or he practices, they are cheating the competition, defrauding other athletes, who have a
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talent, a technique, and the willpower to devote their lives to training and competition. Can
you imagine anything more unfair? The larger mission of the ABCD is to protect all athletes
who compete cleanly, only with their talent, technique, strength, and will. Not coincidentally,
the motto of the World Anti-Doping Agency – WADA – is Fairness (Play True, Jogo limpo).
When talking about WADA, people usually believe that international institutions to combat
doping are a relatively recent development in the history of global sports. However, the
present situation is the result of many years of antidoping efforts in the athlete's life as well
as in the entire sports community that surrounds them. Brazil was one of the first signatories
of the International Convention against Doping in Sport – CICDE, held in Paris on October
19, 2005, during the 33th General Convention of UNESCO. Inspired by the experience of the
best institutions in the world for doping control, the ABCD was founded on November 30,
2011, fulfilling one of the commitments made by Brazil on the occasion of the candidacy of
Rio de Janeiro to host the Olympic and Paralympic Games in 2016 and, in addition, to meet
the requirement established by WADA that all countries have specific and independent plans
for the fight against doping in sport. By accepting the invitation of the Brazilian Minister of
Sports, Aldo Rebelo, to build and implement the ABCD, I began to research the issue and it
became clear to me that it was fundamental for us to develop our strategic planning. In order
to propose feasible solutions to meet the challenges presented by mega sporting events, and
the day-to-day life of Brazilian sport, it was necessary to dive into the problem. And we did
just that! Five key points which guide the strategic planning and all of the actions of the
ABCD emerged from that effort; Information, Education, Prevention, Intelligence, and Action.
With the appropriate and necessary information it was possible to build the best Education
programs to achieve Prevention; allowing Intelligence to assess what has been done and
what needs to be done, therefore defining the path for Action. Much of this work relied upon
a broad survey conducted in early 2013, during the enrollment of beneficiaries of the Bolsa-
Atleta Program (Athlete Grant Program). At that time, 100 percent of Brazil's athletes who
had been awarded a scholarship, and who were enrolled through the portal of the Brazilian
Ministry of Sport, replied to the ABCD questionnaire. This enabled the necessary accurate
evaluation of doping control conducted by sports authorities in Brazil, since the sample
universe included all of the athletes interviewed. It is noteworthy that all who responded to
the ABCD survey are elite, high performance athletes in Brazil, representing all of the sports
of the 2016 Rio Olympic and Paralympic programs in their various categories. The survey
results revealed the need for more effective participation by the sports entities regarding
prevention measures and doping control, with a strong emphasis on guidance and education.
Among the most disturbing findings revealed by the survey was, that only two out of 10
athletes in Brazil already had undergone some sort of doping control test and, that the
majority did not seek the advice of authorities when taking a restricted drug. Moreover, only a
few Brazilian athletes looked to The World Anti-Doping Agency's (WADA) website for help.
Unfortunately, the web site has very little content in Portuguese, a situation which we at
ABCD plan to remedy later this year by working together with WADA to increase the site's
usefulness to Portuguese language speakers. Based on this information, the establishment
of the ABCD quickly became mandatory. It was elected to adopt the “best practice” as our
reference. Thus, to define the model for the structure and functioning of the ABCD, several
national and international meetings were held with those responsible for policies to control
and combat doping in various countries of the world. We worked together on several
agreements of international cooperation which provide exchanges in strategic areas of
intelligence, education programs, and the training of agents for doping control programs.
Today, the ABCD attends major events of the international antidoping community, bringing to
Brazil the best world practices in the fight against doping in sport. The mission of the ABCD,
the result of much internal discussion, reflects our philosophy of performance, that is,
protecting doping-free athletes: “Consolidate doping awareness and advocate at the national
level, the fundamental right of athletes to participate in sports competitions free from all forms
of doping”. In practice, it means that we have to promote and coordinate the fight against
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doping in sport in an independent and organized manner, in and out of competition,
according to the guidelines established by WADA, and the protocols and commitments made
by Brazil. When ABCD began its operation, one of the main difficulties faced by Brazil in the
fight against doping was the lack of adequate procedures for the importation of controlled
substances, in small quantities, for use as calibration standards and equipment in the
Laboratório Brasileiro de Controle de Dopagem/Laboratório de Apoio ao Desenvolvimento
Tecnológico—LBCD/LADETEC. Moreover, the lack of defined processes for transit of
controlled and biological samples used in the analyses performed by the Laboratory, as well
as the importation of sample collection kits, represented at the time, a huge obstacle to
antidoping activities in Brazil. To resolve this problem, we participated, together with the
Laboratory, in the development of revised guidelines and regulations with the Agência
Nacional de Vigilância Sanitária—ANVISA (National Agency of Sanitary Vigilance), resulting
in simplification of the importation of the materials used in the antidoping testing process.
The creation of specific processes and the establishment of operational procedures brought
about improvement in Brazil's capacity to deal with the basic needs for effectively intervening
against doping. Joint action with ANVISA resulted in changes implemented in the Bolsa-
Atleta Program, the largest program of individual sponsorship of athletes in the world and
managed by the Brazilian Ministry of Sport. Established in 2005, the Bolsa-Atleta Program is
targeted at high performance athletes who obtained the best results in national and
international competitions in their sport. The Bolsa-Atleta Program ensures support for
athletes training in their respective competitions. Currently, the Bolsa-Atleta Program has
more than 7,000 beneficiaries. Reaffirming the Brazilian commitment to fair play, all athletes,
starting in 2012, when they joined the Bolsa Atleta program, also agreed to submit to doping
control whenever notified by the federation of their sport or by the Brazilian Ministry of Sports
through the ABCD, at any time of the year, in or out-of-competition. It was also prepared a
Terms of Membership where the beneficiary (athlete) affirms in writing his commitment to
know and to avoid substance use and/or methods that constitute a breach of the rules as
described in the World Anti-Doping Code, which integrates the International Convention
Against Doping in Sport (under penalty of suspension of financial support provided by the
Bolsa-Atleta Program at the time of communication of the first adverse analytical finding).
When doping is proven, the athlete may have their benefits cancelled and need to return
funds already transferred, as well as other applicable penalties depending on the individual
case, after final judgment by the Justice of Sport (a courts that judge all National Sports
cases). Along with the Terms of Membership, every athlete receives a copy of WADA's
annually revised List of Prohibited Substances and Methods, a set of guidelines for the
athlete's safety, and information that the athlete should share with their family, team support,
and medical staff, as well as club officials, consulting them before they take any medication.
In 2012, the ABCD held its first out of competition doping control testing with the support of
LBCD/LADETEC for the analysis of samples. This pilot action served to gather important
information that assisted in the development of our Action Plan for 2014. Doping control
testing was performed on members of the Bolsa-Atleta Program in more than 20 different
sports. This preventive and health action was conducted in Brasilia, Rio de Janeiro, and São
Paulo and performed under the supervision of the Autoridade Antidopagem de Portugal
(ADoP) and included the participation of the most senior doping control officers of ADoP. In
2014, our Test Distribution Plan provides for the implementation of doping controls
encompassing all sports entities. The cost for conducting the tests will be fully subsidized by
the ABCD. Twenty percent of the tests (urine and blood) will be conducted out of competition
with athletes from the Bolsa-Atleta Program and the Bolsa-Podio Program (Athlete Grant for
a selected group of athletes govern is providing huge supporting), and 80% of tests (urine)
will be conducted during competition. For out-of-competition testing, we will use a national
whereabout system to be implemented throughout the year. Further testing of additional
urine (37.5% of total tests), involving EPO, growth hormone (hGH), and isotope, with the
selection of the athletes, will be performed using Intelligence based analysis. Also, we will
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dedicate part of our resources to begin following the “Athlete Biological Passport” model, in
which national and international authorities record the urine and blood profiles of athletes.
The ABCD will train and certify Doping Control Agents (DCOs) for the biennium 2014–2015.
The best of these professionals will be selected to work as DCOs in official test events, for
the Rio2016 Olympic and Paralympic Games. Further work will include training officers from
other countries in South America, who may also participate in the Rio2016 Games, as part of
a government policy presented to the South American Sports Council – CONSUDE.
Education is a priority for the ABCD. We want to promote the dissemination of information to
athletes, coaches, technical staff, and family. Therefore, the ABCD, in 2014-2015 biennium is
planning seminars and lectures geared toward athletes, educators, technicians,
administrators, physical education teachers, physicians, pharmacists, nutritionists, lawyers
and jurists, and sports organizations, aiming to spread the message of antidoping and the
rights of the athlete in Brazil. The National Anti-Doping Program is the crowning achievement
of the Strategic Plan prepared by the ABCD. It is inserted as a relevant public policy within
the Federal Government, having been included in the proposed Federal Budget, with an
expected budget of BRL12 million (USD 5,269,764) for the fiscal year 2014. The actions
listed in the National Anti-Doping Program provide a structured view for combating doping in
sport in Brazil, seeking fair play, and drug-free competitions. Further hearings to discuss and
enrich the program with other sport management bodies are also provided for. Our goal is to
have zero doping cases among the Brazilian delegation at the Rio2016 Olympic and
Paralympic Games. Our dream is that during 2016 we have zero cases of doping in sport
throughout Brazil. The new international order to prevent and fight against doping in sport,
based on the World Anti-Doping Code, reflects the growing awareness for ethical decisions
in compliance with the Core Principles of the Olympic Charter. Therefore, it is expected from
us that we demonstrate antidoping-enhancing values such as self-effort, good example,
respect for fundamental ethical principles, and especially, protection of the Clean Athlete
[14623].
Organization in football
UEFA
UEFA has many years of experience with the planning of in- and out-of-competition doping
controls in elite football and operates an annual anti-doping programme for all of its national
and club competitions. The programme uses an experienced team of doping control officers
(DCOs), established sample transport procedures and a network of World Anti-Doping
Agency (WADA)-accredited laboratories across Europe to ensure maximum effectiveness.
As the final round of the UEFA European Football Championship is one of the world's top
sporting events and UEFA's flagship tournament at national level, it is imperative that an
effective anti-doping programme is in place to deter and detect doping, seeking to ensure
that all results are achieved fairly and without the use of prohibited performance-enhancing
substances. Consequently, the aim for UEFA at UEFA EURO 2012 was to adapt its
established anti-doping processes to ensure an effectively planned and executed anti-doping
programme at the tournament. This was a significant challenge, with the tournament taking
place in two host countries, each with four host cities spread over a wide geographical area,
and only one WADA accredited laboratory in the two countries at which to analyse samples.
The tournament's anti-doping programme involved both pre-tournament out-of-competition
testing of competing squads at their preparatory training camps and a full programme of in-
competition testing at all matches in the tournament. Testing was supplemented by a pre-
tournament education and information programme for participating teams and players. The
final tournament of the UEFA European Football Championship is one of the top sporting
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events in the world, and a high-profile event of this kind requires a well-planned and well-
executed anti-doping programme to ensure the integrity of results in the competition. UEFA
EURO 2012 presented a unique logistical challenge, with the tournament spread across two
countries, both covering a large geographical area. This paper discusses the planning and
delivery of both the pre tournament out-of-competition (OOC) testing programme and the in-
competition (IC) programme, as well as reviewing the activities of doping control officers
(DCOs), the whereabouts programme and assessing the sample collection and transport
process. The analytical approach applied is also discussed, along with an overview of the
distribution of T/E ratios and blood parameters. The final round of the UEFA European
Football Championship is a tournament for the top national teams in European men's football
and is held every four years. The 2012 tournament was contested by 16 teams, which
reached the final round via a series of qualification matches held over the preceding two
years. That tournament was the 14th to be staged by UEFA and the first to be staged in the
neighbouring countries of Poland and Ukraine. The tournament began in Warsaw on 8 June
2012 and ended with the final in Kiev on 1 July 2012. A total of 367 players were registered
to take part (23 from each country, with the exception of one team that registered only 22
players). UEFA regulations specify that the tournament's in-competition period commences
24 hours before the first match of the tournament and ends 24 hours after the final match.
This meant that all pre-tournament samples were analysed on the basis of an out-of-
competition analytical menu and all tournament samples (including those collected between
matches) were analysed on the basis of an in-competition analytical menu [14446].
Collection of test samples

The collection of samples for the pre-tournament and tournament programmes was
conducted solely by UEFA DCOs and blood collection officers (BCOs). DCOs are only
qualified to collect urine samples, while BCOs are qualified to collect both urine and blood
samples. UEFA manages a team of approximately 40 DCOs across Europe, all of whom are
medical doctors with many years of experience in conducting doping controls for UEFA,
national anti-doping organisations (NADOs) and other international sports federations. DCOs
were selected to collect samples for the tournament and pre-tournament programmes on the
basis of criteria such as doping control experience and aptitude, proximity to the test venue,
nationality (to limit suggestions of bias), recent blood collection experience, and languages
spoken. For the pre-tournament programme, nine BCOs and 12 DCOs were used to collect
samples, with some undertaking multiple assignments on consecutive days. Doping controls
were normally conducted during teams’ scheduled training sessions (with samples collected
in places as diverse as Dublin, Moscow and Visby), and the DCOs/BCOs then delivered the
samples to the WADA accredited laboratory in Warsaw by plane. For the in-competition
programme, a team comprising six DCOs and six BCOs was selected, with officers working
in the same designated pairs at all of their appointed matches. The DCOs/BCOs were based
permanently in Warsaw, close to the tournament laboratory, and travelled by plane to each
game on the morning of the match, before returning on the first flight the following morning
[14446].
All UEFA DCOs are trained to collect urine samples in accordance with the WADA
International Standard for Testing (IST) as part of their role in UEFA's annual anti-doping
programme, so no new procedural training was required for the tournament programme. For
BCOs, specific instructions on procedures and the use of blood sampling equipment were
provided in advance of their first assignment in connection with the tournament. In addition,
all DCOs were briefed collectively on arrival in Warsaw for the in-competition programme by
means of a preparatory workshop covering documentation, equipment, player selection and
logistical procedures. During the tournament, DCOs were required to attend a debriefing
session after each doping control to review procedures and provide feedback in the event
that improvements could be made for the next match. During the tournament, DCOs’
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performance was monitored by a team of observers consisting of members of UEFA's anti-
doping unit, the UEFA Anti-Doping Panel and the UEFA Medical Committee. In addition, the
Chairman of the UEFA Anti-Doping Panel was present for the full duration of the tournament
to review the DCOs’ activities [14416].
Collecting and transporting samples, particularly blood samples, always presents a unique
challenge, with the need to ensure that the integrity of samples is maintained during the
journey from the collection site to the laboratory. This is particularly difficult where travel over
longer distances is required. The kit used has to be reliable, trusted by the players and
teams, and – perhaps most importantly – easy for DCOs and BCOs to use, store and
transport [14446].
Management of Therapeutic Use Exemptions (TUEs)

Management of TUEs followed UEFA's usual procedure, which complies with WADA's
International Standard for TUEs. All TUE applications for players had to be submitted to
UEFA 21 days before the start of the tournament (with the exception of emergency cases,
which could be reviewed at shorter notice if they occurred just prior to the start of the
tournament). Between 1 May 2012 and 2 July 2012, UEFA granted one TUE for
glucocorticosteroids. This was the only TUE applied for in connection with the tournament
out-of-competition testing programme [14446].
As is standard practice for doping controls, players were required to declare any medication
that had recently been taken or administered prior to the control. Of the 124 players tested at
the tournament, 65 (52 %) declared that they had taken some sort of medicine during the last
three months, while 59 (48 %) declared that they had not taken any medicine, or only
vitamins or minerals. Of the players who declared the recent use of medication, 4 percent
declared that they had been given a local cortisone injection during the last three months and
54 percent (i.e. 35 of those 65) declared that they had taken (or were taking) NSAIDs. Thus,
28 percent of the players tested at the tournament had taken NSAIDS during the previous
three months. These findings suggest that treatment of musculoskeletal problems with local
corticosteroids is uncommon in football, whereas treatment with NSAIDs is more frequent.
However, the figure for NSAIDs is lower than at UEFA EURO 2008, where 44 percent of
players tested reported having taken NSAIDs. Ten percent of the players tested declared
that they had taken sleeping pills on account of insomnia during the last three months
[14446].
FIFA
The fight against doping in sport receives considerable media interest and results in much
speculation regarding the ability of athletes to compete on a level playing field. Football was
one of the sports that took early leadership in this fight when the Fédération Internationale de
Football Association (FIFA) introduced doping controls in football in 1970 as part of a wider
strategy to ensure that the results of representative matches were a fair reflection of the
ability of those taking part. As a result of the collaborative effort between FIFA and regional
confederations and their member associations in conjunction with national anti‐doping
organisations, more than 20 000 doping controls are performed annually on football players.
The overall incidence of positive doping samples for prohibited substances accounts for 0.4
percent of all tests. Most of the positive drug tests are due to cannabis and cocaine, the
so‐called social drugs. Only a few individual cases (0.07 % of the positive tests in 2004) were
positive for anabolic steroids, such as nandrolone and testosterone. The majority of doping
controls have been carried out in competition. FIFA, the Union of European Football
Associations (UEFA), and some of the national anti‐doping organisations also perform
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unannounced, out‐of‐competition controls at training venues during the football season. Prior
to the 2006 FIFA World Cup being held in Germany, unannounced doping controls have
been performed in the friendly matches between nations. Doping controls have also been
performed during the training camps prior to the opening match on June 2006. All tests to
date have proved negative. UEFA has also performed unannounced testing in the 2005–06
football season in all of the teams participating in the UEFA Champions League and UEFA
Cup. Ten players were randomly selected from each of the 38 European top professional
teams and were subjected to testing. No prohibited substances were found in any of the 380
samples tested. Since 1994, FIFA has followed a similar strategy in international
competitions for both men and women. In these tests, two randomly selected players per
team are tested after each finals match and a total of 3327 tests have been performed in 32
tournaments to date. Only three samples have tested positive since testing commenced: one
for ephedrine, one for cannabis, and one for nandrolone. One sample tested positive for
ephedrine during the qualifying matches for the 2006 FIFA World Cup being held in
Germany. The incidence of positive tests in FIFA competitions over the past 12 years is 0.1
percent. During the 2000 Olympic Games in Sydney and the 2004 Olympic Games in
Athens, none of the football players tested positive for any prohibited substance. The internal
surveys of all Olympic team sports federations revealed that none of the team sports athletes
tested positive for prohibited substances. The comparison of positive drug tests among
different sports is currently not possible as the World Anti‐Doping Agency (WADA) presents
only adverse analytical findings in their published statistics rather than true positive results.
The statistics include “therapeutic use exemptions” as well as elevated (>4) testosterone to
epitestosterone (T/E) ratio which may be seen in normal athletes. Football accounts for the
majority of doping controls performed worldwide. The current doping statistics demonstrate a
very low incidence of positive tests and justifies the assumption that there is no evidence for
systematic doping in football and most probably in any of the other Olympic team sports.
Although no clear data are available from WADA about the distribution of in‐competition and
out‐of‐competition drug testing, it can be assumed that the majority are performed in
competition. It has to be remembered that the professional football season in which the
footballers are subject to random testing runs for 49 weeks a year in most football playing
nations. There are several possible explanations for the low incidence of the positive findings
of prohibited substances among football players:
- the stringent drug testing programme occurs during the entire football season in
most countries
- football players worldwide understand that prohibited substances in sport will
neither improve their physical performance nor their football specific skills and
hence are reluctant to use agents that are not effective and subject to possible
sanctions
- ongoing education campaigns by FIFA for doctors, administrators, officials and
players have encouraged a drug‐free culture in football
It is also possible that both in‐competition and out‐of‐competition testing is insufficient to

detect drug use. This is unlikely, given the large number of in‐competition and out‐of‐
competition drug tests occurring at all levels of professional sport over many years with
relatively few positive results. Over the past six years FIFA, realising that the dimension of
misuse of prohibited substances is different from that in individual Olympic sports, has also
developed close collaboration with the medical representatives of other Olympic team sports
federations, as well as with the International Rugby Board. The medical representatives of
these bodies expressed their collective opinion during the WADA meeting in March 2003 in
Copenhagen, suggesting a possible revision of the World Anti‐Doping Code given the
different needs of international team sports federations and the lack of evidence of
systematic doping in those sports. Furthermore, given the testing of over 20 000 doping
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controls in football annually worldwide, it became obvious that a close collaboration had to be
developed with the accredited testing laboratories to understand the different examination
methods and to keep abreast of new scientific developments. The close collaboration with
the laboratories has resulted in these laboratories being considered equal partners in the
global strategy against doping. It has also resulted in a number of research studies being
performed on controversial issues such as nandrolone metabolism, analysis of the T/E ratio
and the influence of age and ethnic differences on testosterone metabolism. It seems likely
that the constantly increasing number of drug tests will not alter the incidence of positive
findings. Unannounced testing at training grounds following the impressive example of UEFA
with the Champions League teams could be introduced in all FIFA confederations to provide
more information from possible misuse of prohibited substances between official matches.
The absence of any positive tests in the UEFA testing to date makes it unlikely that this
strategy will identify a significant number of drug cheats who are currently not being
detected. Given these findings, the question that arises is whether there is a need for
fundamental change in the strategy to fight doping in football? The FIFA Medical Committee
is of the opinion that the educational process has to be intensified with the help of national
associations and in particular, through team physicians. Team physicians play a central role
in the educational programme as they have direct influence over player behaviour and have
the knowledge to advise players, not only on the potential risks to health, but also of the
effect that sanctions may have on a player's career if he or she is caught. The 32 team
physicians of the 2006 finalists have once again confirmed their unconditional support of
FIFA's strategy by signing their joint declaration prior to the 2006 FIFA World Cup Germany.
The doping control officer at testing controls can also reinforce the educative aspect of the
fight against doping [06009].
The doping control proceure

The full details of the FIFA doping control procedure are set out in the annually updated FIFA
Doping Control Regulations (www.fifa.com/en/regulations/regulation/0,1584,9,00.html). With
regard to the medicolegal aspects of doping control procedures, the process is as follows:
- once an A sample has tested positive, then the FIFA Doping Control Sub‐
Committee investigates the documentation of the case and prepares a report for
the FIFA Chief Doping Control Officer. The FIFA Chief Doping Control Officer has
to verify that the correct doping control procedures have been completed according
to the doping control regulations. This process usually involves contacting the
testing laboratory as well as the original doping control coordinator where the
athlete was tested.
- if the analysis of the A sample is confirmed as positive by the FIFA Doping Control
Sub‐Committee's report, the FIFA General Secretary shall at once confidentially
notify the chairman of the Disciplinary Committee, the Sports Medical Committee
and the national association of the player concerned, which shall have the right to
request a second analysis using the B sample within 24 hours of being notified.
- if a second analysis is requested, FIFA shall communicate this request immediately
to the head of the laboratory where the B sample is being kept. An analysis of the
B sample shall be carried out as soon as possible, by personnel who were not
directly involved with the analysis of the A sample. The association concerned shall
have the right to have a representative present, in addition to the player concerned.
The results of the analysis of the B sample shall be sent immediately to the FIFA
Chief Doping Control Officer responsible, by fax or e‐mail. If no request for a
second test is made, the laboratory shall dispose off sample B after 30 days have
elapsed.
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In addition to the procedural roles above, the FIFA Chief Medical Officer and the Doping
Control Sub‐Committee also have to estimate the seriousness of the individual case from the
medical point of view as to whether the violation was intentional (partially autonomous but
not fully self‐responsible), deliberate (fully autonomous) or negligent and examine whether
any exceptional circumstances may apply. Finally, a written statement about the medical
analysis of the case including an estimation of the medicolegal aspects has to be submitted
to the FIFA Disciplinary Committee for consideration of sanctions. In cases where FIFA is
asked by a national federation or a confederation to take over the sanction or decide about a
sanction for the international level, the same procedure is carried out. The individual case
management as outlined above is an integral part of FIFA's approach to doping control and
based on Swiss sanction law. This means that there must be evidence that the player is
personally guilty of the offence being sanctioned and the unjustness of his behaviour has to
be obvious to him. Thus, every sanction inevitably contains a distinctive individual
component [06002].
FIFA Medical Assessment and Research Centre (F‐MARC)
The ongoing media debate surrounding the issue of doping in sport has raised public
awareness of a problem that been steadily developing over many years. This controversy
reflects both the rapid development of various sports disciplines as well as the evolution of
new doping methods and agents. The Fédération Internationale de Football Association
(FIFA) introduced doping controls in football in 1970 to ensure that the results of national and
international matches were a fair reflection of the ability of those taking part. Over the past 12
years, the FIFA Medical Assessment and Research Centre (F‐MARC) has developed a
worldwide network of specialists who are involved in the educational process within the
regional football confederations and national associations as one facet of global anti‐doping
strategies. F‐MARC has also been involved in the practical implementation of doping controls
for FIFA competitions at all levels. FIFA has developed close collaboration not only with the
confederations and member associations, but also with other team sports federations,
particularly with the accredited drug testing laboratories. These relationships have helped to
understand the extent of doping, which in turn forms the basis of a global, harmonised
strategy in the fight against doping in football [06010].
FIFA's approach to doping in football.
FIFA's anti-doping strategy relies on education and prevention. A worldwide network of

physicians guarantees doping control procedures that are straightforward and leave no place
for cheating. FIFA actively acknowledges its responsibility to protect players from harm and
ensure equal chances for all competitors by stringent doping control regulations, data
collection of positive samples, support of research, and collaboration with other
organisations. One article aimed to outline FIFA's approach to doping in football. Data on
positive doping samples per substance and confederation/nation documented at the FIFA
medical office from 1994 to 2005 are provided. According to the FIFA database, the
incidence of positive cases over the past 11 years was 0.12 percent, with about 0.42 percent
in 2004 (based on the assumption of 20,750 samples per year) and 0.37 percent in 2005.
Especially important in this regard is the extremely low incidence of the true performance
enhancing drugs such as anabolic steroids and stimulants. However, there is a need for
more consistent data collection and cross checks among international anti-doping agencies
as well as for further studies on specific substances, methods, and procedures. With regard
to general health impairments in players, FIFA suggests that principles of occupational
medicine should be considered and treatment with banned substances for purely medical
reasons should be permitted to enable players to carry out their profession. At the same
328
time, a firm stand has to be taken against suppression of symptoms by medication with the
aim of meeting the ever increasing demands on football players. It was concluded that the
incidence of doping in football seems to be low, but much closer collaboration and further
investigation is needed with regard to banned substances, detection methods, and data
collection worldwide [06001].
FIFA introduced doping controls in 1970 to ensure that the results of national and
international matches were a fair reflection of the ability of those taking part. The FIFA Sports
Medical Committee is responsible for implementing doping controls at all FIFA competitions
and also for coordinating with confederations and member associations. The overall
management of doping controls is conducted by the FIFA administration (Medical Office and
the FIFA Sports Medical Committee). Over the past 12 years, the FIFA Medical Assessment
and Research Centre (F‐MARC) has developed a worldwide network of specialists who are
involved in the educational process within the confederations and national associations as
well as in practical performance of doping controls for national, international, and FIFA
competitions. The medical doctors/sports physicians, following their Hippocratic Oath as well
as their professional and ethical values, play key roles in FIFA's long term strategy in the
fight against doping. Many of these doctors are also team physicians in their national
associations. The fight against doping in football focuses on education and prevention with
regular in‐competition and out‐of‐competition controls. In past years, approximately 15 000
doping controls were performed annually on footballers, with over 20 000 performed in both
2004 and 2005. FIFA articulated its unyielding position in the fight against doping prior to the
world cup competition in both 1998 and 2002 and reinforced its strategy in the FIFA
Magazine in March 2004. Physicians demonstrated their strong support of the FIFA long
term strategy in its fight against doping before the 2002 FIFA World Cup Japan/Korea. The
team physicians of all 32 finalists signed a joint declaration in the fight against doping,
supporting FIFA's decision to introduce routine blood sampling to analyse for blood doping
and erythropoietin (EPO). This was a firm message to the football community and
demonstrated the excellent collaboration and cooperation between the FIFA Sports Medical
Committee and the team physicians taking care of the players before and during the
competition. The team physicians of all the finalists of the 2006 FIFA World Cup Germany
again reinforced the fight against doping with a joint declaration signed on 5 March 2006 to
keep this unique event free of doping [06001].
FIFA is a global organisation that unites over 250 million footballers in 207 countries. Around
40 million of these players are female. Currently, confederations, national associations, or
both that fall under FIFA's management, carry out their own doping controls at the
competitions they stage. However, the urine or blood samples, or both must be analysed at
laboratories accredited by FIFA/World Anti‐Doping Agency (WADA). These laboratories send
reports on any “chemically positive” A samples to the member associations, and FIFA
headquarters for management and WADA for information. Once the FIFA medical office
receives a positive A sample report, it requires follow up information from the national
association/confederation in question, or both – that is, the results of the possible B sample
decision made by the particular disciplinary committee. If the information is not provided, the
FIFA disciplinary committee takes appropriate action. Since the 1994 FIFA World Cup in the
USA, the FIFA Medical Office has undertaken stringent registration of analysed samples. A
new doping control policy for FIFA competitions was introduced at the FIFA U-17 World
Championship in New Zealand in 1999. Since then, during tournaments, two players from
each team are randomly selected to undergo doping tests after each match. Between 1994
and 2005, 3327 doping controls (men and women) were performed during three consecutive
FIFA world cups (USA, France, Korea/Japan), two consecutive Olympic games (Sydney,
Athens) as well as the last Women's World Cup (USA, 2003), the FIFA U‐19 in Thailand, the
FIFA U‐17 World Cup in Peru, the FIFA Confederations Cup in Germany, the FIFA Club
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World Cup in Japan, the FIFA Beach Soccer World Cup in Brazil, the FIFA U‐20 World Cup
in the Netherlands, and FIFA World Championship in Futsal, Chinese Taipei, as well as
during the World Cup 2006 preliminaries. Only four samples tested positive during this
period: one for ephedrine and pseudoephedrine in 1994 one for cannabis and one for
nandrolone during the FIFA World Youth Championship 2003 held in the United Arab
Emirates, and one for ephedrine in Angola. This reflects an overall incidence of 0.12%
positive cases over the past 11 years. The extremely low incidence of positive cases during
FIFA competitions indirectly confirms the FIFA long term strategy in the fight against doping:
that education and prevention play a key role in keeping high profile competitions free of
doping [06001].
It can only be assumed that team sports such as football are not as prone to misuse of
performance enhancing substances as are individual sports. During the 2004 Olympic
Games in Athens, there were 27 positive cases – all in individual athletes and none in any
team sport participants. It might be hypothesised that the close collaboration of the team
sport medical committees since the 2000 Olympic Games in Sydney, positively influenced
the attitude of fairplay among team sports during the Olympic Games in Athens. Close
collaboration between accredited laboratories, the reporting system, and the central control
system is an important tool for statistical recording of the extent of doping in football in the
future. Although several prominent footballers have tested positive for drugs in recent
decades, the true extent of the problem is unknown. Even if we assume that doping is still
not a major issue in team sports such as football, any estimation of the problem can be
considered as merely an unscientific hypothesis or speculation. To meet the challenges
brought about by this situation, FIFA has taken action to develop closer collaboration
between the medical committees of the various confederations. In October 1999, the FIFA
Sports Medical Committee and the Union of European Football Associations (UEFA) Medical
Committee met to discuss the latest sports medicine issues with the aim of not only
combating doping but also developing educational programmes designed to meet the
fundamental objectives outlined above. Similar meetings have been conducted between the
representatives of the FIFA Sports Medical Committee and the medical committees of the
Confederation of North, Central American and Caribbean Association Football (CONCACAF)
(North and Central America, 2000, 2001), Asian Football Confederation (AFC) (Asia, 2001,
2002, 2005), and Confederation Africaine de Football (CAF) (Africa, 2003, 2004). During
2005, meetings were conducted with the newly established Oceania Football Confederation
(OFC) Sports Medical Committee and Confederación sudamericana de Fútbol (CONMEBOL)
with the aim of harmonising doping control procedures, improving the understanding of the
scientific background of doping, and enhancing the FIFA network of doping control officers
(DCOs) who fulfil educational duties as a part of their responsibilities [06001].
According to the statistics of the International Olympic Committee (IOC) (until 2003) and
WADA accredited laboratories (as of 2004), approximately 20 750 doping controls are
performed annually on football players. The majority of the controls are done in Europe and
North and South America. The numbers of doping controls continue to increase in the other
confederations. In this respect, FIFA developed its own database to keep records on the
substances being reported as positive to allow online control of management of these
samples within the different confederations and member associations. During 2004 and
2005, 88 (0.42 % based on the assumption of 20 750 samples per year) and 78 (0.37 %)
positive samples, respectively, were registered at FIFA. The increase is probably because of
improved reporting systems used by the laboratories as a result of the implementation of
WADA (March 2004). The majority of the positive cases were detected or reported by the
European laboratories which receive most of their samples from the European national
associations [06001].
330
The FIFA database will allow a continuous cross‐check with the WADA database (ADAMS,
Anti‐Doping And Management System), once that is operational, not only to control the
reporting system of the WADA accredited laboratories, but also to allow prospective studies
on sanctions related to the different substances, the severity of the violation, or both. Just
before the FIFA World Cup in France in 1998, a number of well known players tested positive
for small amounts of nandrolone metabolites in their urine. Nandrolone (chemical name
nortestosterone) is an anabolic steroid often encountered in bodybuilding doping cases. In
general, this compound is taken in high doses and its degradation products (metabolites)
remain detectable in urine for up to several months. Before the 1998 World Cup, FIFA
commissioned an independent anti‐doping laboratory (LAD) to carry out a collaborative study
to obtain a true picture of the situation in football. With the agreement of national and
international bodies, every player from every team in the top national leagues in Switzerland
(A and B leagues) was tested after a game (356 players in total over two weekends) in
collaboration with the Swiss anti‐doping committee. The results were compared with those
obtained by testing amateur footballers and students. Without revealing anything about the
origin of these products, the study showed some players had nandrolone metabolites in their
urine after the game. The traces of metabolites in the urine of those players were very small,
and all were below the limits of a positive reading. On the basis of this study, FIFA was able
to organise the anti‐doping programme for the 1998 World Cup with a degree of assurance
to provide reliable information to the competing teams to rule out any occurrence of false
positive tests. With FIFA's support, this study into nandrolone and its derivate substances
continued. Extraordinary variability in excretion was shown, making the relation between
dosage, time delay, and urine concentration critical. Involvement of a world governing body
in such a research programme is essential if any worthwhile progress is to be made in this
area. The players can also be given the assurance that, scientifically and ethically, they start
a match on an equal “playing field” with their opponents as far as doping is concerned
[06001].
Future challenges
In 2006, FIFA launched a new developmental programme, the Futuro III. The FIFA Medical
Committee undertook to implement the mandate of Mr Joseph S Blatter, President of FIFA,
and the FIFA Executive Committee, to educate more than 3000 physicians worldwide in
football medicine over the next three years. Anti‐doping education is an integral part of the
instructional courses, which were launched in February 2006 in Oceania and then held again
at the CONMEBOL confederation (South America) in April 2006. The active participation
within the instructional courses will entitle physicians to become members of the worldwide
network of FIFA medical officers, not only to deal with optimal management and prevention
of injuries, but also to act as FIFA doping control officers throughout the 207 member
associations of FIFA in collaboration with their national anti‐doping organisations. In this
respect, FIFA is of the opinion that the doping control programmes have to be carried out by
the members of the international sports federations and are obligatory for physicians. There
is no need to delegate this important work to commercial companies. The experience of FIFA
clearly indicates that employing physicians to perform the doping controls is not only effective
but can be done at low cost and most probably will reduce the risk of potential corruption as
the physicians have to follow their professional ethical codes of conduct and have
medicolegal constraints. Another challenge is the continuous search for identification of new
performance enhancing drugs being distributed on the market via the internet and in this
respect medical science, in close collaboration with laboratory experts and the scientific
committee of the World Anti‐Doping Agency, might help to identify possible new drugs and
sanction their misuse accordingly. Arguably the major challenge for the future lies in genetic
doping and its detection. There is no doubt that we cannot stop the development of medical
science as the development of altered genetic information seeks to benefit the many patients
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with incurable diseases. Yet it could be hypothesised that this scientific advancement might
be misused for performance enhancement in sport. In this regard, the education and
cooperation of team physicians forms a crucial link in the chain to prevent athletes adopting
such strategies [06009].
The lowering of the threshold for the ratio of testosterone to epitestosterone (T/E) from 6 to 4
has led to intense discussion with the accredited laboratories and raised concerns on behalf
of FIFA. According to the FIFA database 2005 none of the samples with elevated ratios
between 4 and 6 showed evidence of exogenous intake in the gas chromatography isotope
ratio mass spectrometry (GC‐IRMS) tests. In face of the logistic impact and additional costs
FIFA should strongly advocate detailed statistical analysis of the WADA data, examining the
incidence of exogenous intake of testosterone in samples with T/E ratios between 4 and 6.
Furthermore, legal difficulties arise in cases where the T/E ratio is between 4 and 6 but
GC‐IRMS does not verify exogenous intake [06002].
Recent years have shown a constant increase of positive tests for recreational drugs. While
this finding reveals rather a social than a doping problem in the sense of the word, an
important legal aspect has to be considered too: the consumption of marihuana presents a
severe offence against the law in some countries especially in Africa and Asia, even if
consumed abroad. Here, the publication of a positive result may lead to serious
consequences for the respective player including a prison sentence. Anti‐doping bodies
should therefore carefully reconsider the unconditioned ban of recreational drugs, preferably
based on a juridical expert's opinion [06002].
While the World Anti‐Doping Code and the Doping Control Regulations of FIFA offer a
comprehensive basis for the fight against doping, the permanent progress in the
development of new substances as well as laboratory methods calls for regular review and
update of adopted policies. Whereas harmonisation of the strategies of national and
international anti‐doping agencies is reinforced, the legislation and politics of different
countries constitute a permanent obstacle. Any regulation concerning medicolegal aspects
should therefore be based on scientific evidence and juridical expertise and has to be
supported by close collaboration of national and international bodies [06002].
Costs of the anti-doping organization
WADA estimates the 2006 average cost of an A-sample analysis at USD 330 and the cost of
a B-sample analysis at USD 574. Some 9 percent of doping tests are EPO analyses
(calculated on the basis of WADA 2012) which cost an additional USD 424, on average.
These costs include the “silent” subsidies by other institutions (e.g. governments,
universities, hospitals). Also the costs of IRMS (Isotope mass ratio spectrometry) and the
costs of obligatory storage of urine samples should be included. Currently, approximately 6
percent of doping tests are blood tests (calculated on the basis of the test statistics of WADA
2012 which – according to the Australian Sports Anti-Doping Authority – are about 50
percent more expensive than urine tests. Taking into account general inflation and assuming
an average 2014 cost of some USD 600 per doping test, the laboratory bills total about
USD161 million. In addition, other aspects of the anti-doping programs such as (normal and
abbreviated) therapeutic use exemptions, whereabouts programs, and education about the
prohibited lists are estimated to require involvement of the federations′staff of 2.49 Full Time
Equivalents (FTE) plus USD 26,750 out-of-pocket costs per International Federation (IF). Per
National Anti-Doping Agency (NADO), the respective costs amount to 4.85 FTE plus USD
80,600. Setting a FTE at USD 50,000, the costs of the NADOs, representing 62 countries,
and the 70 IOC-recognized IFs total to about USD 30.6 million. WADA's annual expenditures
332
are approximately USD 26 million, of which about one million is for testing fees and should
be excluded in order to avoid double-counting. In addition to the costs mentioned above, he
and his staff have also included “costs of result management, legal advice etc. In addition the
IOC and the Organising Committees bear substantial costs of the Anti-Doping-Laboratories
during the Olympic Games. Also the costs for research for new testing methods have to be
included [14416].
The increase in the number of individual drug tests conducted between 2005 and 2012 is
approximately 90 000, an increase of about 50 percent, yet the number of adverse analytical
findings has remained broadly the same. Similarly, the reported adverse analytical findings
for anabolic steroids between 2005 and 2012 range from 715 in 2007 to 1038 in 20128 – a
modest increase. The implications of these important statistics are that increasing the
number of doping tests will not necessarily result in a corresponding increase in the number
of adverse analytical findings. In 2012, WADA reported 4,723 adverse analytical findings
(AFA) for the use of prohibited substances or methods (1.76 %). If the enumerated costs are
applied only to AFAs, every “finding” gives rise to a cost of about USD 34,650. AFAs should
not be confused with adjudicated or sanctioned Anti-Doping Rules Violations (ADRV)
because they may contain findings that underwent the “Therapeutic Use Exemption”
approval process. Some AFAs may equal multiple measurements performed on the same
athlete that is, tracking the testosterone level of one athlete over a period of time. No
worldwide statistics are available about the ratio of ADRV to AFAs. In the case of Germany,
for 2012, its share was about 50 percent. Assuming that share worldwide, each ADRV costs
about USD 69,300 [14416].
Large financial resources are needed to implement the current antidoping policy. The cost of
organising, conducting, analysing and managing a single doping test is estimated at
approximately USD 1000 on average. Consequently, given the number of doping tests
conducted on average each year globally, the current estimate for the annual cost for the
fight against doping is USD 30 million per year. Based on these statistics, it costs around
USD3 million to catch one cheat in football for anabolic steroids. Similarly, catching a cheat
for any other prohibited substance, including marijuana, could cost up to USD 300 000. While
this analysis is clearly overly simplistic, it serves to highlight the need for considered risk
assessments that take into account the specifics of the sport (i.e. individual or team) as well
as the standard of the athletes (i.e. professional, semiprofessional, amateur) as these factors
significantly influence the likelihood of doping. Thus, there is need for a comparative study of
the prevalence of adverse analytical findings and positive cases among different sports with
particular emphasis on high-profile competitions such as World Cups, Olympic Games,
World Championships and other major competitions that attract the attention of the public
and substantial sponsorship (e.g. Tour de France, Giro d'Italia, Super Bowl). A similar
approach was recently recommended by the Australian Sports Commission following clear
evidence of systematic doping in Australian Rule Football. This notion is further reinforced by
the 2015 World Anti-Doping Code that specifies the need to consider sport-specific doping
patterns when implementing effective prevention [14429].
Where-abouts
The costs of the anti-doping system go far beyond these pecuniary costs. The current anti-
doping system implies an intensive surveillance of athletes which has – perhaps with the
exception of prisoners – no parallel for other groups of individuals. Using a web-based
application (ADAMS), elite athletes have to report their daily activities. They have to “specify
one hour each day (between 6 a.m. and 11 p.m.) during which they can be located at a
specified location for testing. If they are not at the indicated location at the specified time,
333
they expose themselves to the risk of a missed test. In addition, they are required to indicate
their regular activities for testing purposes. This information does not have to cover every
24/7 movement of the athlete but only recurring or regular activities: for example, overnight
home (address), morning training (address), 14:00–15:00 training (available for testing).” If
such rules were applied to other individuals (employees, students, etc.) it would be
prosecuted as a violation of privacy and other human rights. There have been protests of
elite athletes over this procedure. Nevertheless, to date, this has not been examined by any
national or international human rights organization [14416].
Sports federations
However, the federations are stopping only halfway. Apart from the fact that the penalty
amount seems arbitrary and too low: additionally, it must be ensured that the athletes can
hedge themselves against undesired manipulation by their supervisors. Recipients of the
penalties should ultimately and actually be the damaged parties themselves – not
necessarily their own sports federation. Damaged parties are the fellow competitors, but also
the athletes of other disciplines who also lose credibility because of doping scandals.
Speaking of the financial responsibility of associations, in Germany, every leading
organization has to make a financial contribution to the National Anti-Doping Agency (NADA)
which is proportional to the number of national squad members. This leads to the “perverse”
situation that a successful association, with many top-level athletes, such as the Rowing
Federation, needs to pay a relatively large amount, although there was no single case of
doping during its more than 125 years of existence. The Rowing Federation “forks” out for
the doping offenses of other associations. What if the financial contributions to NADAs are
regulated proportional to the number of doping offenses of each association during the last
four years? Doping-prone federations would certainly feel encouraged to undertake greater
efforts against doping [14416].
Cost reduction proposals
In recent years, organized sport has taken extensive measures to increase the probability of
detection by establishing an unparalleled international laboratory testing industry. The
relevant testing stakeholders include a range of visible, hidden, and unknowable individuals
and institutions, each with their sources of influence, agendas, and goals. This industry – as
any industry – can be expected to pursue its own (income) interests. A typical tool in such
self-interest-led strategies is public relations which warns that any insufficient increase of its
funding will harm the consumers. The anti-doping system, under the guidance of WADA,
costs at least USD 228 million per year, mostly to cover the cost of performing about 270,000
doping tests. However, "testing has not proven to be particularly effective in detecting
dopers/cheats" (WADA). It is suggested, competitions of doping-endangered disciplines be
redesigned. Sports with numerous doping cases should be temporarily excluded from the
Olympic program and not be televised. Pecuniary fines should be higher and collection
guaranteed by a deferred compensation model. Sports with multiple doping offenses should
bear most of the anti-doping costs. Finally, appropriate tenders should guarantee fees of
anti-doping laboratories develop more competitively [14416].
The anti-doping system, under the guidance of WADA, costs at least USD228 million per
year, mostly to cover the cost of performing about 270,000 doping tests. However, "testing
has not proven to be particularly effective in detecting dopers/cheats" (WADA). It is
suggested, competitions of doping-endangered disciplines be redesigned. Sports with
numerous doping cases should be temporarily excluded from the Olympic program and not
be televised. Pecuniary fines should be higher and collection guaranteed by a deferred
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compensation model. Sports with multiple doping offenses should bear most of the anti-
doping costs. Finally, appropriate tenders should guarantee fees of anti-doping laboratories
develop more competitively [14624].
Narrowing the gap between doped and anti-doping controllers
The analytical methods developed and applied by the antidoping laboratories have been
continuously evolving over the past 50 years, with the aim of keeping pace with the constant
evolution of doping strategies. Despite this, the number of adverse analytical tests reported
worldwide by the network of the WADA-accredited laboratories still seems to underestimate
the actual number of doped athletes. It was investigated the most likely causes for this gap
between the likely doping rate and the detection of athletes with positive doping tests. We
consider laboratory and non-laboratory reasons that contribute to this gap. More specifically,
laboratory issues are focused not only on those doping practices that may still be invisible at
the time of a doping test, but also on the possible role of non-conventional masking
strategies. These include
- the intake of banned drugs by specific novel drug delivery systems

- the coadministration of prohibited and non-prohibited drugs, taking advantage of the
capacity of the latter to affect the metabolism, and consequently the detection, of the
former
Non-laboratory issues include the lack of a sufficient level of “intelligent testing”, with the
result that, even in the cases of doped athletes, the biological samples delivered to the
antidoping laboratories for analysis may not contain those target analytes whose detection
(and if necessary quantification above a decision limit) constitutes an adverse analytical
finding. It was present proposals to improve the efficacy of the doping control policies based
on the analysis of biological samples and suggest how to constantly keep up with the
continuous developments of new forms of doping [14414].
WADA-accredited doping laboratories
In 2013 there were 35 WADA-accredited laboratories. Their locations were [13003]:
Continent (n) County (city)

Asia (6) China (Beijing), India (New Delhi), Japan (Tokyo), Korea (Seoul), Malaysia
(Penang), Thailand (Bangkok)
Africa (2) Republic of South Africa (Bloemfontein), Tunisia (Tunis)
Australia (1) Australia (Sydney)
Europe (20) Austria (Seibersdorf), Belgium (Ghent), Czech Republic (Prague), Finland
(Helsinki), France (Paris), Germany (Cologne and Kreischa), Great Britain
(London), Greece (Athens), Italy (Rome), Norway (Oslo), Poland (Warsaw),
Portugal (Lisbon), Romania (Bucharest), Russia (Moscow), Spain (Barcelona
and Madrid), Sweden (Stockholm), Switzerland (Lausanne), Turkey (Ankara)
North Canada (Montreal), United States (Los Angeles and Salt Lake City)
America (3)
South and
Central Brazil (Rio), Colombia (Bogota), Cuba (Havana)
America (3)
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Some antidoping organization
United Nations Educational, Scientific and Cultural Organization (UNESCO)
UNESCO Headquarters is located in Paris, France. Offices are located in two locations: 7
Place de Fontenoy 75352 Paris 07 SP France and 1 rue Miollis 75732 Paris Cedex 15
France. General phone: +33 (0)1 45 68 10 00. UNESCO contributes to the development of
antidoping education and prevention programs aimed at promoting sport values and
informing young people on the moral, legal, and health consequences of doping.
http://www.unesco.org/new/en/socialand-human-sciences/themes/anti-doping/ [14447].
The World Anti-Doping Agency (WADA)
WADA headquarters is located in Montreal, Canada. WADA, Stock Exchange Tower, 800
Place Victoria (Suite 1700), P.O. Box 120, Montreal, Quebec H4Z 1B7 Canada. Telephone:
+1 514 904 9232, Fax:+1 514 904 8650.WADA’s mission is to lead a collaborative worldwide
campaign for doping-free sport. WADA was established as an international independent
agency composed and funded equally by the sport movement and governments of the world.
Its key activities include scientific research, education, development of antidoping capacities,
and monitoring of the World Anti-Doping Code – the document harmonizing antidoping
policies in all sports and all countries. WADA is a Swiss private law foundation. Its seat is in
Lausanne, Switzerland, and its headquarters is in Montreal, Canada. WADA works toward a
vision of a world where all athletes compete in a doping-free sporting environment.
Approximately 90 National Anti-Doping Organizations are associated with WADA.
http://www.wada-ama.org/en [14447].
United States Anti-Doping Agency (USADA)
USADA is located in Colorado Springs, Colorado. USADA, 5555 Tech Center Drive, Suite
200, Colorado Springs, Colorado 80919-2372 USA. Telephone: Main: 719-785-2000, Athlete
Express: 719-785-2000, Toll free: 866-601-2632, Fax: 719-785-2001. The Goals of USADA
are to: 1) preserve the value and integrity of athletic competition through just initiatives that
prevent, deter, and detect violations of true sport; 2) inspire present and future generations of
US athletes through initiatives that impart the core principles of true sport – fairplay, respect
for one’s competitor, and respect for the fundamental fairness of competition; and 3) protect
the right of US Olympic and Paralympic athletes to compete healthy and clean – to achieve
their own personal victories as a result of unwavering commitment and hard work – to be
celebrated as true heroes. http://www.usada.org/ [14447].
Statistics
Statistics from testing in all IOC laboratories [01002].
Year Total number of samples Number positive for steroids % positive
1999 118 259 973 0.82

1998 105 250 856 0.81
1997 106 561 967 0.91
1996 96 454 1131 1.17
336
1995 93 938 986 1.05
1994 93 680 891 0.95
1993 89 166 940 1.05
1992 87 808 717 0.82
1991 84 088 552 0.66
1990 71 941 579 0.81
1989 52 371 611 1.17
1988 47 069 791 1.68
Statistics from hormone testing in the UK Sport programme [01002].
95/96 96/97 97/98 98/99 99/00
T/E ratio 4 16 4 6 5
Methandienone 4 1 1 4
Nandrolone 4 3 4 8 20
Total 15 28 13 20 36
The Sochi Olympics 2014
The laboratory anti-doping services during XXII Winter Olympic and XI Paralympic games in
Sochi in 2014 were provided by a satellite laboratory facility located within the strictly secured
Olympic Park. This laboratory, established and operated by the personnel of Antidoping
Center, Moscow, has been authorized by the World Anti-Doping Agency (WADA) to conduct
doping control analyses. The 4-floor building accommodated the most advanced analytical
instrumentation and became a place of attraction for more than 50 Russian specialists and
25 foreign experts, including independent observers. In total, 2134 urine and 479 blood
samples were delivered to the laboratory and analyzed during the Olympic Games (OG), and
403 urine and 108 blood samples – during the Paralympic Games (PG). The number of
erythropoietin tests requested in urine was 946 and 166 at the OG and PG, respectively.
Though included in the test distribution plan, a growth hormone analysis was cancelled by
the Organizing Committee just before the Games. Several adverse analytical findings have
been reported including pseudoephedrine (1 case), methylhexaneamine (4 cases),
trimetazidine (1 case), dehydrochloromethyltestosterone (1 case), clostebol (1 case), and a
designer stimulant N-ethyl-1-phenylbutan-2-amine (1 case) [14704].
Laboratory equipment
After a lengthy period of preparation, a satellite anti-doping laboratory in Sochi was

authorized by the World Anti-Doping Agency (WADA) to conduct doping control analyses
starting from 27 January to 15 April 2014. The analytical instrumentation, all from Thermo
Fisher Scientific (San José, CA, USA and Bremen, Germany), included four gas
chromatography-triple quadrupole mass spectrometry (GC-MS/MS) systems Trace 1310 /
TSQ Quantum XLS Ultra (steroid analysis), five ultra high performance liquid
chromatography-triple quadrupole mass spectrometry (UHPLC-MS/MS) systems Dionex
Ultimate 3000RS/TSQ Vantage (dilute-and-shoot and total fraction analysis), two gas
chromatography-mass spectrometry (GC-MS) systems Trace 1310/ISQ (one for
glycerol/HES screening and the second for isotope ratio mass spectrometry (IRMS)
identification purposes), two liquid chromatography (LC) systems Dionex Ultimate 3000
(IRMS sample preparation), and one gas chromatography-combustion-carbon isotope ratio
337
mass spectrometry (GC-C-IRMS) system Trace 1310/GC Isolink/ConFlo IV/Delta V Plus.
The third floor of the laboratory was focused on bioanalytical methods and mass
spectrometry of peptidic drugs, including insulins. The mass spectrometric equipment
consisted of an UHPLC-MS/MS system Dionex Ultimate 3000RS/TSQ Quantiva for growth
hormone releasing peptides and a nano-LC OrbitrapMS system Dionex Ultimate
3000RSLCnano/Q Exactive for insulins. The non-chromatographic equipment included one
Cobas E411 immunoanalyzer from Roche Diagnostics GmbH (Mannheim, Germany) for
analysis of human chorionic gonadotropin (hCG) and luteinizing hormone (LH), two Sysmex
XT2000i (Kobe, Japan) systems for athlete blood passports, a flow cytometer Navios from
Beckman Coulter (Lismeehan, Co. Clare, Ireland) for homologous blood transfusion, and
immunoanalyzer Personal Lab from Adaltis (Rome, Italy) for continuous erythropoietin
receptor activator (CERA) analysis. Representatives and service engineers from Thermo
Fisher Scientific were on duty during the whole period of the Games [14704].
Maximum expected number of samples per day was exceeded, peaking at 183 urine
samples in the middle of the Games. This inevitably led to delays with reporting analysis
results to ADAMS with median reporting time 33 h (negative), 43 h (EPO), and 60 h (insulin)
after sample reception. It is worth saying that all samples delivered before and during the OG
were considered as pre- or in-competition tests, meaning that they have to be analyzed using
a full menu. On the contrary, by the agreement with the IPC, some of the samples collected
before and during the PG were considered as out-of-competition tests and were analyzed
accordingly [14704].
Analytical findings during the Games were mainly related to the permitted therapeutic use of
prohibited compounds (TUE), such as corticosteroids and diuretics. Several double blind
quality control samples were introduced with other samples to the laboratory and
appropriately identified. It is interesting to note that one of the reported adverse analytical
findings (AAF) was trimetazidine, a compound added to the Prohibited list just before the
Games in 2014. The AAF for dehydrochloromethyltestosterone (oralturinabol) was the most
challenging, as the concentration of the long-term metabolite was approximately 0.05 ng/mL.
For confirmation purposes, three aliquots of this urine were taken (3 mL each), and after
extraction with n-pentane the organic extracts were combined and evaporated in a single
tube before derivatization. The resulting signal was abundant enough to meet the
identification criteria calculated against the reference collection urine [14704].
The testing
A most significant finding was an increase in EPO analyses, which was requested by the
IOC. The growth hormone releasing peptides were analyzed in approximately half of the
samples, while growth hormone (hGH) analysis was excluded from the testing menu at all.
All test that were performed:
Urine Olympics
ITP 2134
EPO 946
Insulin 70
GHRPs 908
IRMS 74
Paraolympics
ITP 403
EPO 166
Insulin 0
GHRPs 168
338
IRMS 13
Blood Olympics
hGH 0
CERA 324
ABP 62
HBOCs 92
Transfusion 64
Paraolympics
hGH 0
CERA 76
ABP 44
HBOCs 45
Transfusion 1
After the Games, all urine and blood samples were resealed and packed into special
transport boxes designed to accommodate 60 Berlinger bottles for urine and 96 bottles for
blood per box, and shipped frozen with a proper temperature control to Lausanne for a long-
term storage in the IOC repository [14704].
339
EPIDEMIOLOGY OF DOPING
Given the profile of drug use in high-performance sport, it is left to wonder about the use of
anabolic steroids in the playgrounds, gymnasia and arenas of our neighbourhoods. However,
several years ago, it was noted that the use of anabolic steroids had crept into community
sport [014]. A recent survey documented that 3.7 percent of grade 12 Canadian students
reported having used steroids [08020].
The use of anabolic-androgenic steroids (AAS) by young athletes has been a primary
concern of sports governing bodies because of the implications for unfair advantage in
performance and the potential for adverse side effects. Research over several decades
indicated a lifetime prevalence of AAS use for adolescent males of 4-6 percent and for
females of 1½-3 percent, indicating a problem involving millions of athletes and a potential
epidemic of AAS-related pathologies. However, recent studies have questioned the
presumption that participation in organised sport is the primary risk factor for AAS use in
adolescents as well as the extant estimates of the magnitude of the problem. Increasing
evidence indicates that AAS use is associated with non-athletes and is linked to a broader
syndrome of problem behaviours rather than efforts to achieve sporting success, and that
sports participation may be protective against AAS use. Moreover, employing lifetime
prevalence to gauge AAS use limits accurate evaluation of the personal and public health
risk as the majority of respondents are not habitual users. Previous studies may have also
inflated prevalence values through ambiguously worded survey questions and other design
flaws, and few data are available on actual dosages. Prevention efforts need to be focused
beyond organised sport and target the general adolescent population rather than athletes
and should be founded on interventions with demonstrated efficacy for delinquent, antisocial
and self-destructive behaviours rather than the ethical imperative of fair play [10017].
There is evidence to suggest that the prevalence of anabolic-androgenic steroids (AAS) is

higher among young people than the general population. The purpose of one study was to
examine the proportion of students who reported lifetime and past-year AAS use, explore
other drug use among those who reported AAS use, and investigate demographic correlates
of AAS use. Data was taken from a cross-sectional survey of a representative sample of
Australian secondary students. A stratified two-stage probability sampling methodology was
employed and schools were randomly sampled from each Australian State and Territory. A
total of 376 schools participated in the survey. Lifetime AAS use was reported by 2.4% of 12-
17-year-old students; use was more common among 12-15-year olds then 16-17-year olds.
Regardless of age, being male, speaking a language other than English at home, not be at
school on the previous school day, and rating own scholastic ability as below average were
all associated with a greater likelihood of using AAS in their lifetime and in the past year.
Those who reported AAS use also reported the use of a range of other substances,
suggesting that AAS use may be part of a broader experimentation with substances.
Interventions towards these groups regarding AAS may best be placed within a larger
substance use intervention rather than being AAS-specific. In light of the low levels of AAS
use among this group, more detailed research into AAS use among adolescent sporting
groups may be warranted [10317].
Doping in sport, particularly in track and field, is a reality. The WADA condemned a rife
doping culture in Russian Athletics implicating athletes, coaches, doctors, managers,
federations and even the Russian minister of sport. The prevalence of blood doping ranged
from 1 to 48 percent for subpopulation samples (country, endurance, non-endurance) of a
blood-testing programme by the international governing body for athletics (IAAF). In a study
on Doping in Elite Sports Assessed by Randomized-Response Surveys, the prevalence of
340
reported past-year doping was 29 percent at the 13th IAAF World Championships in Athletics
in Daegu, South Korea, and 45 percent at the 12th Quadrennial Pan-Arab Games in Doha,
Qatar. WADA published a list of 113 coaches, physicians and other support staff, guilty of
violating anti-doping rules – athletes are not allowed to associate with any of these
individuals [150010].
Anabolic-androgenic steroids use and abuse are important concerns for the medical
community, as their utilization has been steadily increasing over the past four decades, with
a global lifetime prevalence rate for AAS use of 3.3 percent; use by males (6.4 %) was
significantly higher than that by females (1.6 %). Professional athletes are a population
classically associated with AAS utilization due to the potential for an increase in muscle
strength, as well as fat-free muscle mass. In addition, the abuse of AAS has expanded
beyond the scope of professional athletes to include high school students (1-5 %), individuals
(i.e. recreational body builders), focused on physique (14 %), as well as certain occupations
(i.e. police officers) [150011].
Scientific considerations of determing prevalence of doping
Both the general public and non-sports medicine health professionals have recently been
made aware of a large use of performance enhancing drugs among sports practicing
subjects. It has been suggested that this behavior is similar to that of substance dependence.
Also some have reported that practice of a sport could be in itself an addictive behavior. The
main objective was to address the following question: is performance enhancing drug use in
sports an addictive behavior? It was first reviewed the definition of performance enhancing
drug use in sports and the diagnostic criteria of substance dependence as they are currently
accepted and attempted to determine a possible common factor. Secondly it was reviewed
epidemiological data from the literature according to three approaches: Use of performance
enhancing drugs is an important and increasing phenomenon among adolescents. It is
sometimes associated to risk taking behaviors for health (syringe use and sharing).
Competition participants are at increased risk (up to 20 % according to some authors) and
some substances (anabolic steroids) are also used by non-sports practicing individuals. It
has not been shown that sports practicing subjects were more at risk of using addictive
substances compared to non-sports practicing subjects. It is not established that practice of a
sport is by itself a risk factor for substance use. However, it could be that a sub-group of
individuals that practice certain types of sports in an intensive way, that use both
performance enhancing drugs and addictive substances and that engage in health risk taking
behaviors have an increased risk for developing a dependence syndrome to both addictive
and performance enhancing drugs. This sub-group is even more at risk because some
performance enhancing drugs (anabolic steroids) could increase the risk for occurrence of a
substance dependence syndrome through neurobiological actions. Yet, the few available
clinical studies show that at most only half of regular users actually meet criteria for
dependence. Also, one study has reported an overrepresentation of sports professionals
among patients seeking treatment for heroin addiction. The large majority of sports practicing
subjects have no dependence to either performance enhancing or addictive drugs. However,
a subgroup of individuals that practice sports intensely and makes use of both addictive and
performance enhancing drugs appear to be at increased risk for developing a substance
dependence syndrome [01005].
The prevalence of doping in elite sports is relevant for all those involved in sports, particularly
for evaluating anti-doping policy measures. Remarkably, few scientific articles have
addressed this subject so far, and the last review dates back to 1997. As a consequence, the
341
true prevalence of doping in elite sports is unknown. Even though it is virtually impossible to
uncover the exact prevalence of a prohibited activity such as doping, various methods are
available to uncover parts of this particular problem, which enables the circumvention (to a
certain degree) of the issues of truthfulness, definition problems and the limits of
pharmacological evidence. One review outlined the various methods that exist and presents
the scarce data available in this area. It is concluded that a combination of questionnaires
using the Randomised Response Technique and models of biological parameters is able to
provide the statistical possibilities to reveal accurate estimates of this often undisclosed
practice. Data gathered in this way yield an estimation of 14-39 percent of current adult elite
athletes who intentionally used doping. These period prevalences have been found in
specific sub-groups of elite athletes, and the available data suggest that the prevalence of
doping is considerably different between sub-groups with varying types of sport, levels and
nationalities. The above-mentioned figure of 14-39 percent is likely to be a more accurate
reflection of the prevalence of intentional doping in elite sports than that provided by doping
control test results (estimate of doping: 1-2 % annually) or questionnaire-based research
(estimations between 1 and 70 % depending on sport, level and exact definitions of intent
and doping). In the future, analytical science may play a more important role in this topic if it
may become feasible to detect very low concentrations of prohibited substances in sewage
systems downstream of major sporting events. However, it is clear that current doping
control test results show a distinct underestimation of true doping prevalence. It does not
seem feasible to distil better estimates of the prevalence of doping based on performance
indicators or ego documents because of the various existing effects that influence athletic
performance. Such information can only be used as extra information to augment the
accuracy of prevalence rates that have been found by using other techniques. True doping
prevalence studies have been scarce in elite sports so far. With the correct application of the
available scientific methods, preferably using harmonised definitions of the terms “doping”
and “elite sports”, more information on this topic may be gathered in a relatively short time.
This would assist anti-doping professionals in the future in order to evaluate the effects of
possible anti-doping measures, and better anti-doping policies would serve athletes who
compete without doping. The existing anti-doping measures seriously impact the lives of elite
athletes and their immediate entourage, which imposes a moral burden to evaluate these
measures in the best possible way [14616].
Available evidence
Over the last two decades a growing amount of evidence has become available about the
extent of illicit drug use in many sports. Although much of this evidence has been anecdotal,
some has come from relatively reliable sources – for instance, evidence given to
parliamentary or judicial inquiries under oath. For example, in Canada the Dubin Inquiry,
established after the positive test of Ben Johnson at the 1988 Seoul Olympics, provided
detailed information about the use of banned substances in many sports, particularly in
Canada but also in several other countries. Similarly detailed and relatively reliable
information about the use of performance enhancing drugs in sport was provided by the US
Senate Judiciary Committee Hearing on Steroid Abuse in America in 1989 and by a report on
Drugs in Sport presented to the Australian Parliament in 1989. More recently, the revelations
by the French customs and police in the 1998 Tour de France, and the subsequent criminal
trials in France, have provided incontrovertible evidence that the use of drugs in cycling is
widespread, systematic, and organised. Although there has recently been a study of drug
use in amateur footballers in Cameroon, there are almost no systematic or reliable data
about the extent of drug use in professional football [05005].
342
Estimated number of unreported cases
Recent studies have suggested that the use of doping substances and particularly of
anabolic androgenic steroids (AAS) is often practised by fitness centre visitors. These
studies employed direct interview techniques and questionnaires to assess the estimated
number of unreported cases of doping. Because people hesitate to provide compromising
information about themselves, these techniques are subject to response errors. In one study
it was applied an alternative interview technique to assess more accurately unreported cases
of doping in fitness centres. The investigation employed the randomized response technique
(RRT) to reduce response errors. A cohort of 500 people from 49 fitness centres participated
in this study. The RRT revealed a high prevalence of doping (13 %). In addition, and most
importantly, the present RRT study revealed an alarmingly high prevalence of illicit drug use,
specifically of cocaine use, that has been severely underestimated by previous studies. The
RRT confirmed previously estimated rates of AAS use assessed by direct interview
techniques and voluntary questionnaires, but uncovered a much higher usage rate of illicit
drugs among fitness centre visitors. This outcome enabled us to construct a 'probability'
rating for the use of doping substances in fitness centre visitors. Given its high prevalence
and the predominant use of AAS, doping among fitness centre visitors is an issue of extreme
relevance for the health care system. The study may help to characterize further doping
substance users and to develop and apply prevention and intervention programmes
specifically to individuals at high risk [06019].
Doping in the community
Prevalence of abuse of androgens in individuals of the general population has reached

alarming dimensions. Use of androgens is no longer limited to competitive sports, but has
spread to leisure and fitness sports, bodybuilding, and nonathletes motivated to increase
muscular mass and physical attractiveness. Alarming studies from Germany demonstrated
that members of the healthcare systems provide illegal androgens to 48 percent of abusers
visiting fitness centers. The new trend to combine androgens with growth hormone, insulin,
and insulinotropic milk protein-fortified drinks may potentiate health risks of androgen abuse.
The use of androgens has changed from being a problem restricted to sports to one of public
health concern. The potential health hazards of androgen abuse are underestimated in the
medical community, which unfortunately contributes to illegal distribution of androgens. Both
the adverse effects of current androgen abuse especially in young men as well as the
chronic toxicity from past long-term abuse of now middle-aged men has to be considered as
a growing public health problem. In the future, an increasing prevalence of androgen misuse
in combination with other growth-promoting hormones and insulinotropic milk protein
products has to be expected, which may have further promoting effects on the prevalence of
chronic western diseases [09018].
The incidence of the abuse of illicit drugs in sport may be a useful indicator of the extent of
the phenomenon among the youth population. Many drugs of abuse are included in the
WADA (World Anti-Doping Agency) Prohibited List, and are therefore routinely tested for in
antidoping controls. This study presents the data obtained in tests carried out in the period
2003- 2007 at the Antidoping Laboratory of Rome, on 44781 samples analysed. The
methods used are those developed by the Laboratory for routine antidoping analyses. The
percentage of positive test results ranges from 1.1 to 2.0 percent, with a high incidence of
stimulants and drugs of abuse. The substance most frequently found is THC metabolite,
343
which accounts for 0.2-0.4 percent of the total, followed by cocaine metabolites, accounting
for 0.1 percent. Other stimulants found are ephedrines, carphedon, modafinil, and anorexic
compounds such as phendimetrazine and norfenfluramine. No amphetamines or
amphetaminelike designer drugs have been detected. These data may be indicative of the
widespread incidence of cocaine and cannabis abuse among the young Italian population,
bearing in mind that the phenomenon is underestimated in this study, due to the fact that
drugs of abuse are investigated only in samples involved in competitive sport, and especially
to the fact that people doing such activities take more care of their health and are subject to
greater control in their behaviour and habits with respect to the “normal” population [09019].
Pioneering studies regarding epidemiology of doping in the US were done in the early 1980s,
when it was interviewed 3403 male high school seniors nationwide [08021]. The report in
1988 indicated that 6.6 percent of respondents had used steroids and more than two-thirds
of the group started using steroids when they were 16 years old or younger. Twenty percent
reported that health professionals were the primary source for obtaining steroids and 38
percent used injectable steroids. Pope et al studied 1,010 college men for use of steroids
and also reported their findings in 1988 [08022]. The study found that only 2 percent of the
respondents reported using steroids. The authors qualified their finding as potentially
underestimating the true prevalence of steroid abuse. A review of published reports
concluded that 3-12 percent of high school students in the 1990s used steroids, and of the
group of abusers about half were adolescent females [08023, 08024].Contrary to popular
belief and supported by Pope's early findings, steroid abuse is not exclusively related to
performance enhancement. DuRant et al reported in 1993 that steroid abuse in ninth graders
was associated with use of cocaine, injected drugs, alcohol, marijuana, cigarettes and
smokeless tobacco [08023]. They then reviewed the 1991 Centers for Disease Control and
Prevention Youth Risk Behavior Survey of over 12,272 male and female public and private
high school students, and confirmed the earlier finding that there is an association between
steroid abuse and multiple drug abuse. In a later review of the 1997 Centers for Disease
Control and Prevention Youth Risk Behavior Survey of 16,262 high school students, Miller et
al reported no significant correlation in male or female steroid-abusing high school students
with physical activity, nor were athletic participation or strength conditioning alone associated
with lifetime steroid abuse [08024]. Steroid abuse may also include a wider population of
non-athletes who have behavioral problems and may experiment with these now easily
available performance-enhancing drugs. Their motivation may not be athletic enhancement,
but rather cosmetic and body shaping purposes. To maintain youthful appearances,
weekend athletes may experiment with hormones encouraged by "anti-aging" marketing,
while adolescent females desirous of the long, lean female media images of "adult women"
may use steroids and growth hormon to reduce fat and increase muscle tone [08025].
Drug abuse by adolescents has been investigated in various surveys that reported
correlations between age, gender, and activity. However, none of these studies included
chemical analyses to help substantiate the statements of participants. In one study, the urine
specimens of 964 students (439 females, 525 males; mean age 22 years), who applied to
study sports sciences at university, were assessed for anabolic steroids, stimulants, and
selected drugs prohibited in sports. In total, 11 percent of the urine specimens provided
contained drugs covered by doping controls. The most frequently detected compound was
the major metabolite of tetrahydrocannabinol (9.8 %) followed by various stimulants related
to amphetamine and cocaine (1.0 %). Indications of anabolic steroid use were found in 0.4
percent of urine samples but originated from contraceptives containing norethisterone. The
present study provided unambiguous data on the status quo of drug (ab)use by adolescents
hoping for a career related to elite sport or sports sciences. No use of anabolic steroids was
detected. However, evidence for stimulants and tetrahydrocannabinol administration was
obtained, although not reported by any participant, which highlights the issue of under-
344
reporting in surveys based solely on questionnaires [08026].
The 1999 cross-sectional European School Survey Project on Alcohol and Other Drugs
(ESPAD). Data collection by standardized methodology using anonymous self-administered
questionnaires completed in the classroom from national probability samples of a total of
18,430 16-year-old high school students from six European countries (Bulgaria, Croatia,
Cyprus, Greece, the Slovak Republic, and the U.K.) Besides anabolic steroid use and
physical exercise, questionnaire items selected for this study included tobacco, alcohol, and
illicit drug use, indicators of other deviant behavior (self-harming thoughts and behavior,
truancy, aggressive behavior), friends' use of steroids, and perceived availability. Backward
elimination with likelihood ratio tests was used to select the variables to be retained in a
mutlifactorial model. Interactions of other independent variables with country were checked.
Logistic regression analysis of lifetime anabolic steroid users compared to nonusers showed
that the odds of lifetime AS use are 1.4 times higher for students who exercise almost daily
and 1.8 times higher for boys compared to girls. Significant associations of steroid use were
also found with current frequent alcohol use, lifetime use of tranquilizers/sedatives and
cannabis, and with the perceptions of friends' use of anabolic steroid and of easy availability
of the substance. The authors concluded that findings indicate that daily exercising appears
to increase the risk of anabolic steroid use in adolescents. However, a more general pattern
of closely interlinked deviant types of behavior, such as other drug use and aggressive
behavior, is prominent. Preventive interventions are needed targeted towards adolescents
involved in intensive exercise and sport. These should take into account both the
idiosyncrasy and setting of the sporting culture and the special characteristics of this group
[08027].
The types and patterns of PED use
AASs are the most commonly used PEDs, with testosterone, boldenone, and trenbolone
being the most frequently detected drugs among illicit PED users in the United States.
Although boldenone is a veterinary steroid not approved for human use, this fact has not
diminished its popularity among illicit AAS users. In the small subgroup of PED users who
are elite athletes, WADA most commonly detects testosterone, stanozolol, and nandrolone,
and the highest prevalence of positive tests occur in bodybuilding, power lifting, weightlifting,
boxing, and kickboxing. PED users often combine multiple drugs, including classical drugs of
abuse such as opiates. Most AAS users engage in high-intensity exercise to maximize
anabolic gains. The combined use of AAS and opiates enables the user to continue training
despite muscle and joint pain. Inevitably, some individuals develop opioid dependence. In
particular, nalbuphine hydrochloride is popular among weightlifters and is associated with
other substance abuse. It has been suggested that AAS could act as a gateway drug to
opioid dependence. In another study of 223 men entering a drug treatment program, AAS
use was considerably higher (25 %) among opioid users compared with men using other
drugs (5 %). In yet another recent study, 50 percent of dependent AAS users met Diagnostic
and Statistical Manual of Mental Disorders-IV criteria for a lifetime history of opioid abuse or
dependence as compared with 8 nondependent AAS users (19 %) and 5 nonusers (7 %). In
1 case report of a man with AAS dependence, naloxone precipitated symptoms suggestive of
opiate withdrawal, even though the man denied using opiates. AASs may also interact with
heroin in accidental drug overdoses [14426].
Recent studies increasingly suggest that the use of AASs and other PEDs often occurs in
conjunction with use of multiple classical drugs of abuse. PED users are increasingly
encountered in needle-exchange programs, where they may sometimes represent most of
the clientele. AAS use has also been linked to alcohol use in humans and rats. Chronic AAS
use may make rats susceptible for alcohol intake. Steroid-induced alterations in opioid
345
peptides in the brain reward system may explain the increased sensitivity to alcohol. Other
studies have observed an imbalance in dopaminergic pathways in the nucleus accumbens, a
brain area involved in reward, leading to speculation that the alterations in the actual
peptidergic and monoaminergic systems promote the rewarding effects of ethanol, thereby
increasing alcohol intake. Additional studies have reported increased sensitivity to cocaine
and amphetamine in rats exposed to high doses of AAS. Thus, AASs may induce effects on
the brain reward system that may render individuals susceptible to other drugs of abuse
[14426].
Athletes and nonathlete weightlifters that use AASs commonly combine different
steroids (stacking) in cycles of increasing and decreasing concentrations
(pyramiding). Most stacks will include both androgens and nonsteroidal drugs. The
latter are typically chosen to provide further anabolic effects (hGH, IGF-1, and
insulin), to counteract negative side effects of AAS (aromatase inhibitors and
estrogen receptor antagonists), to enhance fat and water loss (diuretics, thyroid
hormones, and beta2-adrenergic receptor agonists), to reactivate endogenous
testosterone production at the end of a cycle (gonadotropins), and to reduce the risk
of detection (diuretics and probenecid). Side effects of these nonsteroidal drugs
include headache, nausea, nervousness, diarrhea, perspiration, hot flushes, and
bone pain. Athletes may add epitestosterone to normalize their testosterone to
epitestosterone (T/E) ratios, thus avoiding testosterone-use detection. Researchers
have not adequately investigated interactions of AAS with nonsteroidal drugs
[14426].
An example (considerable variations exist) of a bodybuilder's 12-week AAS cycle

followed by 4 weeks of post-cycle therapy [14427]
Nandrolone
Testosterone Metadienone hCG Anastrazole Clomiphene Tamoxifen
(Deca-
Week cypionate, (Dianabol), (IU/2– (Arimidex), citrate (Nolvadex),
Durabolin),
mg/wk mg/d 3 d) mg/d (Clomid) mg/d
mg/wk
1 500 500 25
2 750 500 25
3 750 500 25
4 750 500 25
5 1000 500 50
6 1000 500 50
7 1000 500 50
8 1000 500 50
9 1000 500 0 500 0.25
10 1000 500 0 500 0.25
11 750 500 0 500 0.25
12 500 500 0 500 0.25
13 200 40
14 100 40
15 50 20
16 50 20
Athletes and nonathlete weightlifters take AASs orally, transdermally, or by intramuscular

injection; however, the most popular mode is the im route. Oral preparations have a short
half-life and are taken daily, whereas injectable androgens are typically used weekly or
346
biweekly. A number of transdermal testosterone preparations have become available
recently, but it is difficult to deliver large amounts of testosterone using the transdermal
formulations. Users may supplement their program of injections and pills with topical gels to
provide a constant low-level testosterone supply [14426].
Studies in the USA and in Sweden suggest that steroid use may be associated with the use
of recreational drugs. In the UK, prevalence of cocaine use among steroid users is
substantially higher than the comparable general population. However, large scale studies
with an in-depth focus on the causal relationship between the use of steroids and
recreational drugs are required. The complex polypharmacy adopted by many users of these
drugs poses additional risks. Common regimens include the concurrent usage of various
drugs for enhancement purposes including a range of anabolic agents, stimulants and an
array of drugs used as self-treatment of steroid-induced side effects through to the use of
dietary and herbal supplements that may be contaminated with undeclared harmful
substances [14437].
Epidemiological confounding factors and false consensus effect (FCE)
The “False Consensus Effect” (FCE), by which people perceive their own actions as
relatively common behaviour, might be exploited to gauge whether a person engages in
controversial behaviour, such as performance enhancing drug (PED) use. In a study it was
assumed that people's own behaviour, owing to the FCE, affects their estimation of the
prevalence of that behaviour. It was further hypothesised that a person's estimate of PED
population use is a reliable indicator of the doping behaviour of that person, in lieu of self-
reports. Over- or underestimation was calculated from investigating known groups (i.e. users
vs non-users), using a short questionnaire, and a known prevalence rate from official reports
or sample evidence. It is proposed that sample evidence from self-reported behaviour should
be verified using objective biochemical analyses. In order to find proofs of concept for the
existence of false consensus, a pilot study was conducted. Data were collected among
competitive UK student-athletes (n=124) using a web-based anonymous questionnaire. User
(n=9) versus non-user (n=76) groups were established using self-reported information on
doping use and intention to use PEDs in hypothetical situations. Observed differences in the
mean estimation of doping made by the user group exceeded the estimation made by the
non-user group (35 % vs 15 % for general doping and 34 % vs 26 % in hypothetical
situations, respectively), thus providing preliminary evidence in support of the FCE concept in
relation to doping. The presence of the “False Consensus Effect” in estimating doping
prevalence or behaviour in others suggests that the FCE based approach may be an avenue
for developing an indirect self-report mechanism for PED use behaviour. The method may be
successfully adapted to the estimation of prevalence of behaviours where direct self-reports
are assumed to be distorted by socially desirable responding. Thus this method can enhance
available information on socially undesirable, health compromising behaviour (i.e. PED use)
for policy makers and healthcare professionals. The importance of the method lies in its
usefulness in epidemiological studies, not in individual assessments [00828].
The false consensus effect (FCE) is the tendency for people to assume that others share
their attitudes and behaviours to a greater extent than they actually do. The FCE has been
demonstrated for a range of health behaviours, including substance use. The study aimed to
explore the relationship between elite athlete's engagement in recreational drug use and their
consensus estimates (the FCE) and to determine whether those who engage in the
behaviour overestimate the use of others around them. The FCE was investigated among
974 elite Australian athletes who were classified according to their drug use history.
347
Participants tended to report that there was a higher prevalence of drug use among athletes
in general compared with athletes in their sport, and these estimates appeared to be
influenced by participants' drug use history. While overestimation of drug use by participants
was not common, this overestimation also appeared to be influenced by athletes' drug use
history. The results suggest that athletes who have a history of illicit drug use overestimate
the prevalence of drug use among athletes. These findings may be helpful in the formulation
of normative education initiatives [12028].
The false consensus effect (FCE) is the tendency for people to assume that others share
their attitudes and behaviours to a greater extent than they actually do. The FCE has been
demonstrated for a range of health behaviours, including substance use. The study aimed to
explore the relationship between elite athlete's engagement in recreational drug use and their
consensus estimates (the FCE) and to determine whether those who engage in the
behaviour overestimate the use of others around them. The FCE was investigated among
974 elite Australian athletes who were classified according to their drug use history.
Participants tended to report that there was a higher prevalence of drug use among athletes
in general compared with athletes in their sport, and these estimates appeared to be
influenced by participants' drug use history. While overestimation of drug use by participants
was not common, this overestimation also appeared to be influenced by athletes' drug use
history. The results suggest that athletes who have a history of illicit drug use overestimate
the prevalence of drug use among athletes. These findings may be helpful in the formulation
of normative education initiatives [11012].
Despite the growing body of literature and putative links between the use of ergogenic
nutritional supplements, doping and illicit drugs, it remains unclear whether, in athletes'
minds, doping aligns with illicit behaviour or with functional use of chemical or natural
preparations. To date, no attempt has been made to quantitatively explore athletes' mental
representation of doping in relation to illegality and functionality. A convenience sample of
student athletes from a large Australian university responded to an on-line survey.
Competitive athletes (n=46) were grouped based on self-reported use as follows: inone used
(30 %), supplement only (22 %), illicit only (26 %) and both supplements and illicit drug use
(22 %). Whereas no athlete reported doping, data provided on projected supplement-,
doping- and drug use by the four user groups allowed evaluation of doping-related cognition
in the context of self-reported supplement- and illicit drug taking behaviour; and comparison
between these substances. The False Consensus Effect was found for illicit substance use
and was evident as a trend for ergogenic supplement use. It is unclear whether the results
point to a relationship between doping and either or neither of the other substances. The
results associated with respondents who used supplements suggested that doping estimates
may be influenced by ergogenic supplement use. Individuals who used supplements tend to
inflate the percentage of individuals who dope but to a much smaller degree than those who
use other substances. In addition to these main results, illicit drug use and doping were
overestimated. This indicates that, self-report notwithstanding; Australian university athletes
may have unrealistic perceptions of illicit drug use and doping. While the pilot nature of this
study, especially the small sample, curtails generalisability. The results are therefore
interpreted under a generalisability caveat and are intended to inform the broader research
program with regard to the observed trends. The presence of the FCE within rather than
between substances provides an indication that nutritional supplement use and illicit drug
use come from different behavioural domains. Individuals who admitted using one particular
type of drug tend to inflate the percentage of individuals who uses the same drug to a much
larger degree than those who use other substances. This suggests the FCE could provide an
expedient way of identifying when interventions designed to influence one behaviour could
influence another. The results indicate that interventions aimed at illicit drug use are unlikely
to have much effect on supplement use, and vice versa. Estimates of those who used both
348
supplements and illicit drugs were more akin to illicit drug users, suggesting illicit drug use
may be a dominant behavioural domain. Users of “both” illicit drugs and nutrition supplement
gave similar social projections (67 %) for illicit drug use to the projected figure of those who
use illicit drugs only (74 %), compared to a definitely lower estimate (55 %) given by nutrition
supplement-only users, suggesting that users of 'both' may have behaved like illicit drug
users due to that domain requiring a different psychological mechanism. For example, users
of both may do so as a function of substance use, whereas supplement only users may do
so for ergogenic reasons. The dominance of illicit drug use may have implications for the
domain specificity of doping behaviour. A reversed pattern was observed for nutrition
supplement use projection, where nutrition supplement-only users gave higher estimation (46
%) compared to those who use both (34 %), thus providing further evidence that in mixed
behavioural categories, and the self-anchored behavioural domain is context specific. This
pilot study provided an indication that harnessing the FCE might be a fruitful avenue to
further examine whether doping behaviour has more in common with illicit drug use or
ergogenic supplement use. The pilot nature of the study suggests that doping may be an
ergogenic phenomenon, however further testing with an improved research design and
sample is needed to establish any such claim. The importance of having a precise picture of
the mental representation of doping is underscored by the increasing need for effective anti-
doping prevention and intervention. Further research is required to establish if some athletes
project their own behavioural tendencies or actual behavior onto other athletes and assume
that many others feel or do the same and indeed are using prohibited performance
enhancing substances. As a consequence, their own doping tendency or behaviour appears
normal and normative, so that they can follow it without compromising their own self-esteem
and social acceptance. In this vein, FCE has importance beyond being a useful vector to
understanding the position of doping in athletes' minds. These considerations, coupled with
the mental representation of doping in athletes' minds, suggest possible intervention
strategies to increase compliance with anti-doping initiatives [11013].
One of the key constraints in designing effective anti-doping programs is the lack of
conceptual clarity of the psychological mechanisms that influence doping behaviour. For
example, preventing doping use in sport on the basis of fair play versus cheating naturally
lends to prevention and intervention programs that focus on the ethics of anti-doping and
values, coupled with the consequences of being caught – not necessarily limited to sanctions
but including dishonour, shame and guilt. Other programs may emphasise the potential
hazards and detrimental health effects as consequences of doping use, which are
omnipresent, independently from doping testing and sanctioning. In the recent years, a
number of athletes have talked publicly about their reasons and motives for doping use,
contrasting perceived obligations and duty to perform well with guilt and the shame of lying.
Studies conducted among professional athletes, particularly cyclists, offer valuable insight
into how athletes perceive doping; and how this perception varies in different contexts. In
addition to the fact that many athletes consider doping as part of professional sport, most
openly talk about experimentation with non-prohibited substances such as over-the-counter
painkillers and non-steroidal anti-inflammatory drugs, caffeine and other non-prohibited
stimulants. Nutritional supplement use, which has been considered as a gateway to doping
by many is common among emerging and elite athletes and has raised concerns on its own
account owing to potentially harmful interactions from combined use and high dosage. The
question of whether doping behaviour has the character of illicit substance use, ergogenic
substance use, neither or both has been recently raised in connection with anabolic steroids.
The ongoing debate is around whether the use of prohibited ergogenic substances aligns
with behaviours associated with illicit substance use (e.g. psychoactive controlled drugs) or
with nutritional (ergogenic) supplement use. Resolving which behavioural domain doping
belongs in athletes' mind provides valuable insight for primary prevention activity. As doping
has been categorised as an illicit (illegal) activity, it follows illicit drug models. Thus, the
349
current anti-doping prevention follows typical demand control models seen for illicit drug use
that focus on health education. It may be that the behaviour is functional with regard to its
performance enhancing qualities. There is currently little in the way of ergogenic supplement
primary prevention. Finally, doping may be an entirely new class of drug use behaviour,
requiring a new set of primary prevention activities to be developed [11013].
Previous results investigating social projection in performance enhancing and illicit drug use
suggest that projected prevalence of doping and drug use was higher among self-admitted
users respectively but absent for nutritional supplement use, and that this social projection
was domain specific. Domain specificity refers to an observed phenomenon that admitted
doping use came with high estimations of doping use among other athletes with illicit drug
use remaining unaffected; and conversely illicit drug users gave higher estimations of illicit
drug use among others with estimated doping use remaining unaffected. Although
differentiating between cause and effect between social projection and behaviour in data
from cross sectional research is impossible, the relationship is clearly present in self-reported
data. Interestingly, this phenomenon is only observable within the cognitively controlled
information when athletes admitted the use of one or both of these drugs. The importance of
the social project lies with the question of whether an elevated and potentially distorted social
projection leads to a congruent behavioural choice or resulted from it. The fact that social
projection aligns with self-reported behaviour but not necessarily with actual behaviour is
intriguing, but more importantly it reveals something about athletes' cognitive processes
relating to these substances. Thus, this may be used to gain insight into athletes' implicit
mental representation of these, often concomitantly used, substances. Descriptive norms are
individuals' perceptions of how common a particular behaviour is. These norms are likely to
be affected by some degree of projection (i.e. x % of athletes use doping). In particular, the
projection may suffer from a social bias coined the “False Consensus Effect” (FCE) which
peoples' perception of their environment (including the behaviours of others) is distorted, thus
resulting in a higher estimation. The FCE is a perceptual bias where people who engage in
particular behaviours tend to overestimate the proportion of the population who also engage
in that behaviour. People who abstain either underestimate or correctly estimate prevalence.
For example, marijuana users tend to overestimate the proportion of the population who use
marijuana, and non-users are more accurate or underestimatec. A further characteristic is
that the FCE is domain specific as it works within rather than across different categories or
domains of behaviour. Therefore, if doping was an ergogenic phenomenon, users of
nutritional supplements should overestimate doping and vice versa (the positive case).
Conversely if doping belongs to another domain, then the estimates of doping would occur
independently of nutritional supplement use (the negative case). This suggests the
relatedness of doping with either illicit or ergogenic substance use behaviours can be
determined by emergent patterns of the FCE across behaviours. It may be assumed mutually
exclusive categories (i.e. a nutritional supplement user is not a doping user or illicit drug user,
etc.). In reality, it is likely that athletes use substances from two or even all three of these
substance categories, thus making mixed categories with testable differences in their
estimations of drug, doping and nutrition supplement use. For example, athletes construct
doping as illicit drug use and therefore athlete illicit drug users also overestimate doping. The
legality of the substances may have a confounding influence [11013].
Elites
High use of medication and nutritional supplements has been reported in several sports. To
document the use of prescribed medication and nutritional supplements in female and male
junior, youth, and adult track and field athletes depending on their sports discipline a
350
descriptive epidemiology study was performed. Analysis of 3 887 doping control forms
undertaken during 12 International Association of Athletics Federations World
Championships and 1 out-of-competitions season in track and field was done. There were 6
523 nutritional supplements (1.7 per athlete) and 3 237 medications (0.8 per athlete)
reported. Nonsteroidal anti-inflammatory drugs (NSAIDs; 0.27 per athlete, n=884),
respiratory drugs (0.21 per athlete, n=682), and alternative analgesics (0.13, n=423) were
used most frequently. Medication use increased with age (0.33 to 0.87 per athlete) and
decreased with increasing duration of the event (from sprints to endurance events; 1.0 to
0.63 per athlete). African and Asian track and field athletes reported using significantly fewer
supplements (0.85 vs 1.93 per athlete) and medications (0.41 vs 0.96 per athlete) than
athletes from other continents. The final ranking in the championships was unrelated to the
quantity of reported medications or supplements taken. Compared with middle-distance and
long-distance runners, athletes in power and sprint disciplines reported using more NSAIDs,
creatine, and amino acids, and fewer antimicrobial agents. The use of NSAIDs in track and
field is less than that reported for team-sport events. However, nutritional supplements are
used more than twice as often as they are in soccer and other multisport events; this
inadvertently increases the risk of positive results of doping tests. It is essential that an
evidence-based approach to the prescribing of medication and nutritional supplements is
adopted to protect the athletes' health and prevent them from testing positive in doping
controls [09020].
The aim of one study was to describe qualitatively and quantitatively dietary supplements
and medication use in elite athletes. Athletes (n=912) reported medications and dietary
supplements taken within 3 days before doping control. It was analyzed data collected from
2006 to 2008, indentified and classified substances. Total of 75 percent athletes reported use
of at least one substance, 61 percent took dietary supplements (3.2 per user) and 41 percent
took medications. Among users, 21 percent reported the use of six and more different
products, and one took 17 different products at the same time. Majority of medication users
took non-steroidal anti-inflammatory drugs (NSAID) (25 %), and 22 percent used more than
one NSAID. It was found no gender differences in dietary supplements use. Individual sport
athletes used more dietary supplements. The study showed widespread use of dietary
supplements and drugs by elite athletes in Serbia. Consumption of dietary supplements with
no evident performance or health benefits, demonstrated the need for specific educational
programs focused on dietary supplements use. Amount, quantity and combination of the
reported products raised concern about the risk of potential side effects [09021].
Use of drugs during the Olympics
It was gathered data and examined the use by elite Olympic athletes of food supplements
and pharmaceutical preparations in total and per sport, country, and gender in the Athens
2004 Olympic Games. Data from two sources were collected: athletes' declaration of
medications/supplements intake recorded on the Doping Control Official Record during
sample collection for doping control, and athletes' application forms for granting of a
therapeutic use exemption (TUE) and through the abbreviated TUE process (aTUE). 24
percent of the athletes tested for doping control declared no use of medications or food
supplements. Food supplements (45 %) continue to be popular, with vitamins (43 %) and
proteins/aminoacids (14 %) in power sports being most widely used. Nonsteroidal
antiinflammatory agents and analgesics were also commonly used by athletes (11 % and 4
%, respectively). Laboratory analysis data reveal that of the aTUEs received for inhaled
glucocorticosteroids, only budesonide was detectable in significant percentage (10 %). Only
7 percent of the 445 athletes approved to inhale beta2-agonists led to an adverse analytical
finding [09022].
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Age of onset
Although it is widely believed that AAS use is common among teenagers, the great majority
of AAS use begins after the teenage years. Data on high school drug use from the University
of Michigan's Monitoring the Future study provides valuable information concerning the
youngest AAS users. Some 2 percent of American high school students report having used
AAS in the past 12 months. Although the annual prevalence figures may well be inflated as a
result of false-positive responses to the steroid question, the data suggest that AAS use may
have declined since the year 2000 when the media widely publicized adverse Congressional
comments regarding PED abuse. However, it is not possible exclude the possibility that this
might not reflect a true decline in AAS use, but rather a decline in false-positive responses as
students became better informed about AAS and hence less likely to misinterpret the steroid
question on the survey. There are 9 studies from the United States, Australia, and the United
Kingdom since the year 2000 that provide at least some data on age of onset of AAS use.
These included 6 studies that evaluated AAS users in person and 3 Internet surveys of AAS
users. In the largest Internet study, only 1 of 1955 male AAS users (0.05 %) reported starting
AAS use before age 15, and only 6 percent started before age 18. In 5 other studies,
collectively evaluating 801 AAS users, only 12 (1.5 %) started before age 16, and 199 (25 %)
started before age 20. Notably, the median age of onset across all studies consistently fell
into the narrow range of 22 to 24 years. However, the actual median age of onset is probably
higher, because at the time of recruitment, many study candidates had not completed the
age range of risk for starting AAS use [14426].
Student athletes
There is a general perception that use of performance-enhancing substances (PESs) does

not fit the standard profile of substance use. One study sought to determine whether users of
PESs report high-risk patterns of alcohol and other drug use and demonstrate risk behaviors
associated with problematic substance use. Anonymous self-report questionnaires were
administered to a sample of 234 male student athletes. PES users were defined as college
athletes who reported past-year use of a broad array of PESs (including stimulants, hormone
precursors, and nutritional supplements). Male athlete PES users (n=73) compared with
nonusers (n=160) reported more problematic alcohol-use behaviors and more alcohol- and
drug-use-related problems. The former compared with the latter was also more likely to
report past-year use of tobacco products, marijuana, cocaine, psychedelics, and prescription
drugs without a prescription. In addition, PES users demonstrated higher sensation seeking,
and greater coping and enhancement motivations for drinking and marijuana use than non-
PES users. Although banned PESs are not typically viewed as having a high addiction
potential, male athletes who use these drugs may be more likely to participate in other
problematic substance-use behaviors. Importantly, the male athletes in this study who
reported PES use also participated in substance-use behaviors that can have profound
negative effects on athletic performance [09023].
Adolescents
The use of anabolic-androgenic steroids (AAS) by young athletes has been a primary
concern of sports governing bodies because of the implications for unfair advantage in
performance and the potential for adverse side effects. Research over several decades
352
indicated a lifetime prevalence of AAS use for adolescent males of 4-6 percent and for
females of 1.5-3 percent, indicating a problem involving millions of athletes and a potential
epidemic of AAS-related pathologies. However, recent studies have questioned the
presumption that participation in organised sport is the primary risk factor for AAS use in
adolescents as well as the extant estimates of the magnitude of the problem. Increasing
evidence indicates that AAS use is associated with non-athletes and is linked to a broader
syndrome of problem behaviours rather than efforts to achieve sporting success, and that
sports participation may be protective against AAS use. Moreover, employing lifetime
prevalence to gauge AAS use limits accurate evaluation of the personal and public health
risk as the majority of respondents are not habitual users. Previous studies may have also
inflated prevalence values through ambiguously worded survey questions and other design
flaws, and few data are available on actual dosages. Prevention efforts need to be focused
beyond organised sport and target the general adolescent population rather than athletes
and should be founded on interventions with demonstrated efficacy for delinquent, antisocial
and self-destructive behaviours rather than the ethical imperative of fair play. It has been 50
years since the first report of AAS use by a high school athlete, and since then considerable
research has characterised AAS use in young athletes. It appears that illicit use of AAS may
be less problematic in young people in organised sport than in the general adolescent
population and that prevention efforts would be more effective utilising a public health model
rather than one focused on sports-specific concerns such as “fair play”. Yesalis and Bahrke
reviewed anabolic steroid use by adolescents up to 1999 and reported that lifetime
prevalence in 16 regional studies from 1989-1993 in the USA ranged from 1.6 to 4.4 percent
(with higher values for males; in three studies, the prevalence for males was reported as 6.3-
6.5 %); in two sets of USA national studies from 1991 to 1997, prevalence ranged from 1.9 to
3.7 percent (with higher values for males), with the values in a third national survey from
1991 to 1994 indicating prevalence from 0.3 to 0.7 percent. The authors’ summary of nine
studies from 1990 to 1999 of anabolic steroid use among high school-aged students in a
variety of other countries (Canada (three); Sweden (two); South Africa (two); Australia (one);
UK (one)) indicated lifetime prevalence ranging from 0.6 to 3.7 percent (with higher values
for males). In a review including eight studies (seven from the USA; one from Canada) it was
reported prevalence ranging from 1.0 to 5 percent for boys (although two studies reported
values of 11 % and 15 %, respectively) and 0.8-2.8 percentfor girls (with the same study that
reported 15.3 % in boys indicating a prevalence of 6.7% for girls). Of 16 more recent works,
six were conducted in the USA (four national samples; two regional studies); nine were in
Europe (one trans-European study involving six countries; two national studies (Poland,
Norway); six regional (Sweden (three); Norway, Germany, France (one each)); and one
regional study from Brazil. As with all of the previously published work on this topic, the
majority of new studies differed significantly in their methodology, including the time frame of
use (lifetime; previous 30 days; previous 6 months; previous 12 months), sample (middle-
school students; high school students; athletes; community-dwelling youth) and source of
data (most used researcher-developed questionnaires, although few provided the actual
wording of the AAS use questions; internet solicitation). The lack of a standardised approach
makes it difficult to compare across studies or acquire an accurate picture of the
phenomenon. In Sweden, the Center for Alcohol and Narcotic Information (CAN)
questionnaire was used for regional studies in 1995, 1998 and 2000, and indicated
prevalence for males of 2.1 percent, 2.9 percent and 1.2 percent, respectively, and for
females of 0.2 percent and 0.0 percent (1995 and 1998, respectively). Findings from other
studies varied widely. For example, it was reported only 0.8 percent for a national follow-up
study of 8508 Norwegian middle and high school students, whereas in another study it was
found 3.6 percent for males and 0.6 percent for female high school students in a smaller
regional study in Norway [09024].
One article explores the issue of performance-enhancing drug use in adolescent athletes.
353
The article describes current substances that are being used by adolescent athletes,
explains their positive and negative effects, examines factors contributing to their increased
use in adolescent athletes, and discusses approaches to educating adolescents about
alternate means of enhancing their athletic performance. It is hoped that this information will
be useful toward encouraging young athletes to pursue, safe, healthy, and natural means of
performance enhancement, such as practice and strength training, to improve sports
performance in a safe, effective manner [06021].
Ergogenic drugs are substances that are used to enhance athletic performance. These drugs
include illicit substances as well as compounds that are marketed as nutritional supplements.
Many such drugs have been used widely by professional and elite athletes for several
decades. However, in recent years, research indicates that younger athletes are increasingly
experimenting with these drugs to improve both appearance and athletic abilities. Ergogenic
drugs that are commonly used by youths today include anabolic-androgenic steroids, steroid
precursors (androstenedione and dehydroepiandrosterone), growth hormone, creatine, and
ephedra alkaloids. Reviewing the literature to date, it is clear that children are exposed to
these substances at younger ages than in years past, with use starting as early as middle
school. Anabolic steroids and creatine do offer potential gains in body mass and strength but
risk adverse effects to multiple organ systems. Steroid precursors, growth hormone, and
ephedra alkaloids have not been proven to enhance any athletic measures, whereas they do
impart many risks to their users. To combat this drug abuse, there have been recent changes
in the legal status of several substances, changes in the rules of youth athletics including
drug testing of high school students, and educational initiatives designed for the young
athlete. One article summarized the current literature regarding these ergogenic substances
and details their use, effects, risks, and legal standing [06003].
Versus those not participating in sports
One study examined the relationship between high school sports participation and the use of
anabolic steroids (AS) and legal performance-enhancing dietary supplements in young
adulthood. Additionally, the relationship between the use of AS and legal dietary
supplements was explored. Data on approximately 15,000 adolescents from the National
Longitudinal Study of Adolescent Health were used. School sports participation was
assessed when adolescents were in grades 7-12. AS use and legal performance-enhancing
dietary supplement use were assessed six years later. Males were more likely than females
to use AS and legal supplements. A sport by gender interaction emerged for the use of AS,
indicating that the gender differences in AS use were greater for those who participated in
sports during high school. High school sports participation was associated with increased
likelihood that adolescents would use legal supplements in young adulthood. Finally, there
was a positive relationship between the use of legal dietary supplements and AS use. The
study highlights the important role that the social environment during adolescence has on
future health behaviors. Results suggest that the sporting context experienced during early
adolescence may have lasting effects on the use of performance-enhancing substances. The
use of legal performance-enhancing dietary supplements appears to be more prevalent than
the use of AS, and there seems to be a positive relationship between the use of AS and legal
performance-enhancing dietary supplements [06022].
Disableds in sports
Activities concerning the fight against doping with regard to the Paralympic Games have
been initiated in 1984, when first doping controls were conducted. The foundation of the
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International Paralympic Committee exactly 20 years ago (1989) considerably supported
systematic sports drug-testing programs specifically designed to meet the particular
challenges related to disabled sports, which yielded a variety of adverse analytical findings
(e.g.,with anabolic steroids, diuretics, corticosteroids, and stimulants) especially at
Paralympic Summer Games. In Germany, doping controls for handicapped athletes were
established in 1992 and have been conducted since by the National Paralympic Committee
Germany and the National Anti-Doping Agency. Also here, various analogies in terms of
antidoping rule violations were found in comparison to doping controls of nondisabled
athletes. In the one article, available numbers of samples analyzed at Paralympic Summer
and Winter Games as well as within the doping control program for disabled sports in
Germany were summarized, and particularities concerning sample collection and the doping
method termed boosting were presented [09025].
To examine the use of food supplements and pharmaceutical preparations by elite

Paralympic athletes in the Athens 2004 Paralympic Games data obtained from athletes’
declaration of intake of drugs/supplements recorded on the Doping Control Official Record
during sample collection for doping control, and athletes’ application forms for granting of a
therapeutic use exemption. Sixtyfour percent of the athletes tested for doping control
declared use of medications or food supplements, and 81 percent of these athletes declared
intake of fewer than four preparations. Non-invasive routes of administration dominated.
Food supplements (42 %) were popular, and drugs used to treat several pathological
conditions noted. Non-steroidal anti-inflammatory agents and analgesics were commonly
used (10 % and 6 %, respectively). The prevalence of inhaled beta2-agonist use (5 %) was
higher than expected and exceeded that at the Athens Olympic Games. The first
examination of elite Paralympic athletes shows a more rational approach to the use of
medication and food supplements, but a similar consumption pattern to that of athletes at the
Athens Olympic Games. Because of the dearth of such studies, consumption trends among
Paralympic athletes remain unclear. Fewer Paralympic athletes declared the use of
medications and food supplements and, in general, a more rational intake pattern was
recorded than for Olympic athletes. Drugs used to treat several pathological conditions were
recorded, with a higher prevalence of drugs for insulin-dependent diabetes mellitus than in
recent Olympic Games. The prevalence of inhaled beta2-agonist use at the Athens
Paralympic Games was higher than expected and exceeded that at the Athens Olympic
Games. The need to counsel athletes with disabilities on their nutritional needs is confirmed,
and close monitoring by healthcare professionals is recommended [09026].
Issues in paraolympics
Autonomic dysreflexia (AD) is unique to individuals with spinal injuries (SCI) at T6 or above
and can be voluntarily induced. Although AD improves wheelchair racing performance in
some athletes, it also elicits exaggerated blood pressure, which could be dangerous. The
International Paralympic Committee considers AD doping and banned its use. Purpose. The
purpose of one study was to evaluate AD knowledge, incidence and attitudes (KIA) of
Paralympians with SCI. An existing questionnaire was modified to include questions of AD
KIA, validated by three experts and piloted with a small sample. It was administered on-line,
mailed to members of a scientific network and distributed during the Beijing Paralympic
Games. Of 99 participants, 55 percent had previously heard of AD while 39 percent were
unaware; 17 percent, all males, had used AD to enhance performance. Participants reported
that AD was (1) useful for middle (79 %) and long distance (71 %), marathon (64 %) and
wheelchair rugby (64 %); (2) somewhat dangerous (49 %), dangerous (21 %) or very
dangerous (26 %) to health. Results were not influenced by age, injury level or injury
duration. The findings indicate the need for educational programmes directed towards
enhancing the AD knowledge of rehabilitation professionals, coaches and trainers working
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with spinal injuries individuals [10319].
Paralympic medicine describes the health-care issues of those 4500 or so athletes who
gather every 4 years to compete in 20 sports at the Summer Paralympic Games and in five
sports at the Winter Paralympic Games. Paralympic athletes compete within six impairment
groups: amputation or limb deficiencies, cerebral palsy, spinal cord-related disability, visual
impairment, intellectual impairment, or a range of physically impairing disorders that do not
fall into the other classification categories, known as les autres. The variety of impairments,
many of which are severe, fluctuating, or progressive disorders (and are sometimes rare),
makes maintenance of health in thousands of Paralympians while they undertake elite
competition an unusual demand on health-care resources. The increased physical fitness of
athletes with disabilities has important implications for cardiovascular risk reduction in a
population for whom the prevalence of risk factors can be high [12036].
One study reports in detail on the antidoping program of the Paralympic Movement to
improve knowledge and optimize intervention programs, including educational and
awareness initiatives. Data retrieved from annual statistics reports and historical records are
complemented with personal observations. An overall incidence proportion of <1 percent of
antidoping rule violations in the Paralympic Movement is reported, mainly resulting from urine
testing during in-competition periods. This led to a total of 60 antidoping rule violations (of
which 37 in the sport of International Paralympic Committee powerlifting) since 2000. A
critical analysis of these data allows for an assessment of risk factors by sport. An efficient
transfer of knowledge indicates the need to strengthen educational awareness, preferably
imbedded in a multidisciplinary approach toward athletes' health. The particular case of
autonomic dysreflexia is addressed as a separate theme [12037].
Gym users
The use of performance- and image-enhancing drugs/substances (PIED) outside elite sports
appears to be increasing, although the current knowledge of the nature of PIED use among
recreational athletes is scarce. The present study analyzed enquiries that were submitted to
the Danish Anti Doping Agency (ADD) over an 18-month period, to gain knowledge of PIED
use among individuals who exercise recreationally in Denmark. One thousand three hundred
ninety eight queries were examined with respect to the age and gender of the enquirer,
affiliation to sport or exercise and substance in question. The key findings were that the ADD
information service is generally used by males in their mid-20s who exercise in gyms and are
not engaged in competitive sports. Approximately 15 percent of the enquirers were users of
anabolic androgenic steroids (AAS) or other substances banned within elite sports by the
World Anti Doping Agency, and an additional 15 percent considered using such substances.
The present results suggest that there is a pronounced interest in the use of AAS and other
PIEDs among Danish gym members [09027].
State-based sports institute
The purpose of one investigation was to examine the nutritional supplement intake of
athletes from a state-based sports institute. Athletes (n=72) from seven sports (kayaking,
field hockey, rowing, waterpolo, swimming, athletics and netball) completed a questionnaire
detailing their daily usage and rationale therefore. The large majority (63/72; 88 %) of
surveyed athletes reported using nutritional supplements, with no difference between female
and male athletes. Kayakers (6.0) consumed a higher number of nutritional supplements
356
than swimmers (4.0), field hockey (1.5), rowing (2.4), waterpolo (2.3), athletics (2.5) and
netball (1.7) athletes. The athletes believed that nutritional supplements are related to
performance enhancements (65 %), positive doping results (63 %), and that heavy training
increases supplement requirements (65 %). The cohort was equivocal as to their health risks
(56 %) or their need with a balanced diet (53 %). The most popular supplements were
minerals (46 %), vitamins (43 %), other (32 %), iron (31 %), caffeine (22 %), protein (17 %),
protein-carbohydrate mix (14 %), creatine (13 %) and glucosamine (4 %). The majority of
supplementing athletes (n=63) did not know their supplements active ingredient (62 %), side
effects (57 %), or mechanism of action (54 %) and admitted to wanting additional information
(57 %). Only half of the athletes knew the recommended supplement dosages (52 %). The
performance enhancing perception may explain the large proportion of athletes that reported
using nutritional supplements, despite over half of the athletes believing that supplements
are not required with a balanced diet and can cause positive doping violations [10021].
Pharmaco-epidemiology of anabolic steroids
The first report of AAS abuse dates from 1954 when members from the Soviet Union’s world
champion’s weight lifting team were found to abuse these substances inadvertently. As late
as 1975, AAS abuse was classified as doping. According to the world anti-doping agency
report in 2008, AAS were the most commonly identified prohibited drugs among all,
comprising 59 percent of all reported findings. Unfortunately, AAS abuse occurs frequently
not only amongst professional athletes, but also in the general population, particularly high
school students. Also, hypogonadism is considered to affect 2-4 million men in the United
States. In a recent Endocrine Society clinical practice guideline, AAS replacement therapy
was indicated to treat men suffering from hypogonadism correlated with low testosterone
blood levels. Next to relevant clinical indications of AAS in physiological doses (28-56
mg/week), illegal abuse of high doses of AAS as a muscle strengthening agent in eugonadal
men is reported in high rates, which results in blood levels of androgenic compounds 10 to
100 times above the physiologic and therapeutic range [12125].
Non-prescribed use of anabolic androgenic steroids (AAS) has been associated with a
number of physical and psychiatric/behavioural complications, some of which are potentially
lethal. Here, we review both observational and experimental studies on human subjects
concerned with such side-effects. The only physical complication of AAS use that receives
definitive support from such investigations is unfavourable changes in blood lipid profiles.
Support for various psychiatric complications has also been provided by a number of cross-
sectional studies, most involving comparisons between weight-training individuals who use or
do not use AAS. Certain of these complications, in particular hypomania and increased
aggressiveness, have been confirmed in some, but not all, randomized controlled studies.
Epidemiological attempts to determine whether AAS use triggers violent behaviour have
failed, primarily because of high rates of non-participation. Studies regarding the prevalence
of AAS use in different populations typically report life-time prevalences of 1-5% among
adolescents. However, the life-time prevalence (i.e. use on at least one occasion) is of
doubtful relevance in attempting to estimate the number of individuals at risk for side-effects,
as most of these complications appear to develop during prolonged use of AAS.
Furthermore, it is reasonable to assume that the symptoms and signs of AAS use are often
overlooked by healthcare professionals, so that the number of cases of possible AAS-related
complications is virtually unknown. These limitations, together with an apparently low
prevalence of prolonged AAS use among the general population, indicate that future
epidemiological research in this area should focus on retrospective case-control studies and,
357
perhaps, also on prospective cohort studies of populations selected for a high prevalence of
AAS use, rather than attempting to perform large-scale population-based studies [05011].
Comparison with a prison population
Use of anabolic androgenic steroids (AAS) has been associated with adverse psychiatric
effect, violent behavior, and criminality. The aim of one study was to further investigate the
motives for and consequences of AAS use, with focus on violent and antisocial behavior.
Fifty-nine prisoners were interviewed on their use of AAS, and their history was mapped with
Addiction Severity Index interviews. Of these prisoners, 56 percent admitted previous use of
AAS, of whom 24 percent declared to have committed violent crimes in connection with use
of AAS. However, the only significant difference between users and nonusers with regard to
criminal history when measured with the Addiction Severity Index was that the AAS users
more often stated that they had been prosecuted for crimes labeled as "other crimes," which
did not include violent crimes. The reported side effects of AAS corresponded well to those
previously reported. These results indicate that use of AAS is common among Swedish
prisoners and that the motives and consequences of such use are similar to what has been
observed in other AAS-using populations. Furthermore, the study supports earlier notions
that misuse of AAS might cause violent behavior, but only in certain individuals and mainly in
combination with other substances [10025].
Drug Information Database
To analyse enquiries made in the Drug Information Database (DID) to develop a better
understanding of athletes’ interests and concerns regarding the prohibited status of available
substances a retrospective analyses of anonymous enquiries recorded in the DID in 2006
and 2007 of athletes and supporting personnel was performed. The DID recorded 223 717
enquiries with 200 of the >6000 UK licensed pharmaceutical products receiving over 100
enquiries each. The majority (79 %) of these enquiries were in the pharmaceutical product
category, followed by recreational drugs (10 %). A variety of common medications were
subject to enquiry, with anti-inflammatory agents, decongestants and bronchodilators being
most common; a trend in keeping with reported medication use by athletes. Of all enquiries,
42 percent were not found owing to misspelled words or enquiries about unregulated
substances. The proportion of enquiries about substances not listed in the database is
relatively high and has increased over the 24 month observation period. The DID is a well-
used information resource with some 10 000 enquiries being made each month. Of the about
60 percent of successful enquiries, the major focus was on pharmaceutical products. With
some 73 percent of enquiries being made by the athletes themselves, further investigations
are warranted to explore enquiry patterns in relation to specific sports. Of the unsuccessful
enquiries, a large number were related to nutritional supplements, which warrants further
investigation. The DID database appears to be a valid mirror of athletes’ chemically assisted
practices and may be successfully used to inform health professionals as well as antidoping
prevention programmes [09028].
The widespread use of chemically assisted performance enhancements in sport is a growing

concern, with the emphasis on the need for harm reduction policies and intervention,
underpinned by empirical evidence from clinical trials. Considerable advances have been,
and continue to be, made in the development and application of the analytical sciences that
underlie the detection of prohibited performance-enhancing substances (PES) both in and
out of competition. Practices of chemically assisted performance enhancements reach
358
beyond using prohibited drugs and methods. Recent studies show that a noteworthy
proportion of athletes take nutritional supplements daily and the majority of users of
supplements in sports fail to take appropriate supplements to achieve their desired health-
maintenance or performance-related outcomes. This mismatch between rationale underlying
supplement use and the outcomes of the chosen supplements could form part of an
educational programme to eradicate misplaced supplement use in sports. However, these
literature reports may also be useful in designing new trials and testing programmes to
evaluate the side effects of supplements. In addition to supplements, athletes competing at
international sport events reported an alarmingly high use of a number and combination of
nutritional supplements, over-the-counter (OTC) and prescription-only medications, a pattern
that is mirrored in sub-elite athlete populations. Among Belgian athletes, the reported use of
OTC medication has increased from 20 percent to 25 percent in 3 years (2002-2005), with
the proportion of users above 35 percent in certain sports (such as corticosteroids in cycling).
Whilst antidoping prevention programmes target high-performing athletes, the use of
performance-enhancing substances has spread beyond elite sports. The complexities of
ranges of prescription and over-the-counter medicines, social drugs and supplements, along
with the broad range of products which contain them, necessitate an ever-watchful eye from
athletes and coaching teams so that inadvertent doping does not occur. The risk to athletes
is twofold: in addition to the scenario of inadvertently failing a doping test, several
prescription and OTC medicines have known side effects that may hinder an athlete’s
performance. This concern is exemplified by the well-known gastric irritation caused by some
non-steroidal anti-inflammatory drugs (NSAIDs). Owing to the enormous number and variety
of supplements, prescription and OTC medicines available, along with a lack of clear
information regarding doping, further research is required for information on which studies
should be undertaken in terms of drug types in order to inform WADA testing and antidoping
prevention programmes. One approach to this conundrum is to utilise the drug information
databases to study patterns of athlete enquiries, assuming that enquiries are true reflections
of interest and behaviour or behavioural intention. While enquiries do not necessarily equate
to uses, it is reasonable to assume that athletes make enquiries about the prohibited status
of the drugs or substances they are taking or considering for medical or performance-
enhancing reasons [09028].
Specifically, the most frequent enquiries were on variants of salbutamol (1882), caffeine
(1702) and ingredients of pain/fever relievers (ibuprofen and paracetamol, 1412 and 1349,
respectively) and cold medications (ephedrine, 1151). Both ephedrine and salbutamol are
bronchodilators and their in-competition use is prohibited. However, salbutamol may be used
under the therapeutic use exemption (TUE) scheme. The high frequency of enquiries about
NSAIDs and bronchodilators is in keeping with the literature precedents. One possible
explanation for the relatively high prevalence of decongestants and asthma medications
among the enquiries is the exercise-induced asthma that has been documented among
winter endurance athletes. It is equally possible that these substances have performance-
enhancing effects but are allowed if prescribed with evidence of medical need under the TUE
scheme. Therefore, further investigation is warranted into sport-specific enquiries about
potentially performance-enhancing substances (i.e. salbutamol, corticosteroids, etc). The
most frequent “unlisted” enquiry was creatine (24 %), followed by various commercial
products for muscle building (HMB, Maximuscle, Methoxyisoflavone) and some form of sugar
(dextrose, glucosamine) at 9 percent and 8 percent, respectively. In this survey among UK
athletes, respondents believed that unintentional doping offences are mainly caused by
inadequate labelling, changes in composition of supplements and lack of information, and felt
that increased awareness of the UK Sport website and regularly updated list of acceptable
supplements would help to prevent inadvertent doping. However, nutritional supplements,
herbal remedies and other non-herb non-mineral substances are not covered in the DID
[09028].
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Global lifetime prevalence rate
To estimate the global lifetime prevalence rate of anabolic-androgenic steroid (AAS) use and
investigate moderators of the prevalence rate a meta-analysis and meta-regression analysis
was performed using studies gathered from searches in PsycINFO, PubMed, ISI Web of
Science, and Google Scholar among others. Included were 187 studies that provided original
data on 271 lifetime prevalence rates. Studies were coded for publication year, region,
sample type, age range, sample size, assessment method, and sampling method.
Heterogeneity was assessed by the I2 index and the Q-statistic. Random effect-size modeling
was used. Subgroup comparisons were conducted using Bonferroni correction. The global
lifetime prevalence rate obtained was 3.3 percent (95 % confidence interval 2.8 to 3.8 %).
The prevalence rate for males, 6.4 percent was significantly higher than the rate for females,
1.6 percent. Sample type (athletes), assessment method (interviews only and interviews and
questionnaires), sampling method, and male sample percentage were significant predictors
of AAS use prevalence. There was no indication of publication bias [14027].
Different countries
The most common steroids involved withdoping are testosterone and nandrolone based on
WADA data and retrospective autopsy data. In 2006, 34 WADA-accredited laboratories
analyzed almost 200,000 urine samples with positive findings in approximately 2 percent. Of
the positive samples, AASs accounted for 45 percent of the adverse findings with the most
common AASs being testosterone, nandrolone, stanozolol, and methandienone [13003].
The use of performance-enhancing drugs is not limited to the US high rates have been
consistently documented in Scandinavia, Brazil, and British Commonwealth countries and
more recently in continental Europe. By contrast, AAS use is rare in East Asian countries
such as China, Korea, and Japan, perhaps because these cultures place less emphasis on
male muscularity, as explained in recent reports [14017].
Sweden
To evaluate the effects of an appearance programme in preventing the misuse of androgenic

anabolic steroids among male adolescents in a primary health care area in Sweden.
Attitudes to steroid hormones among 16-17 years old male and female adolescents are
discussed. A well-established anonymous multiple-choice questionnaire was answered by
921 adolescents and statistically analysed. The misuse of androgenic anabolic steroids
tended to decrease after the intervention. It was concluded that the misuse of androgenic
anabolic steroids did not increase, and even tended to decrease, after the intervention,
indicating that drug-abuse among male adolescents can be decreased through discussions
about appearance and attitudes. Repeat and prospective studies have to be done before this
intervention programme can be generalised [01029].
The prevalence of the use of androgenic anabolic steroids has been poorly studied in
Europe. One study was undertaken to examine the prevalence of the misuse – the non-
medical use – of androgenic anabolic steroids among adolescents in a county of Sweden.
The total population of 16 and 17 year old male and female adolescents in a county on the
south-west coast of Sweden was studied. The investigation was done by an anonymous
multiple-choice questionnaire. The questionnaire was completed by 5,827 pupils and
360
statistically analysed. The participation rate was 95 percent. Among male adolescents 16
and 17 years old, 3.6% and 2.8% had misused androgenic anabolic steroids, respectively.
These male adolescents had also misused alcohol, growth hormones and narcotic drugs
more than the steroid hormone non-users. Among female adolescents there was no
recorded misuse of these drugs (0.0 %). It was concluded that yhe misuse of androgenic
anabolic steroids is a reality in both small and large municipalities in Sweden. The
prevalence figures are higher among 16 year old compared to 17 year old male adolescents.
There is an association between this drug misuse and other substance misuse such as
narcotic drugs. Female adolescents do not misuse steroid hormones. The findings indicate
the need for preventive work among male adolescents in order to induce adolescents not to
start misusing androgenic anabolic steroids [01006].
A recent doping report performed by the Swedish National Institute of Public Health
estimated that at least 10,000 people in Sweden, among 9 million people, have used AAS in
2008. However, higher estimations of 50,000–100,000 users have also been made. Different
questionnaires among gym customers showed a lifetime prevalence between 2.7 and 4.8
percent. Among the males 3.8-6.0 percent had tested AAS/doping agents at some time and
0-4 percent of the females. In an investigation the prevalence of abuse among young
adolescents, 16 and 17 years old, in one Swedish county, by an anonymous multiple-choice
questionnaire, indicating a misuse of 2.9 percent among the male population. The Swedish
Customs and the National Criminal Police in 2010 reported an increasing illegal import of
AAS into Sweden. In particularly, the import of pure substances, used in local production of
ampoules and tablets for the black market, has increased markedly over the last years
[12025].
14-18 years old

The aim of one study was to investigate attitudes towards androgenic anabolic steroids
among male adolescents who have used anabolics compared to those who have not. A
cross-sectional survey was performed in the year 2000 in all secondary schools in the county
of Halland on the west coast of Sweden. An anonymous multiple-choice questionnaire was
distributed to all classes with 14-, 16-, and 18-year-old male adolescents. The response rate
was 93 percent (n=4049). Those who admitted having used androgenic anabolic steroids
differed in several ways from those who had not. Fewer believed androgenic anabolic
steroids to be harmful (odds ratio 0.15) and more believed that girls preferred boys with large
muscles (OR 6.1). They trained more often at gyms (OR 5.6), drank more alcohol (OR 4.2),
and had used narcotic drugs more often (OR 15.3) than the other male adolescents. More
immigrants than native-born adolescents had used anabolics (OR 4.2). Attitudes towards
anabolics differ between users and nonusers. These aspects may be beneficial to focus on
as one part of a more complex intervention program in order to change these attitudes and
decrease the misuse of androgenic anabolic steroids [05012].
Forensic study in Sweden

Anabolic Androgenic Steroids (AAS) are controlled substances in Sweden. The law was
passed in 1992 and concerns synthetic anabolic steroids, testosterone and its derivatives,
growth hormones and chemical substances that increase the production or release of
testosterone and its derivatives or of growth hormones. Even the use of AAS is prohibited in
Sweden since 1999, which is quite unique compared to other countries in Scandinavia and
also worldwide. Traditionally AAS have been used by elite athletes to enhance performance,
but in recent years it has become an increasing problem outside elite sport among athletes,
bodybuilders and criminals. Use of AAS is associated with psychiatric side effects such as
aggression, depression and violent behavior. Supraphysiological doses and long term use
can cause serious physical harm such as cardiovascular toxicity and even premature death.
It was investigated and evaluated the drug analytical findings in forensic cases from
361
suspected perpetrators in cases from the police where a screening for AAS was requested to
get information about the prevalence of AAS use and the occurrence of poly-drug abuse. The
study was based on samples submitted from the police authorities to the Department of
Forensic Toxicology in Sweden during the period 1999–2009. Urines were analyzed by
methods based on GC–MS and LC–MS–MS. It was also analyzed the prevalence of AAS
use at the prison and probation services. A total number of 12,141 urine samples (6362
police cases and 5779 inmates) were analyzed and 34 percent of the cases from the police
and 12 percent of the inmates were tested positive for AAS. Nandrolone was the most
frequently detected AAS, found in 1249 (62 %) of the positive cases followed by testosterone
found in 719 (36 %) cases. Methandienone, stanozolol, boldenone, trenbolone and
drostanolone are also commonly misused. Thirteen different AAS substances were detected
in 2009, an addition of eight new substances since 1999 when only five different types of
AAS were detected. Including oxymesterone, which only has been identified once in 2003,
gives a total number of fourteen different AAS identified during the study period. Nandrolone,
testosterone, methandienone and stanozolol have overall been the most frequently used
AAS and are currently still in lead. 1120 (60 %) of the positive cases were found to have
used more than one type of AAS. The number of AAS found simultaneously has increased
from 1 (48 %) or 2 (52 %) in 1999 up to eight different AAS in year 2009. High concentration
levels, more than 30,000 ng/mL urine, were measured for some of the metabolites and even
unchanged steroids were detected at several thousand ng/mL. The most commonly illegal
drugs found were cannabis and amphetamines (including methamphetamine and MDMA),
both of which were detected in 440 (37 %) cases each. Benzodiazepines were detected in
339 (28 %) cases, cocaine in 218 (18 %) cases, opiates (morphine and codeine) in 77 (6 %)
cases with 6- acetylmorphine present in 9 of these cases indicating intake of heroin,
ephedrine in 68 (6 %) and GHB in 61 (6 %) cases. It was also investigated the drug abuse
pattern of the AAS positives (n=921) compared to the 148,585 cases of petty drug offences,
analyzed 1999–2009, where AAS were not measured. The drug abuse pattern for the
confirmed AAS users corresponds to the pattern for other drug addicts with some minor
differences. Amphetamine and cannabis were most frequently used by both groups, but to a
wider extent noted for the non-AAS group. Also opiates were most commonly used within the
non-AAS group. Cocaine and benzodiazepines were on the other hand used more broadly
by the AAS-users. Thus, the illegal use of AAS in Sweden has increased over the last eleven
years. Of the individuals suspected of doping offence, more than 30 percent were tested
positive for AAS. At the correctional institutions, 11 percent of the inmates tested, were using
AAS. The AAS users in Sweden are primarily young men (99 %) with a median age of 25
years. The high concentrations of AAS and their corresponding metabolites measured in
urine, reveals that supraphysiological doses of AAS are administered. The incidence of poly-
drug abuse of 60 percent unequivocally demonstrates that AAS are commonly used together
with other drugs of abuse, which is in accordance with the drug abuse pattern of the average
drug addict. AAS have to be considered as an equally serious social problem as narcotics in
Sweden [12026].
In Swedish prisons
Anabolic Androgenic Steroids (AAS) are considered drugs of abuse and are controlled
substances in Sweden since 1999. Traditionally AAS have been used by elite athletes to
enhance performance, but in recent years it has become an increasing problem outside elite
sport among athletes, bodybuilders and criminals. Use of AAS is associated with psychiatric
side effects such as aggression, depression and violent behavior. Supraphysiological doses
and long term use can cause serious physical harm such as cardiovascular toxicity and even
premature death. It was investigated and evaluated the drug analytical findings in forensic
cases from suspected perpetrators in cases from the police where a screening for AAS was
requested to get information about the prevalence of AAS use and the occurrence of poly-
drug abuse. The study was based on samples submitted from the police authorities to the
362
Department of Forensic Toxicology in Sweden during the period 1999-2009. Urines were
analyzed by methods based on GC-MS and LC-MS-MS. We also analyzed the prevalence of
AAS use at the prison and probation services. A total number of 12,141 urine samples (6362
police cases and 5779 inmates) were analyzed and 33.5% of the cases from the police and
12 percent of the inmates were tested positive for AAS. The users of AAS were mainly in 99
percent men with a mean age of 26 years whereas the women were 30 years old. The most
frequently used AAS was nandrolone followed by testosterone and methandienone. Other
illicit and licit drugs were detected in 60 percent of the cases from the police, strongly
indicating a frequent poly-drug abuse among users of AAS [12118].
Health-compromising behavior among 18-year-old Swedish males

The aim of one study was to determine whether there are differences regarding well-being,
health habits and health-compromising behaviour between Swedish young men who had
never had sexual intercourse (group I), those who were sexually active but without a
pregnancy record (group II), and those who had caused pregnancies (group III). A self-report
questionnaire was used covering questions regarding health, health habits and risk
behaviour. Tests of significance for differences between the three groups were performed
with chi 2-tests. Differences were found in health, health behaviour and risk-taking behaviour
between the three groups of young men, with an apparent tendency towards riskier lifestyles
from group I, through group II, to group III. Two especially striking findings were the more
frequent use of anabolic steroids and the reported high rate of sexual offences in group III
compared with groups I and II. The study highlights serious health-compromising behaviours
in 18-y-old men involved in pregnancies in a medium-sized Swedish city. This group has
been largely ignored both in clinical practice and in scientific studies, in contrast to the huge
efforts directed towards pregnant adolescent females. Public health workers, school health
programmes and adolescent clinics need to acknowledge this and work accordingly [02008].
Denmark
From 2003 to 2009, the number of cases testing positive in Denmark for use of doping
increased from 11 to 181. The cases were observed exclusively among individuals
exercising in fitness centers. Consequently Anti Doping Danmark initiated a population based
survey about individual knowledge, attitudes and use of muscle enhancing drugs. The survey
focused in particular on young males aged 15 to 25 years old exercising in fitness centers.
5.010 individuals aged 15-60 years were selected at random and asked to respond to a
postal or web-based questionnaire. 1.703 individuals (34 %) responded to the questionnaire.
The most important results from the survey included:
- 1,5% reports that they currently use or have been using muscle enhancing drugs.
The proportion amounts to 44.000 individuals aged 15-60 years in the general
population. The prevalence of use among individuals that exercises in a fitness center
was 3,3 %, which amounts to approximately 16.500 individuals
- 6% have considered using muscle enhancing drugs. This proportion amounts to
150.000 individuals
- more than half of those that have considered using muscle enhancing drugs knows of
other people in their social network, that uses the drugs
- a substantial proportion of the population opposes the use of muscle enhancing drugs
- attitudes to muscle enhancing drugs among individuals exercising in fitness centers
are no different from others
- 19 % of the population knows where to acquire muscle enhancing drugs. Among
those that have considered using the drugs, 49 % report that they know where to get
the drugs
363
- the majority of those using anabolic steroids do not discuss their abuse with their own
doctor.
In addition, results showed a more relaxed attitude to muscle enhancing drugs and a higher
level of knowledge about the effect of the drugs among the 6 percent of the respondents that
at some point have considered using the drugs. Users of muscle enhancing drugs are found
more frequently in fitness centers than other sport facilities. Muscle enhancing drugs are
more frequently a topic of discussion among respondents exercising in fitness centers, and
several report about supervision in use of muscle enhancing drugs being available in the
fitness center. At the same time, significant more individuals in the fitness environment report
knowing about someone in their social network that use muscle enhancing drugs, compared
to other respondents. A positive results is the rarity of offers of muscle enhancing drugs
among individuals exercising in fitness centers, which occurs equally seldom as among other
respondents exercising elsewhere. Also positive is the opposing attitude to muscle
enhancing drugs among respondents exercising in fitness centers, as among others. The
rarity of offers and the opposing attitudes among respondents exercising in fitness centers
indicates that the occurrence of muscle enhancing drugs is limited to narrow groups of
individuals. Finally, a significant higher proportion of those exercising in fitness centers points
to a good social environment. In sum, the positive results observed should be included in the
preventive activities [10429].
Individuals exercising to for a more muscular and athletic body or to gain more self
confidence, report that use of muscle enhancing drugs is acceptable if use of drugs was not
associated with health hazards. Individuals that exercise to increase their muscle volume are
more frequently observed in fitness centers and other areas where excessive weight training
is a supplement to the main sport. The positive attitude to muscle enhancing drugs among
individuals exercising for a more muscular and athletic body is contrasting the attitude among
individuals for whom such motives for exercising are of no importance. For this reason,
information and education about the health hazards associated with use of muscle
enhancing drugs should be continued and increased in strength – both within and outside
commercial fitness centers. As argued above, information about short- and long term health
hazards associated with use of muscle enhancing drugs should be included as an integrated
part of the preventive initiatives. In contrast, information on legal consequences should be
included to a lower degree – if at all. Narratives that illustrate the health and social risks
associated with use of muscle enhancing drugs could form the core of the information and
education parts of the preventive work. The prevalence of attitudes to and use of muscle
enhancing drugs is documented in the present survey. Results indicate a possible higher
prevalence of use in some environments – including fitness centers. Since the latest survey
on use of muscle enhancing drugs in Denmark published in 1999, other surveys have been
conducted. Any results and conclusions that are based on self-reported behavior that is
perceived a criminal act and social unacceptable will necessarily face major methodological
challenges. The results of the now presented survey are no exception. Notwithstanding these
caveats, the overall conclusion of the surveys conducted within the past ten years is that the
abuse of muscle enhancing drugs at best remains stable or worse is increasing. Nothing
points to a decrease in use of the drugs [10429].
In general practice
The goal of one study was to investigate the use and side effects of anabolic androgenic
steroids (AASs), growth hormone (GH), erythropoietin (EPO) and other enhancing drugs by
patients in general practice. In a questionnaire, 702 general practitioners (GPs) in Denmark
were asked to estimate the number of their patients who within the preceding year had
admitted to using or were highly suspected of having used AASs, GH, EPO or other
enhancing drugs. In addition, they were asked to describe the possible side effects of their
364
use. Of 571 eligible GPs, 119 had within the preceding year treated patients who had
admitted to or were highly suspected of having used AASs, GH, EPO or other enhancing
drugs. 182 users were reported by the GPs during that period; 180 (99 %) were males, and
156 (86 %) were between 20 and 40 years of age. 125 of the patients (69 %) had admitted
the use to the GP; AASs had been used by 123 patients (98 %). EPO was not reported in
any case, but GH had been used by 9 patients (7 %). 127 (70 %) of the patients had
experienced side effects, and 87 (49%) had contacted the GP due to these side effects. The
use and side effects of AAS are commonly reported in general practice, whereas the use and
side effects of GH and EPO seem to be uncommon. Their use is mainly by men under 40
years of age. The use of AAS is often connected with dermatological and musculoskeletal
side effects. It is recommended also to consider possible use of AASs when consulted by a
patient with unexplained symptoms of cardiovascular disease, psychological or sexual
dysfunction, gynaecomastia or liver dysfunction [06026].
Norway
To investigate the prevalence of anabolic-androgenic steroid (AAS) use among Norwegian

adolescents and to contrast three perspectives on AAS use: performance enhancement in
sports competition, body image and eating concerns, and AAS-use as belonging to a cluster
of problem behaviors. A nationally representative sample of 8,877 (54 % female) Norwegian
youths (15-22 years of age) were surveyed (response rate 78 %). Sports participation
included measures of participation in strength sports, participation in competitive sports,
strength training and perceived athletic competence. Body image and eating concerns
included measures of disordered eating, perceived physical appearance and satisfaction with
body parts. Problem behavior was measured by three dimensions of conduct problems (overt
destruction, overt nondestruction and covert destruction), illicit drug use and sexual
involvement. Information about AAS was obtained from 8,508 subjects. Lifetime AAS use
was 0.8 percent (1.2 % male and 0.6 % female), 12-month prevalence was 0.3 percent and
5.1 percent had been offered AAS. AAS use did not vary according to sports involvement
and demographics. Logistic regression analyses showed that AAS use was associated with
such problem behavior as marijuana (cannabis) involvement and overt nondestruction (e.g.,
aggressive-type conduct problems) and, to some extent, with involvement in power sports
and disordered eating. AAS users differed little from those who had been offered but
refrained from using AAS, except that they were more likely to be current marijuana users.
Adolescent AAS use seems primarily to be another type of problem behavior and only
secondarily is it associated with strength-sport participation and disordered eating [01007].
AAS users and contemplators were investigated for differences in aggression and body
image concern. Prevalence rates were sought as a secondary aim. 396 male adolescents at
Norwegian high schools completed a questionnaire battery during school hours. Prevalence
of AAS use showed 4.0 percent; AAS contemplation showed 5.1 percent. No significant
differences between the AAS users and contemplators were found on levels of aggression
and body image concern. AAS users and contemplators reported significantly higher levels of
aggression and body image concern compared nonusing controls. AAS contemplators
enhance understanding of AAS use by representing psychosocial factors contributing to
increased aggression, and AAS use or risk thereof indicative of an aggressive personality
profile. Body image concerns for AAS users and contemplators may indicate that AAS use
does not diminish body image concern, and that body image concern is a risk factor for AAS
use. This is supportive of previous research [14617].
Anabolic-androgenic steroid (AAS) use has been identified as a serious public health
problem. One study investigates the prevalence and correlates of AAS use among
Norwegian adolescents. In 2012, a nationally representative sample of 2,055 17-year-old
365
adolescents (963 males and 1,088 females) participated in a survey. The response rate was
70 percent. In addition to questions about AAS use, participants completed the Parental
Monitoring Scale, the Family Relations/Cohesion Scale, the Alcohol Use Disorders
Identification Test C, the Mini-International Personality Item Pool-Five-Factor Model, the
Eysenck Narrow Impulsiveness Subscale, the Arnett Inventory of Sensation Seeking, the
Short-Form Buss-Perry Aggression Questionnaire, the Hospital Anxiety and Depression
Scale, and the UCLA Loneliness Scale. They also answered questions about demography,
gambling, smoking, snus, and narcotic use. Descriptive statistics and logistic regression were
used to analyze the data. The lifetime prevalence of AAS use was 0.30 percent (0.52 % in
males and 0.09 % in females), while current prevalence was 0.25 percent. Moreover, 19.4
percent of the sample reported having an acquaintance who used or had used AAS. Having
an acquaintance who used or had used AAS was significantly related to snus use,
depression, aggression, extraversion, and conscientiousness in both univariate and
multivariate logistic regression analyses. The findings suggest a high prevalence of AAS use
among Norwegian adolescents and denote the significance of social, personality, and health
factors in adolescents' exposure to AAS milieu [150013].
AAS users and contemplators were investigated for differences in aggression and body
image concern. Prevalence rates were sought as a secondary aim. 396 male adolescents at
Norwegian high schools completed a questionnaire battery during school hours. Prevalence
of AAS use showed 4.0 percent; AAS contemplation showed 5.1 percent. No significant
differences between the AAS users and contemplators were found on levels of aggression
and body image concern. AAS users and contemplators reported significantly higher levels of
aggression and body image concern compared nonusing controls. AAS contemplators
enhance understanding of AAS use by representing psychosocial factors contributing to
increased aggression, and AAS use or risk thereof indicative of an aggressive personality
profile. Body image concerns for AAS users and contemplators may indicate that AAS use
does not diminish body image concern, and that body image concern is a risk factor for AAS
use. This is supportive of previous research [150014].
Adolescents
To prospectively study the stability of anabolic androgenic steroid (AAS) use and predictors
of AAS use, and to investigate whether AAS use alters the risk of later emotional and
behavioral problems a survey of a national sample of Norwegian high school students (age
15-19) in 1994 was followed up in 1999 (n=2924). Measures of frequent alcohol intoxication
(50+ times per 12 months), cannabis use (12 months), hard drug use (12 months), being
offered cannabis, eating problems, conduct problems, sexual debut before age 15, BMI,
involvement in power sports, perceived physical appearance, and satisfaction with body
parts were obtained. Life-time prevalence of AAS use were 1.9 and 0.8 percent in the follow-
up period. Multivariate logistic regression revealed that future AAS use was predicted by
young age, male gender, previous AAS use, involvement in power sports, and frequent
alcohol intoxication. AAS use did not predict future emotional or behavioral problems other
than reducing the risk of future frequent alcohol intoxication. It was concluded that frequent
alcohol intoxication and involvement in power sports appear to predict future AAS use. At the
population level there was little stability in individual AAS use from adolescence to early
adulthood. No detrimental effects of AAS use could be detected in this study, but low
statistical power limits this conclusion [06024].
A total of 1351 high school students (52 % males, 48 % females) with mean age 18 years
from randomized school classes in Hordaland County, Norway, participated in an Internet
survey conducted in 2004 about the lifetime use of anabolic steroids and personal
acquaintance with at least one user of anabolic steroids. In addition to questions about
anabolic steroids the participants completed the Hospital Anxiety and Depression Scale and
366
the Alcohol Use Disorders Identification Test. They also answered questions about
demography, smoking, and narcotic use. The lifetime prevalence for use of anabolic steroids
was 3.6 percent for males and 0.6 percent for females. In all, 28 percent of the respondents
reported having at least one acquaintance that used or had used anabolic steroids. Use of
anabolic steroids and having acquaintances using such drugs were strongly related to use of
other drugs such as alcohol, nicotine, and narcotics. Implications for prevention are
discussed and the study's limitations are noted [06025].
Finland
The aim of one study was to describe the lifetime occurrence and associated factors of
anabolic-androgenic steroids (AAS) among young Finnish males. Of the 10 829 male
conscripts (median age 19), 10 396 (96 %) answered a questionnaire during the first days of
their conscription in the years 2001-2007. The main outcome was lifetime AAS use. It was
also studied associations between 13 socioeconomic, health, and health behavioral
background variables and AAS use by logistic regression. Eighty-nine (0.9 %) respondents
reported having used AAS. In addition, 26 (0.3 %) respondents reported that they would use
AAS if they could obtain them. In multivariate analysis, which included all significant variables
and age, the strongest associated factors were weight training at fitness centers more than
three times a week (odds ratio 11.8; 95 % confidence interval 7.1 to 19.6), low educational
status (odds ratio 3.7; 95 % confidence interval 2.0 to 7.0), and weekly drunkenness as
drinking style (odds ratio 2.4; 95 % confidence interval 1.4 to 4.5). Sports other than weight
training were not associated with AAS in the sample. The use of AAS is relatively uncommon
among Finnish males. It is strongly associated with weight training at fitness centers but also
with lower educational status and a drunkenness-oriented lifestyle. Prevention should be
targeted at those males participating in weight training [10024].
The Netherlands
Studies on the use of performance enhancing drugs (PED) in fitness centres rely
predominately on conventional survey methods using direct questioning. However, research
indicates that direct questioning of sensitive information is characterized by under-reporting.
The aim of the present study was to contrast direct questioning of different types of PED use
by Dutch fitness centre members with results obtained with the randomized response
technique (RRT). Questionnaires were conducted among members of fitness centres. PED
were classified into the following categories: anabolic steroids, prohormones, substances to
counteract side-effects, growth hormone and/or insulin, stimulants (to reduce weight), and
miscellaneous substances. A total of 718 athletes from 92 fitness centres completed the
questionnaire. The conventional method resulted in prevalences varying between 0 percent
and 0.4 percent for the different types of PED with an overall prevalence of 0.4 percent. RRT
resulted in prevalences varying between 0.8 and 4.8 percent for the different types of PED
with an overall prevalence of 8.2 percent. The overall prevalence of the two survey methods
differed significantly. The current study showed that the conventional survey method using
direct questioning led to an underestimation of the prevalence. Based on the RRT results,
the percentage of users of PED among members of fitness centres is approximately 8.2
percent. Stimulants to lose weight had the highest prevalence, even higher than anabolic
steroids. The key task for future preventive health work is to not only focus on anabolic
steroid use, but also include interventions focusing on the use of stimulants to lose weight
[14435].
UK
367
In an English report the House of Commons Science and Technology Committee says that
more needs to be done on every level to combat the use of illegal substances by athletes.
The cross party group of MPs says that athletes caught cheating by using chemicals or
biological agents should be banned from sport for four years and ordered to repay any
financial gains they have made since their last clean test. Athletes should also have to state
where they obtained the banned substances before they are allowed to return to competitive
sport. The committee concluded that official figures on the incidence of illegal doping may not
accurately reflect the problem, and it called for more research into the true scale of the
problem. Figures from the World Anti-Doping Agency show that 2.1 percent of tests for
banned substances resulted in “adverse analytical findings” in 2005; in the UK 1.3 percent of
7968 tests proved positive in 2005-6. To make it easier to detect performances improved by
illegal substances all UK athletes should be made to compete on the international circuit
during the 12 months before the Olympics, says the report. And a new agency that is
independent of UK Sport and other national sporting bodies should be set up to test athletes
for drug use. The agency should also monitor and evaluate potential new illegal substances
and methods as they are developed. The report also recommends a pilot project to examine
the feasibility of a physiological or doping passport to be carried by all athletes. This would
record the results of doping tests and natural concentrations of hormones such as
erythropoietin during their careers, which would make it easier to detect any substance
abuse, the committee says. The committee also expressed concern at the ease in which
banned and potentially dangerous substances can be obtained for use by athletes. It
recommended that the government review regulation in this area [07035].
Figures from the Department of Health in the United Kingdom showed that 0.2 percent of
young people had tried anabolic steroids in 2001-4 and 0.5 percent in 2006. A questionnaire
study of 3403 12th grade students (final year of secondary school) in the United States found
that 6.6 percent admitted to taking anabolic steroids. Worryingly, two thirds of those had
started using anabolic steroids when they were aged 16 or younger. A study of homosexual
men who regularly attended gyms in London in 2000 found that 15 percent of the 792 men
surveyed had used anabolic steroids in the preceding year, with 12 percent of them having
injected the drugs. Two thirds of the respondents used more than one agent (so called
“stacking”). The high prevalence of use of injectable anabolic steroids correlates with a local
experience, where 43 percent of new registrations for needle exchanges were users of
anabolic steroids. This figure might be an underestimate as many users of anabolic steroids
will collect needles and syringes for friends and other users as well [12025].
British army
The use of supplements is widespread at all levels of civilian sport and a prevalence of 60-
90 percent is reported among high-performance UK athletes, including juniors. The
prevalence of supplement use among UK-based British Army personnel is not known. The
aim of the present study was to establish the point prevalence of supplement use in UK-
based British Army soldiers under training (SuTs) and associated staff. A cross-sectional
anonymous survey was carried out in 3168 British Army SuTs and soldiers, equating to
3.1 percent of regular Army strength, based at eleven Phase 1, 2 and 3 UK Army training
sites. Overall, 38 percent of the respondents reported current use of supplements, but
prevalence varied according to the course attended by the respondents. The number of
different supplements used was 4,7. Supplements most commonly used were protein bars,
powders and drinks (66 %), isotonic carbohydrate-electrolyte sports drinks (49 %), creatine
(38 %), recovery sports drinks (35 %), multivitamins (31 %) and vitamin C (25 %). A small
proportion of respondents reported the use of amphetamines and similar compounds
(1.6 %), cocaine (0.8 %), anabolic androgenic steroids (1.1 %), growth hormone (2.0 %), and
other anabolic agents, e.g. testosterone (4.2 %). Logistic regression modelling indicated that,
for current users, younger age, being female, smoking and undergoing Officer Cadet training
368
were associated with greater supplement use. This is the first study to investigate the
prevalence of dietary and training supplement use in UK-based British military personnel.
Self-administration of a wide range of supplements is reported by British military personnel in
training, which is at least as great as that reported by those on deployment, and has
implications for Defence policy and educational needs [14618].
British gay men

To examine, by HIV status, the use of anabolic steroids among London gay men and their
effect on physical and mental health a cross-sectional survey using self-administered
questionnaire were performed in six gyms in central London with 772 gay men using the
gyms in 2000. Of the 772 gay men, 117 (15 %) had used and 90 (12 %) had injected steroids
in the previous 12 months: HIV positive men (steroid use) 32 percent (40/126), HIV negative
men 14.5% (69/477), never-tested for HIV 5 percent (8/169). No one reported sharing
needles or syringes. HIV positive men were more likely to have used steroids for medical
reasons than other men (24 % vs 6 %). Nearly all steroid users (96 %) reported side effects
including testicular atrophy (51 %), insomnia (48 %), depression between cycles (25 %) and
hypertension (19 %). Steroid users were more likely to have had suicidal thoughts in the
previous 6 months than non-users (23 % vs 11 %, adjusted odds ratio after controlling for
HIV status 1.84) or to have felt depressed (49 % vs 39 %, adjusted OR 1.52. It was
concluded that one in seven gay men surveyed in central London gyms in 2000 said they
had used steroids in the previous 12 months. HIV positive men were more likely to have
used steroids than other men, some therapeutically. Side effects were reported widely and
steroid use was associated with having had suicidal thoughts and feeling depressed,
although cause and effect could not be established. The findings suggest that steroid use
among gay men may have serious consequences for both physical and mental health
[02006].
South Asian males

Consumerism of image and performance enhancement drugs (IPEDs) is a world-wide public
health concern. Given anecdotal reporting of increased normalisation of IPED use and
uptake of British South Asian male IPED users at UK needle and syringe exchange services,
the study aimed to explore use of IPEDs among this under-researched ethnic group. Twenty
in depth interviews were conducted with a purposive sample of British South Asian males
attending harm reduction outreach in the North East of England. The interviews explored
motives for use of IPEDs, sourcing routes, information seeking, injecting behaviours and
cultural and community sensitivities around IPED use among this group. The data was
collected and analysed using the Interpretative Phenomenological Analysis approach (IPA).
Motives for use centred on the achievement of enhanced definition and density of muscle,
and improved recovery from training and injuries. All participants reported initial stimulation of
interest and triggers to seek information on IPEDs due to social media, community and peer
messages. Diverse forms of IPED use were described, with rational and moderated use
common among older participants. In contrast younger participants adopted more excessive
use in seeking short cuts to attaining muscle size. Sourcing of androgenic-anabolic steroids
(AAS) and growth hormones from originating countries (Pakistan, India) was reported, along
with diversification of entrepreneurial activity into IPED dealing networks. Sellers were
generally reported to provide effective and reliable products and mentoring to inexperienced
users. Group injecting practices were common. IPED use was observed by some as health
promotion medium within religious contexts. Crime deterrence and drug abstinence occurred
for some while involved in AAS cycles. The study is intended to contribute to health policy
and practice debate around the targeting of dedicated education, outreach and harm
reduction for ethnic groups engaged in IPED use [150015].
Pooled urine samples

369
Analysis of anonymous pooled urine samples from street urinals has been used to
demonstrate time-trends in the detection of classical recreational drugs and novel
psychoactive substances (NPS). One study aimed to expand this to undertake a
geographical trend analysis of classical recreational drugs/NPS across UK. Samples of
anonymous pooled urine were collected from street urinals that had been in place for one
night in April 2014 in nine cities across the UK. Collected samples were then analysed for the
presence of recreational drugs, NPS anabolic steroids using high-performance liquid
chromatography coupled to high-resolution accurate mass full-scan mass spectrometry and
gas chromatography coupled to electron impact ionization mass spectrometry operating in
selected ion monitoring and full-scan modes. Ten classical recreational drugs, nine NPS and
four anabolic steroids were detected across the nine cities; the range of detection was from 1
in Leeds to 14 in London. The most common classical drugs were cocaine (9 cities) and 3,4-
methylenedioxy-methamphetamine (8 cities); the most common NPS was 4-
methylmethcathinone (5 cities). In addition there was variation in the detection of NPS, with
methylhexaneamine detected only in Bristol and London, piperazines (3-
trifluoromethylphenylpiperazine and 1-benzylpiperazine) and pentedrone only detected in
Birmingham and the cathinone methylone only detected in London. There is variability in the
detection of classical recreational drugs, NPS and anabolic steroids across UK, likely
reflecting variation in their use. This technique can be used to supplement drug use surveys
to determine geographical and time trends in the use of these substances. This is important
to ensure appropriate targeting of drug-related interventions [150022].
Greece
Doping use is an ongoing problem in contemporary sports. Despite efforts to detect and
control doping, research on its etiology is limited, especially among elite-level athletes. One
study used an integrated social cognition model to examine the predictors of doping
intentions. Structured anonymous questionnaires were completed by 1075 Greek adult elite-
level athletes (medium age = 25 years, 36 % females) from both team and individual sports.
Multiple regression and mediation analyses showed that attitudes, normative beliefs,
situational temptation, and behavioral control significantly predicted doping intentions. A
normative process was identified whereby situational temptation mediated the effects of
normative beliefs on intentions. The findings provide the basis for future social cognition
research in doping use, and set the framework for the development of evidence-based
preventive interventions [10436].
One article concerns the analysis of the adverse analytical findings (AAFs) and the
appropriate alterations made during the period 2005-2011, so that the Doping Control
Laboratory of Athens (DCLA) obeys the updated World Anti-Doping Agency (WADA) List of
Prohibited Substances. The percentage AAFs of the DCLA was compared with those of
WADA-Accredited Laboratories. In 2008, the term Atypical Finding was introduced by the
WADA representing a reported but inconclusive result. A characteristic example is when a
testosterone-to-epitestosterone ratio is >4 followed by a negative gas chromatography/
combustion/isotope ratio mass spectrometry result. In a total of about 30,000 athlete
samples, 136 athletes were found with an increased testosterone/epitestosterone ratio and
43 with tetrahydrocannabinol metabolite (THCCOOH) of 427 reported AAFs. Twenty-one
athletes in total were found positive with methylhexaneamine, the 11 found after a batch of
1000 samples was reprocessed. Besides, there were AAFs below their Minimum Required
Performance Level (MRPL). The increasing need for higher detectability imposed new
apparatus, e.g., liquid chromatography/quadrupole/time-of-flight mass spectrometry, whereas
that for lowering the capital costs and reporting times led to the unification of the screening
method which includes stimulants, diuretics, anabolics and other substances [0125].
370
Within the context of problem-behaviour theory, one study investigated the intra-relationship
between attitudes and behaviours towards exercise, sport involvement, violence in sport-
related events, eating fruits, smoking and hashish or ecstasy use in a sample of Greek
adolescents. Age and gender patterns are considered. Participants were 5991 Greek school
pupils who responded to questionnaires assessing behaviour and attitudes towards health-
related behaviours. Positive associations were found between pupils' reports of violence in
sport-related events, smoking and hashish or ecstasy use on the one hand, and eating fruits
and participation in sport and exercise on the other. In contrast, small positive association
was observed between sport involvement and violence in sport-related events. Attitudes
towards health risk behaviours were inversely related to attitudes towards health-promoting
behaviours, and attitudes were positively related to corresponding behaviours. Sport
involvement and regular exercise decreased but smoking and use of hashish or ecstasy
increased with age. More males than females participated in organized sport and violent acts
in sport-related events. Males' involvement in sport violence increased with age. Sport is a
suitable context for the promotion of several health-related behaviours apart from exercise.
Nevertheless, the present sport structure excludes most young people and is positively
linked with sport violence. A less demanding sport context should be provided for the
majority of young people, particularly for females. Sport programmes designed to promote
health behaviours should be encouraged. More concentrated actions to combat sport
violence are required [14015].
Italy
Physical activity, diet plans, the mantainment of a certain Body Mass Index (BMI) and the
use of various types of supplementation are common elements in the search for disease
prevention, health promotion and well-being. It was analyzed the data regarding Italian
university students' BMI, dieting behavior, personal body perception, exercise habits, and
use of dietary supplements and of doping substances. 0.9 percent admitted consuming
doping substances. The most important concern arising from the questionnaire is
represented by physical inactivity. Indeed, it is necessary to encourage and plan initiatives
aimed at promoting physical activity in university students [150017].
The abuse of anabolic steroids is emerging as a psychosocially significant issue. In the last
few years the use of the substances has shifted from professional sports to amateur sports
and certain occupations (bouncers, models, etc.). In the literature, steroid users are
portrayed as multidrug users who engage in dangerous and aggressive behavior towards
themselves and others. One study looked into the habits, lifestyles and psychological profiles
of a group of subjects who make regular use of sports centres in the city of Palermo, Italy,
with the aim of establishing how the abuse of anabolic substances is associated with a
specific lifestyle and particular psychosocial behaviour. A revision of the American
Massachusetts Youth Risk Survey questionnaire (1993), adapted for the Italian context, and
a personality assessment scale, The Adjective Check List (1980), were administered to a
group of 71 subjects. Fifteen of these subjects admitted taking steroids with differing
frequencies. Using Spearman's rho rank correlation, repeated use of anabolic steroids was
found to be correlated with abuse of other types of drugs, risk behavior and a distinct
personality pattern. Steroid abuse was found to be significantly correlated with illegal drug
use (LSD, cocaine and heroin). It is therefore imperative to develop studies and analyses to
investigate more thoroughly the phenomenon and its related psychological and social context
in order to lay the foundations for a targeted prevention programme, especially in countries
such as Italy where this type of drug abuse is still largely unrecognised and risks
degenerating into a new, full-blown social disease [05002].
371
The objective of one study was to assess the prevalence of illicit drugs use among young
adults, in particular elite athletes. The study considers the data obtained from anti-doping
analyses performed on nearly 100,000 urine samples from 2000 to 2009 by the World Anti-
Doping Agency accredited Italian Anti-Doping Laboratory. The percentage of adverse
analytical findings varies on a yearly basis, but it is in the range 1.0-1.8 percent (not
considering atypical findings, such as an altered endogenous steroid profile). Among positive
results, there is a high prevalence of stimulants and drugs of abuse. The drug of abuse found
most frequently is the tetrahydrocannabinol (cannabis) metabolite, accounting for 0.2-0.4
percent of the total samples analysed (18 % of the positive results). The second most
frequently encountered drug is cocaine, as detected from cocaine metabolites, accounting for
0.1 percent of the total samples analysed (7 % of positive results). Other stimulants found
included amphetamines, ephedrines, carphedon, modafinil, and anorexic compounds. No
amphetamine-like designer drugs were detected. These data are indicative of the
widespread prevalence of cocaine and cannabis use among the young adult population.
However, due to the particular population studied, it must be considered an underestimation
of the phenomenon among elite athletes with respect to the general population [11016].
Promoting physical activity is one of the main goals of health-promotion policies. The period
of adolescence is characterised by a high rate of abandonment of any physical activity. In
this age range, moreover, the risk of assuming substances in order to improve muscular-
mass or athletic results is concrete. This study quantifies the involvement in physical
activities and substance assumption in a sample of 6915 students aged 14 to 18 years and
living in 7 different areas, mostly in northern Italy. The survey's tool is an adapted and
modified vision of the Youth Risk Behaviour Surveillance questionnaire, created by US
Centers for Disease Control and Prevention (CDC). The study showed a high percentage of
the sample not involved in any form of physical activity out of school (34 %), more among
girls (44 %) than boys (21 %). Between 14 and 18 years, a continuous reduction of
involvement in physical activity is evident, while the percentage of totally physically inactive
subjects rises from 30 to 43 percent. Finally, 6 percent in the sample admitted to have been
using substances to improve muscular-mass or athletic results at least once in the past.
According to this study, only a minority of the interviewed adolescents is involved in a regular
physical activity. In males, using substances to improve physical strength showed to be
rather diffused. Specific health promotion projects are suggested [04012].
Promoting physical activity is one of the main goals of health-promotion policies. The period
of adolescence is characterised by a high rate of abandonment of any physical activity. In
this age range, moreover, the risk of assuming substances in order to improve muscular-
mass or athletic results is concrete. One study quantified the involvement in physical
activities and substance assumption in a sample of 6915 students aged 14 to 18 years and
living in 7 different areas, mostly in northern Italy. The survey's tool was an adapted and
modified vision of the Youth Risk Behaviour Surveillance questionnaire, created by US
Centers for Disease Control and Prevention (CDC). The study showed a high percentage of
the sample not involved in any form of physical activity out of school (34 %), more among
girls (44 %) than boys (21 %). Between 14 and 18 years, a continuous reduction of
involvement in physical activity is evident, while the percentage of totally physically inactive
subjects rises from 30 to 43 percent. Finally, 5.6 percent in the sample admitted to have
been using substances to improve muscular-mass or athletic results at least once in the past.
According to this study, only a minority of the interviewed adolescents is involved in a regular
physical activity. In males, using substances to improve physical strength showed to be
rather diffused. Specific health promotion projects are suggested [04013].
372
France
Whatever method is used (observation, interviews, questionnaire, laboratory tests), it is

difficult to collect epidemiological data on doping. Particularly difficult problems are related to
the definitions of sports players and the drugs involved as well as the often illicit nature of
drug use. The prevalence of doping in children and adolescents participating in sports is
estimated at 3 to 5 percent with higher percentages in boys, older adolescents and those
playing at a competition level. Use of anabolic steroids, as early as 8 years of age, has
increased since 1990, especially in girls. All studies have emphasized how easy it is for
adolescents to procure any prohibited drug. In adults participating in amateur sports, the
prevalence of doping would be 5 to 15 percent. All sports are involved with higher prevalence
in men, age ranges 20-25 years and 35-39 years, and competitive level players. The main
drugs used are stimulants, narcotics, corticosteroids and anabolic steroids. Combination of at
least 2 drugs is frequent with an increase of mean daily dose over the last 15 years.
According to users, the drugs are obtained with a medical prescription, on the underground
market, or from other participants. Few data are available on doping outside sports activities.
In the French department Meurthe-et-Moselle, 15 percent of the inhabitants use drugs to
improve their occupational performance. It was concluded that doping is more widespread
than would be expected from antidoping control data. Other studies are needed to acquire
more precise epidemiological data [00007].
Early February 1999, the French Ministère de la Jeunesse et des Sports (Youth and Sports
Ministry) sponsored three different studies, aiming to prevent harmful behavior in the area of
sport practices among youth. Two years earlier, our health care team working with drug
users published reports on the meaningfulness of intensive sports activities in the history of
our patients. The present work was performed to highlight the midterm results of one of these
studies, to better understand and quantify the importance of physical training in the history of
a group of outpatients seen for addictive disorders and comorbid pathologies. For 20
consecutive weeks, 3,040 self-administered questionnaires were available for persons
consulting 20 health centers, 2 self-help groups and a general practitioner network working in
the field of alcohol or heroine abuse. One thousand one hundred and eleven questionnaires
were filled out (36 %) and returned by mail for complete analysis: 86 percent of the
answering persons had practiced at least one sports activity or participated in physical
training, 11 percent had participated in a national or international level competition, and 11
percent reported stress fractures. In the intensive sports group, 36 percent had used illicit
drugs intravenously and 16 percent said they had already used doping substances. Only 28
percent said they experienced dependence during their period of intensive sports activities
compared with 15 percent before this time, and a majority (56 %) thereafter. Intensive sports
or physical training should not be seen as a protective factor nor as a way of improving
addictive behaviors. More studies are needed to evaluate individual vulnerability factors and
specific harm of overtraining and to determine the exact periods when men and women
participating in sports activities are likely to abuse drugs, especially at the end of their career
[00008].
To describe the prevalence of doping and its progression in a cohort of preadolescent

athletes during a 4-year follow-up a prospective cohort study was performed. It was a self-
questionnaire survey where all of the pupils entering the first year of secondary school (sixth
grade) in the Vosges Département (east France) and followed for 4 years was asked about
drug use (prohibited substances, tobacco, alcohol, cannabis), intention to use, reported
health hazards, perceived drug effectiveness, self-esteem, trait anxiety. At the beginning of
the study, 1.2 percent (95 % confidence interval 0.8 to 1.6) stated that they had taken doping
agents at least once in the preceding 6 months, and this had risen to 3.0 percent four years
later, which was a statistically significant difference. Of those who had used doping agents, 4
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percent reported that they had experienced a health problem related to doping, and 44
percent reported that they had won at least one sports event as a result of using the drug.
Use of doping agents is linked to the number of hours of practice per week, intention to use,
use of other drugs, self-esteem and trait anxiety. The results show that doping does exist in
preadolescent athletes who train every day [07034].
The French report of the National Academy of Medicine named "Sport and Health"
underlines the medical, social and educational dimensions of sporting activities. Various
kinds of sporting practices are described: they concern the approximately 7,000 high level
athletes, around 8,000 professional (licensed) sportsmen, and sporting club members
(approximately 15 millions people). A large number of amateurs do not practice in any
structure and therefore are neither managed in their activities nor medically followed. Some
characteristics of sporting practice at various stages of life have been documented. Around
50 percent of the teenagers from 12 to 17 years have a sporting practice out-of-school
besides the weekly three hours applied at school or college; however, the withdrawal of
sporting practice by a high number of teenagers results in a sedentary lifestyle with
overweight and obesity, major risks factors for health. Elderly people take a profit from a
regular and medically controlled physical activity. Functional capacities are thus improved,
cardiovascular risks factors among other, which results in better quality of life of the aged
and delays their dependence. The benefit upon public health of sporting practice has been
pointed out in the primary prevention of cardiovascular and respiratory diseases,
osteoporosis, obesity, diabetes, breast and colon cancer, and mood disturbances. It is
currently well acknowledged that sporting practice is an important component of public health
in both primary and secondary prevention of many diseases. Deleterious effects of which the
most serious is the sudden death related to a cardiovascular anomaly, which generally
occurs during an important physical effort. An important sport drift is the practice of doping to
improve performances through the use of hormones, anabolics, EPO, transfusions, ... When
a person exceeds his/her capacities of adaptation, because of a badly adapted or a too
intense drive, this overtraining results in a reduction in physical capabilities, stress,
behavioral issues and sleep-wake disorders. All of those issues often lead sportsmen to
doping with the aim to improve their capabilities, rapidly installing an overtiredness state
resulting in a fall of performances. A major aim from the view point of public health is to
reinforce the fight against doping since it concerns a large number of people, sportsmen and
amateurs, with teenagers among them. Lastly, the report underlines that sport medicine is
practically not taught in the initial training of medical doctors [09029].
The purpose of one investigation was to determine the substances used, and the attitudes
towards doping of high school athletes. A four-page, self-completed questionnaire was
designed to determine the drugs used (licit, illicit and doping substances) along with beliefs
about doping and the psychosociological factors associated with their consumption. The
questionnaire was distributed to all the high school students enrolled in a school sports
association in the Lorraine region in Eastern France. The completed forms were received
from 1459 athletes: 4 percent stated that they had used doping agents at least once in their
life (their main source of supply being peers and health professionals). Thirty-four percent of
the sample smoked some tobacco, 66 percent used alcohol, 19 percent cannabis, 4 percent
ecstasy, 10 percent tranquillizers, 9 percent hypnotics, 4 percent creatine and 41 percent
used vitamins against fatigue. Beliefs about doping did not differ among doping agent users
and non-users, except for the associated health risks which were minimized by users. Users
of doping agents stated that the quality of the relations that they maintain with their parents is
sharply degraded, and they reported that they are susceptible to influence and difficult to live
with. More often than non-doping agent users, these adolescents are neither happy, nor
healthy, while paradoxically, they seem less anxious and they are more self-confident. The
findings suggest that doping prevention among young athletes cannot be limited uniquely to
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the list of banned drugs [10441].
healthy, while paradoxically, they seem less anxious and they are more self-confident. The
Few studies have analyzed in the general population psychoactive substance use among
athletes, especially among females. In fact, sporting activity is often promoted in prevention
actions, as an alternative to addiction or alcohol, tobacco or other substance misuse. So, we
propose an analysis of the ESPAD 1999 sample among students (16-18 years old), focused
on the relationship between sporting activities and substance use. Boys play sport more
frequently than girls (72 % vs 50 %) and report 8 hours and more a week 4 more times than
girls (14 % vs 4 %). Sixty-eight percent of boys and 36 percent of girls have already
participated in sport competitions, more often at a local, departmental or regional level; a
minority of them (26 % of boys and 20 % of girls) have already participated in sport
competitions at a national or international level. Sporting activity is decreasing with age
among girls, students from general lycée play sport more frequently than others do
(vocational lycée); the higher the father's education level, the more frequently the students
play sport. Moderate sporting activity (1-8 hours a week) is a protective factor against regular
smoking (OR=0.54 in boys and OR=0.60 in girls) and against regular cannabis use among
boys (OR=0.64). Intensive sporting activity (>8 hours a week) is a risk factor for illicit drugs
(except cannabis) use (OR=2.74) and sleeping drugs/tranquillizers (OR=1.82) only among
girls. Competition level is the most important risk factor for substance misuse as well in boys
(except sleeping drugs/tranquillizers) as in girls. Practical implications are: adjusting health
policy concerning the beneficial effects of sporting activity, raising sports associations
abilities and avoiding doping and addiction in high-level sporting activities [03015].
Belgium
For many years, doping has been considered a major problem in sports. Recent doping
cases have shocked the general public and press reports have further generated the idea
that a great number of athletes are doped. In this study statistical data provided by the
International Olympic Committee (1996-2000) to IOC accredited laboratories and results
from the Flemish anti-doping program (1993-2000) are discussed. During these periods, the
average percentage positive samples in the IOC accredited laboratories and in Flanders
were 1.8 and 4.1 percent, respectively. The percentage of positive samples was significantly
higher for in-competition than for out-of-competition samples. During the period 1993-2000,
375
doping was detected in all sports in Flanders, for which a representative number of samples
(n > 50) was tested except mini-soccer, where no positive doping samples were found. The
use of doping among male athletes is significantly higher than for female athletes.
Bodybuilding and power lifting had the highest incidence of positive cases in Flanders. The
distribution of detected drugs among the different groups of prohibited substances shows a
significant increase in the number of samples containing cannabis over the last years. The
occurrence of cannabis in all sports and the high frequency of detection in Flanders, indicate
that cannabis is predominantly misused as a "social" drug rather than for doping purposes. In
Flanders, multiple prohibited substances were detected in 41 percent of all positive cases. At
least 28 percent out of those were due to co-administration of drugs [03016].
Germany
Goal-directed measures to prevent doping and drug abuse in sports requires empirical data.
In this connection, a cross-sectional analysis was carried out in 2004. The purpose of the
study, on the one hand, was to register reliable data of the current situation in Thuringia, and,
on the other hand it was to give information on possible interventional steps with scientific
support. Within three months, 2319 adolescents from 16 Thuringian schools (5 regular
schools, 4 secondary schools, 3 sport schools and 4 vocational schools) were surveyed.
Three hundred and forty-six (15 %) students out of 2287 students (26 students without a
statement) indicated use of prohibited substances from the WADA list in the previous year:
16 (0.7 %) anabolic-androgenic steroids (AAS), 10 (0.4 %) growth hormones, 56 (2.4 %)
stimulants, 305 (13.2 %) cannabis, 2 (0.1 %) diuretics, 52 (2.2 %) cocaine/heroin and 6 (0.3
%) erythropoeitin. Moreover, nonathletes (n=490) reported a substance use that was
approximately 5 percent higher than that of recreational athletes (n=1254) and nearly three
times higher than that of competitive athletes (n=497). All three groups (nonathletes,
recreational athletes and competitive athletes) performed poorly on a knowledge test
regarding doping in general with an average below 60 percent in each case. Another main
aspect of the study was to determine factors influencing substance use in sports. Besides the
doping specific knowledge, age contributed as well as anti-doping attitude, to the resulting
variance. Gender, however, played no role. The findings of the study point towards the need
for improvement of specific knowledge of doping among students and that their attitude
towards doping must be altered. The goal in this case is to test the effectiveness of
appropriate scientific intervention [07033].
Doping controls are key factors for fair and clean sports. The developments of German
activities in the national antidoping fight were evaluated over a period of 18 years (1989-
2006) with regard to in-competition and out-of-competition testing. The quantity of respective
controls conducted by federations and antidoping organizations, the ratio of in- and out-of-
competition controls, the number of athletes per squad (and thus per-capita tests) as well as
adverse analytical findings were summarized in a review. The available data demonstrated a
constantly increasing effort, particularly regarding the amount of out-of-competition controls,
but also discrepancies in per-capita analyses between different federations. In light of recent
doping scandals and confessions in 2007, a critical review of national antidoping actions is
considered necessary [08031].
In the context of house searches in Germany, numerous drugs were confiscated and
subjected to chemical analysis, including anabolic agents such as various anabolic-
androgenic steroids (stanozolol, testosterone derivatives, trenbolone esters, etc.) and
clenbuterol, as well as agents with anti-estrogenic activity (tamoxifen, clomiphene), drugs
stimulating virility (sildenafil, tadalafil), and unlabeled plastic bags. Liquid chromatography-
tandem mass spectrometry, gas chromatography-mass spectrometry with nitrogen-
phosphorus specific detection, gel electrophoresis, and immunological tests were employed
376
to test for the effective content of 70 products. In 18 cases (26 %), the declared ingredients
differed from the actual content, in particular concerning anabolic-androgenic steroids.
Nandrolone and trenbolone esters, for instance, were frequently substituted or
complemented by various testosterone derivatives, and several testosterone depot
formulations originally composed of four different esters were found to contain fewer or
wrong components. Except for those drugs supposedly originating from so-called
underground labs, fake packings were hardly or not distinguishable from original boxes by
visual inspektion [08032].
Anabolic ergogenic substance use, in particular the use of anabolic androgenic steroids, is a
serious problem in general. Nevertheless, it is subject to debate whether ergogenic
substance users exhibit similar features as multiple substance users or whether they
constitute a discrete group. 1802 standardized, anonymous questionnaires were distributed
among visitors of 113 fitness centers. Questions were asked concerning biometric
parameters, social indicators, physical fitness, use of natural stimulants, general illicit drugs
and ergogenic substances. With logistic regression analysis, multivariate odds ratios were
estimated to investigate the association of anabolic ergogenic substance or general illicit
drug use with other parameters. Fourteen percent of all participants confessed to having
used anabolic ergogenic substances at some point in time. Anabolic ergogenic substance
use was positively related with cocaine use, training years, training frequency, negatively
related to the level of education, alcohol intake and less frequently used by Germans than by
non-Germans. General illicit drug use, however, was positively related with alcohol intake,
smoking and a university degree and negatively with having children. In addition, anabolic
ergogenic substance use was significantly related with the use of general illicit drugs based
on the strong relation with the use of cocaine, which is an ergogenic substance itself. The
health care system supplies 48 percent of the anaolic ergogenic substance users with their
substances and 32 percent are even monitored by a physician. The results of the study
strengthen the notion that anabolic ergogenic substance users constitute a specific body-
oriented substance user group. Uncommon for general illicit drug use, the health care system
is a major sponsor of anabolic ergogenic substance users. These findings suggest the need
for alternative approaches for successful prevention and intervention programs [05013].
Rural gyms
To investigate epidemiology of doping in rural gyms, a total of 206 persons doing exercise in
5 different gyms in the district of Traunstein (Germany) were interviewed. Nearly 5.0 percent
of the surveyed persons take performance-enhancing drugs, mostly for aesthetic reasons or
to rapidly build up strength. There are no gender or education-specific, yet agerelated
significant differences in using doping substances. Significant factors influencing the intake of
doping agents are the motivation for competition participation, the additional intake of protein
supplements and nutritional supplements. Sixty-two percent of the drug-abusers take
anabolic steroids (testosterone). One athlete consumes growth hormones. Forty percent of
drug-abusers administer medication in the form of an injection, 60 percent in the form of
tablets. Costs per intake cycle are between 100 and 150 Euros. The average body weight
gain through the muscle mass increase is approximately 15 kg. Suppliers were friends (36
%), the Internet (27 %) and physicians (18 %). The potential side effects are important
cornerstones for the educational work in the prevention of doping [150016].
Adolescents
Goal-directed measures to prevent doping and drug abuse in sports requires empirical data.
In this connection, a cross-sectional analysis was carried out in 2004. The purpose of the
study, on the one hand, was to register reliable data of the current situation in Thuringia, and,
on the other hand it was to give information on possible interventional steps with scientific
support. Within three months, 2319 adolescents from 16 Thuringian schools (5 regular
377
schools, 4 secondary schools, 3 sport schools and 4 vocational schools) were surveyed.
Three hundred and forty-six (15 %) students out of 2287 students (26 students without a
statement) indicated use of prohibited substances from the WADA list in the previous year:
16 (0.7 %) anabolic-androgenic steroids (AAS), 10 (0.4 %) growth hormones, 56 (2.4 %)
stimulants, 305 (13.2 %) cannabis, 2 (0.1 %) diuretics, 52 (2.2 %) cocaine/heroin and 6 (0.3
%) erythropoeitin. Moreover, nonathletes (n=490) reported a substance use that was
approximately 5 percent higher than that of recreational athletes (n=1254) and nearly three
times higher than that of competitive athletes (n=497). All three groups (nonathletes,
recreational athletes and competitive athletes) performed poorly on a knowledge test
regarding doping in general with an average below 60 percent in each case. Another main
aspect of the study was to determine factors influencing substance use in sports. Besides the
doping specific knowledge, age contributed as well as anti-doping attitude, to the resulting
variance. Gender, however, played no role. The findings of the study point towards the need
for improvement of specific knowledge of doping among students and that their attitude
towards doping must be altered. The goal in this case is to test the effectiveness of
appropriate scientific intervention [06023].
Spain
According to the data reported in the survey on general population, between 1999 and 2011
the consumption of drugs of abuse in the previous 12 months remained stable for the
majority of the substances (alcohol: 75 % vs 77 %; tobacco: 44 % vs 40 %, MDMA: 0.8 % vs
0.7 %, amphetamines: 0.7 % vs 0.6 %, hallucinogens: 0.6 % vs 0.4 % and heroin 0.1 % vs
0.1 %), with a significant increase in cannabis and cocaine consumption (6.8 % vs 9.6 % and
1.5 % vs 2.3 %), respectively. Hair testing is a useful tool to investigate the prevalence of
unsuspected chronic exposure to drugs of abuse in pediatric populations and it has been
applied to three different cohorts of children from Barcelona, Spain along fifteen years to
evaluate eventual changes in this exposure. Children were recruited from three independent
studies performed at Hospital del Mar (Barcelona, Spain) and approved by the local Ethics
Committee. Hair samples were collected from the first 187 children cohort (around 4 years of
age) in 1998, from the second 90 children cohort (1.5-5 years of age) in 2008 and from the
third 114 children cohort (5-14 years of age) in 2013. Hair samples were analysed for the
presence of opiates, cocaine, amphetamines, and cannabis by validated methodologies
using gas or liquid chromatography-mass spectrometry. Familiar sociodemographics and
eventual consumption of drugs of abuse by parents, and caregivers were recorded. Hair
samples from 25 percent children in 1998 were positive for any drug of abuse (23 %
cocaine), 26 percent in 2008 (23 % cocaine), and 28 percent in 2013 (20 % cocaine and 11
% cannabis). In none of the cohorts, parental sociodemographics were associated with
children exposure to drugs of abuse. The results of the three study cohorts demonstrated a
significant prevalence of unsuspected pediatric exposure to drugs of abuse which mainly
involved cocaine maintained along fifteen years in Barcelona, Spain. It was recommended to
be aware about unsuspected passive exposure to drugs of abuse in general population and
to use general or selected hair screening to disclose exposure to drugs of abuse in children
from risky environments to provide the basis for specific social and health interventions
[14619].
Serbia
Socio-economic changes that occured in the wake of dismemberment of former Yugoslavia

resulted in the appearance of social pathology, one of which was the increase in the use of
psychoactive substances. A study was carried out among 1011 elementary school children
(seventh and eighth grades) and secondary school children (all four grades) in the area of
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Belgrade from 2003 to 2004. Out of the total number 457 (45 %) were elementary school
pupils and 554 (55 %) secondary school pupils. There were 524 (52 %) boys and 487 (48 %)
girls, aged from 12 to 18 years (the average age being 15 years). The method used was the
European School Survey Project on Alcohol and Other Drugs Questionaire. Totally 14
percent examinees tried psychoactive substances. The most frequent drug used at the first
contact was marijuana (10.8 %) at the age of 15 tried by 13 percent examinees, inhalants
(4.4 %), amphetamines (4.1 %), sedatives (3.7 %), alcohol combined with marijuana (3.9 %),
then cocaine (2.8 %), heroine (2.3 %), alcohol combined with sedatives (2.2 %), and ecstasy
(1.6 %), followed by anabolic steroids, heroin, diethilamid lisergic acid (LSD) and magic
mushrooms. It was determined that going out in the evening, smoking and binge form
drinking were directly connected with the use of psychoactive substances [08030].
Croatia
Substance use and misuse (SUM) and the relation to physical activity/exercise/athletic
participation (sport factors) and scholastic achievement are rarely studied in Croatia. The aim
of this study was to investigate the SUM habits in Croatian adolescents (17-18 years of age,
254 males, and 218 females), and to study potential gender-specific interrelationships
between scholastic and sport factors in relation to SUM. The testing was done using an
extensive, anonymous, self-administered questionnaire that consisted of scholastic variables,
sport factors, and SUM data. Descriptive statistics, counts, and proportions were calculated.
Gender differences were established using the Kruskal-Wallis test. Gender-specific
correlations within and between studied variables were established using the Spearman's
correlation. The incidence of smoking habits and alcohol consumption among Croatian
adolescents was alarming, and a serious intervention program should be developed to
address this issue. Educational achievement was negatively related to SUM, with no gender-
specific relationships. The data indicated some "protective" effects of the sport factors
against SUM in boys, but a significant positive correlation between alcohol drinking and sport
participation in girls was also noted [11019].
Poland
To estimate the prevalence of anabolic-androgenic steroids (AAS) abuse among adolescent

and young adults in Poland 3,687 men (48 %) and women (52 %), median age 23
(interquartile range 19-30 years) participated in a survey via a "pop-up window" which
appeared on two popular Polish internet portals during one month. Questions concerning
their body image, exercise behaviour, education level and use of anabolic-androgenic
steroids were asked. The prevalence of anabolic-androgenic steroids use was 6.2 percent
among males and 2.9 percent among females. Male AAS users, compared to non-users,
were more often concerned about their physical appearance, were less educated and often
engaged in some sport activity. Among female AAS users, no significant differences
concerning self-body image satisfaction or participation in sports were found. However,
compared to non-users, female AAS users were less educated. It was concluded that the
abuse of AAS is a reality in Poland and may become a serious health concern among
adolescents and young adults [06027].
Europe, combined countries
Data on doping among young non-professional athletes are scarce. In order to estimate the
prevalence and predictors of doping use, a standardized, anonymous questionnaire was self-
administered by 2650 tertiary education students from five European Union countries
(Finland, France, Germany, Greece, Italy) and Israel. The reported usage rate of a doping
379
agent (at least once) was 2.6%, with no significant variation in the frequency of doping
reporting among the participating countries. Doping was, however, less common among
students of biomedical schools (OR: 0.49) and was higher among males (OR: 2.16).
Students, who use to drink coffee or recall frequent occasions of involvement in drunkenness
episodes, were more likely (twice and three times, respectively) to report doping, and
students using nutritional supplements or having participated in a major athletic event were
more likely (four times and twice, respectively) to report doping in comparison with students
who do not. Of note is the high odds ratio for reporting individual doping when having a friend
who uses doping (OR: 8.61). Given the large size of the physically active young individuals in
the population and the small number of professional athletes, doping in the general
population may be, in absolute terms, as sizeable problem as it is among the professional
athletes. There was evidence that high-risk behaviour and supplement use increased the risk
of doping [06028].
Israel
A survey on knowledge and attitudes towards the use of steroids in youth who exercise in
fitness centers was conducted in a national sample of fitness centers in Israel. A total of 528
participants aged 13-18 years completed the questionnaire. Fitness centers were chosen by
cluster samples. Forty-five percent of the respondents believe that the use of steroids
improves physical fitness and 41 percent believe that most well-known athletes use steroids.
Ten percent of respondents believe that the use of steroids is popular among young people
in gym centers. Overall, 7 percent stated that they tended to use steroids and a significantly
lower percentage were aware of the side effects of steroids. The role of the physician is
essential to identify the patients at risk, to deliver accurate information, to minimize harm and
to follow-up. There is also a need for health education in schools, fitness centers and the
community, concerning the use of steroids among young people [04016].
Jordan
One study was conducted to measure the extent of androgenic steroids abuse among two
targeted groups in Jordan, college students and athletes, and the risk factors associated with
this abuse. Five hundred and three Jordanian collegiate students and 154 bodybuilding
athletes completed a three section questionnaire that investigated demographic information,
prevalence of anabolic-androgenic steroids and attitude towards steroids abuse. Of the
investigated collegiate students, 4.2 percent were current users, while the percentage rose to
26 percent among the athletes; the mean age of users in the two groups was 20 and 28
years, respectively. Almost one-third of the students started abusing anabolic steroids before
the age of 15 years while more than half of the athletes started between the ages of 15 and
18 years. Knowing where and how to get the drugs has not been a problem for either the
students or the athletes as their friends and coaches were the major sources. The main
reasons for using anabolic steroids have been found to help improving athletic performance
and physical appearances [08029].
One study investigated the abuse of over-the-counter (OTC) products (e.g., proteins, dietary
supplements) and prescription drugs (e.g. hormones) in gymnasiums in Amman by random
distribution of a structured questionnaire to 375 gym clients (November 2012 to February
2013). Data were analyzed using SPSS for Windows (version 17.0). A total of 31 (9 %)
clients admitted to using 21 products (mentioned 71 times) of anabolic steroids and other
hormones (e.g. growth hormone and thyroxine) to increase muscular power at the gym or
build muscle mass. Abuse of different prescription and OTC drugs among gymnasium clients
380
is present in Jordan, but current methods for controlling the problem are ineffective. Better
methods should be developed [14620].
Kuwait
Considering the recent popularity of bodybuilding and the apparent spread of anabolic
androgenic steroid (AAS) use amongst bodybuilding enthusiasts in Kuwait, there is a relative
lack of scientific investigation into the use, knowledge and attitudes towards AAS amongst
the population at risk of abusing it. Therefore, one study aimed to investigate the frequency,
knowledge, attitudes and practice of AAS use amongst male fitness centre attendees in
Kuwait. A cross sectional survey utilizing a self-administered questionnaire was used.
Information on demographics as well as knowledge and attitude about and towards the use
of AAS was included in the questionnaire. Ten fitness centres in Kuwait were randomly
selected and questionnaires were distributed to all individuals leaving each centre on
randomly selected days and periods of time for each centre. Overall 400 questionnaires were
distributed. A total of 194 questionnaires were returned completed (49 %). Of the responders,
23 percent used AAS. The 19-25 age group had the highest occurrence (47 %) of first-time
AAS use. In contrast with non-users, most (71 %) of AAS users believed that having an
optimally muscular body can only be achieved by using AAS, and a small minority (7 %)
believed that AAS usage would have significant harms to health. Only 18 percent of AAS
users had appropriate knowledge regarding the side effects of AAS. Non-users were as
much uninformed as AAS users regarding the side effects of AAS. The usage of AAS is high
amongst male gym users in Kuwait and is likely to present an additional burden to the health
service. An effective initiative to minimize the burden of AAS abuse should focus on
changing the attitudes towards AAS rather than spreading awareness of their side effects
[150019].
Iraq
The majority of men's sports need high levels of strength and power. The effects of any given
type of performance-enhancing substance are mostly directly related to its ergogenic effects
(enhanced strength, higher energy production, and better recovery), anabolic potential
(increased protein synthesis, especially in muscles), and/or stimulating properties (increased
attention and loss of fear), which give a competitive advantage to athletes. A descriptive
correlational study was conducted to identify bodybuilders' and athletes' perception toward
substance use and to identify the relationship between substance use and those athletes'
sociodemographic characteristics of age, level of education, social status, and monthly
income. A purposive "nonprobability" sample of 172 bodybuilding athletes were recruited
from gym users of Baghdad city. The study found that two fifths of those who used anabolic-
androgenic steroids (AAS) were 19 years old or younger, less than one half were overweight
(body mass index 25-30), two fifths of participants enjoyed exercise/training to an extreme
level, two fifths of study participants highly perceived the improvement of athletic
performance, two fifths of the study participants highly perceived the importance of improving
athletic performance, less than half of the study participants used AAS, one quarter of the
study participants who used AAS had been influenced by their coaches to use such
substances, and more than one third of the study participants who used AAS were using
such substances in the form of oral tablets and intramuscular injection together [12027].
Iran
The high prevalence and potential side effects of anabolic-androgenic steroids (AAS) misuse
by athletes has made it a major public health concern. Epidemiological studies on the abuse
381
of such drugs are mandatory for developing effective preventive drug control programs in
sports community. One study aimed to investigate the prevalence of AAS abuse and their
association with some psycho-socio-demographic factors in Iranian male recreational body-
builders. Between March and October 2011; 906 recreational male body-builders from 103
randomly selected bodybuilding clubs in Tehran, Iran were participated in this study. Some
psycho-sociodemographic factors including age, job, average family income, family size,
sport experience (months), weekly duration of the sporting activity (h), purpose of
participation in sporting activity, mental health as well as body image (via General Health
Questionnaire and Multidimensional Body-Self Relations Questionnaire, respectively), and
history of AAS use were obtained by interviews using questionnaires. Participants were all
recreational male body-builders (mean age 26 years). Self-report of AAS abuse was
registered in 150 body-builders (17 %). Among different psycho-socio-demographic factors,
only family income and sport experience were inversely associated with AAS abuse. Lifetime
prevalence of AAS abuse is relatively high among recreational body-builders based on their
self-report. Some psycho-socio-demographic factors including family income and sport
experience may influence the prevalence of AAS abuse [150020].
One study was conducted to determine the frequency of anabolic-androgenic steroids

consumption in male students studying at the university and their awareness, attitude, and
role of sports activities; the present descriptive study was conducted on 271 volunteers in
2008. The data collected by self-report questionnaires was analyzed by descriptive inferential
statistics. The prevalence of consumption was 3.3 percent, and it was significantly higher in
those with a history of bodybuilding or athletic performance. The overall awareness rate was
low, and the attitude was too optimistic. It seems that unawareness, incorrect attitude, and
history of athletic performance increases the risk of consumption [14621].
Androgenic-anabolic steroids (AAS) are abused by a growing number of bodybuilders. This

descriptive cross-sectional study was conducted to determine prevalence and patterns of
AAS use by bodybuilders in Hamadan, Western Iran. In this cross-sectional study,
participants were recruited from five gym clubs in two area of Hamadan (a total of 10 clubs).
Twenty-five bodybuilders from each club were administered. Questions investigating
demographic information, sport history, education level, general knowledge about AAS, and
their side effects were asked. The frequency of AAS use was 29 percent (72/250). Fifty-four
percent of users were 25 years or younger. AAS abuse showed a significant association with
duration of exercise. The drugs were suggested mostly from peers (43 %) and coaches (36
%). The most commonly consumed anabolic steroid was testosterone (67 %). The most
commonly reported AAS side effect was acne (18 %). There was not significant association
between general knowledge about side effects of ASS and their use. The results of current
survey indicate that frequency of ASS use is high in adolescents and young adult
bodybuilders. Well educated bodybuilders have a higher prevalence of abuse. Awareness
about the side effects of drugs is not deterrent factor for their abuse. Iranian Ministry of Sport
and the Youth, and the National Council for Youth, should be urged to conduct more effective
prevention strategies [14028].
Korea
Athletes report frequent use of various dietary supplements (DSs). The objectives of one
study were to obtain information about Korean Olympians' DS use during the training period
for the Beijing 2008 Summer Olympic Games and immediately before their Olympic events,
to obtain DS-intake reasons and DS providers, and to obtain information on athletes' doping
education, knowledge, and educators. Korean Olympians completed 2 questionnaires 1
week before the opening and within 1 week after the closing of the Beijing 2008 Summer
Olympic Games. Results showed that 79 percent of male and 82 percent of female
382
Olympians take more than 1 dietary supplements during the training period and that vitamins
and Oriental supplements are the 2 top-ranked DSs. Reasons for dietary supplements use
were to improve recovery ability (66 %) and muscle performance (22 %), and sources of
obtaining dietary supplements were parents (36 %) and coaches (35 %). Furthermore, 79
percent of Korean Olympians reported receiving regular education on antidoping regulations
from Olympic-sponsored education classes (64 %) and coaches (15 %) [11021].
USA
The use of drugs and supplements to enhance performance has become a part of
mainstream athletics. Many team physicians and sports medicine practitioners are unfamiliar
with the benefits and risks of these products and thus are unable to educate young athletes
on this topic. In spite of numerous reports on the health risks of anabolic steroid use, 1 to 3
million Americans have used them. Human growth hormone has been tried by up to 5
percent of 10th graders, although no scientific study has shown that it is an effective
performance-enhancing drug. Amphetamines and similar compounds may be the most
widely abused drug in baseball; recently, they have come under increased scrutiny in sport.
Erythropoietin is a highly effective aerobic enhancer that has been linked to multiple deaths
in cyclists and other endurance athletes. The neutraceutical industry, led by supplements
such as creatine, ephedra, and androstenedione, remains unregulated by the Food and Drug
Administration and has serious issues with quality and side effects. An understanding of
these products is essential for the sports medicine practitioner to provide sound, safe advice
to the athlete [04017].
Drug Abuse indicated that there were more than 1 million anabolic-androgenic steroid (AAS)
users in the United States and that the lifetime use was 0.9 percent for males and 0.1
percent for females. Despite the fact that AASs were added to the list of Schedule III
Controlled Substances in 1990, recent data suggest that AAS use has increased. Current
estimates (2004) indicate that there are as many as 3 million AAS users in the United States
and that 2.7 to 2.9 percent of young American adults have taken an AAS at least once in
their lives. Surveys in the field indicate that AAS use among community weight trainers
attending gyms and health clubs is 15 to 30 percent. Furthermore, two thirds of AAS users
are noncompetitive recreational body builders or nonathletes, who use these drugs for
cosmetic purposes rather than to enhance sports performance. An estimated 10 percent of
AAS users are teens, and the prevalence of AAS use among American adolescents is 3 to
12 percent in males and 0.5 to 2 percent in females. Approximately 375,000 adolescent
males and 175,000 females have used an AAS at least once during their lives. Surveys
indicate that ASS use among National Collegiate Athletic Association athletes is
approximately 5 to 14 percent. An estimated 4 million American men take doctor-prescribed
testosterone replacement therapy in 2004 [04018].
Although AAS use is widespread in Western countries, the US appears to have the largest
absolute number of AAS users. This is not surprising since the US is the most populous
country with substantial AAS use, and likely the first country in which AAS use began to
spread from elite athletics to the general population. A recent study based on data from
American surveys of school and youth populations used mathematical models to generate
estimates of the lifetime prevalence of AAS use in the US (this value should technically be
called the “cumulative incidence,” although the term “lifetime prevalence” is generally used in
studies of substance abuse and other psychiatric disorders). Important to note, this study
took into account the fact that anonymous surveys of American high school students almost
always overestimate the prevalence of AAS use because students erroneously answer that
they have used “steroids” when in fact they have used corticosteroids, rather than actual
383
anabolic-androgenic steroids, or have used over-the-counter supplements that the students
incorrectly believe are “steroids”. After adjusting for this source of bias, and applying the
mathematical models, the analysis produced an estimate that 2.9 to 4.0 Americans have
used AAS at some time in their lives. The AAS users at greatest risk for adverse effects are
likely those who develop AAS dependence and accumulate many years of AAS exposure.
Therefore, this same study sought to estimate the number of Americans who had
experienced AAS dependence. To do so, the investigators combined the data from 10
studies that collectively diagnosed AAS dependence in 1,248 AAS users. Applying a random
effects model to these 10 studies, the analysis yielded an estimate that 33 percent (95 %
conference interval 25 to 40 %) of AAS users develop AAS dependence. Applying this
proportion to the above estimates of the overall American AAS-using population, it follows
that in the US alone, about 1 million men have experienced AAS-dependence at some time.
As noted in the analysis, virtually all of these AAS-dependent individuals are likely to be
male, since only two of the 363 cases of AAS dependence found in the 10 pooled studies
described above were female. Thus, the lifetime prevalence of AAS dependence in American
men is likely in the same general range as that of HIV infection or of type I diabetes, both of
which afflict less than 1 million American men [14017].
Although various surveys have tracked the prevalence of anabolic-androgenic steroid (AAS)
use in American teenagers and young adults, no recent surveys have assessed the lifetime
prevalence of AAS use in Americans overall. It was therefore analyzed serial youth-survey
data to derive estimates of the lifetime prevalence of AAS use in the current American
general population. It was first determined the distribution of age of onset of AAS use, based
on pooled data from nine studies. Using this distribution, we then developed equations to
project the eventual lifetime prevalence of AAS use among young survey respondents, once
they aged and completed the period of risk for initiating AAS. It was similarly calculated the
denominator of lifetimes of risk for AAS use in the total American population. It was next
applied these equations to four independent national youth datasets to derive current
American general-population estimates for lifetime AAS use. Finally, using data from 10
pooled studies, it was estimated the lifetime prevalence of AAS dependence among AAS
users. Age-of-onset studies consistently showed that AAS use begins later than most drugs,
with only 22 percent of users (95 % confidence interval 19 to 25 %) starting before age 20.
Applying the age-of-onset findings to national youth datasets, it was estimated that among
Americans currently age 13-50 years, 2.9-4.0 million have used AAS. Within this group,
roughly 1 million may have experienced AAS dependence. Although subject to various
limitations, the estimation techniques suggest a surprisinigly high prevalence of AAS use and
dependence among Americans [13030].
Because 57 percent of all high school students play on formal sports teams, the use of both
illicit and legal ergogenic drugs to enhance performance in amateur athletics is of significant
concern today. Furthermore, up to one third of high school students who use anabolic
steroids are in the population of nonathletes who use steroids to improve their appearance.
Drug and supplement use is not uncommon today. It is estimated today that 1 to 3 million US
athletes are taking steroids, and 2500 tons of creatine were consumed in 1999. Many
substances are being used by today's youths, commonly without recognizing any risks of
such drugs. Even as this problem increases, there are concerns that pediatric residents are
receiving minimal education in the field of sports medicine during both medical school and
residency. Although the focus of pediatric sports medicine is typically proper training and
managing common injuries, an emerging issue is learning about the drugs that are chosen
by young athletes to improve athletic performance [06003].
The US National Institute on Drug Abuse has called for increased research into the use of
physical activity in substance abuse prevention, specifically research into physical activity
384
type and context. One paper examined the relationships between secondary school student
substance use and exercise in general and school athletic team participation, and examines
such relationships over time. Nationally representative cross-sectional samples of 8th-, 10th-,
and 12th-grade students were surveyed each year from 1991 to 2009. Substance use
measures included past 2-week binge drinking and past 30-day alcohol, cigarette, smokeless
tobacco, marijuana, and steroid use. Analyses were conducted during 2009-2010. Across
grades, higher levels of exercise were associated with lower levels of alcohol, cigarette, and
marijuana use. Higher levels of athletic team participation were associated with higher levels
of smokeless tobacco use and lower levels of cigarette and marijuana use across grades and
to higher levels of high school alcohol and steroid use. Exercise helped suppress the
undesired relationship between team participation and alcohol use; exercise and athletic
team participation worked synergistically in lowering cigarette and marijuana use. Observed
relationships were generally stable across time. There appear to be substantive differences
between exercise and team sport participation in relation to adolescent substance use.
These findings from cross-sectional data suggest that interventions to improve levels of
general physical activity should be evaluated to determine if they help delay or reduce
substance use among youth in general as well as among student athletes [11020].
Data on the actual prevalence of AAS use are limited by recall and reporting biases
associated with illicit drug use. Many studies of AAS use focus on adolescents. In a
nationwide study of 3403 12th-grade male students in 46private and public US high schools
during the 1980s, 6.6 percent of the respondents admitted to everusing AASs based on their
response to questionnaires. The individual participation rate was low (50 %). Almost 40
percent of the respondents admitted to the administration of 5 or more cycles of AASs.
Further studies performed at local and statewide eleves have confirmed similar findings
indicating that 3-12 percent of high school male adolescents use AASs at some time in their
lives. These studies indicate that about one-third of the high school students use AASs for
appearance rather than athletic performance. In 1999, the National Institute on Drug Abuse
Monitoring the Future study indicated that approximately 2.7 percent of US 8th and 10th
graders and 2.9 percent of 12th graders admitted to the use of AASs at least once. For 10th
graders, the prevalence of AAS use increased to 2.7 percent in 1999 from 2.0 percent in
1998. For all 3 grades, the 1999 levels represented a significant increase from 1991, when
1.9 percent of 8th graders, 1.8 percent of 10th graders, and 2.1 percent of 12th graders
admitted AAS use at least once. The data on adolescent use of AAS in 2004 and 1999 were
similar with 2.5 percent of 12th graders reporting the use of AASs at least once. Risk factors
for the use o fAASs among adolescents include male gender, participation in strength-related
sports, and use of other illicit drugs. In college and professional athletics, the prevalence of
AAS abuse is less defined because of the ramification of AAS use. However, the prevalence
of AAS use among these groups is probably substantially higher than in high school
students, particularly in football players and male track-andfield-athletes. Between 1972 and
2000, there were 29 athletes disciplined for positive AAS drug tests. However, the abuse of
AASs is probably more widespread among these athlete sbecause of the difficulty detecting
all AAS use and the small number of doping charges compared with the number of positive
drug tests. Current trends suggest that most AAS abusers are non-athletes using these
drugs for cosmetic purposes [13003].
Statistics reporting adolescent use of ergogenic agents are staggering. According to the
Centers for Disease Control and Prevention Youth Risk Behavior Surveillance, 6.1 percent of
students from grades 9 through 12 had taken illegal anabolic steroids without a prescription
one or more times during their lifetime. Additionally, more adolescent athletes are using non-
Food and Drug Administration-regulated herbal supplements that claim ergogenic benefits.
Many athletes either are unaware of or do not consider the possible health risks caused by
these agents. School-based programs for athletes may be successful in preventing the use
385
of ergogenic agents by increasing knowledge about such agents and offering skills in critical
thinking to improve decision-making skills [05014].
Substance abuse is a major public health concern. Among healthcare professionals involved
in sports and exercise, a specific goal is to reduce steroid use among adolescents. According
to the Healthy People 2010 report, the estimated percentage of US male high school seniors
using anabolic-androgenic steroids (AAS) declined from 4.7 percent in 1989 to 4.1 percent in
1997. Estimates for 1998 were lower: 2.8 percent for male and 0.3 percent for female high
school seniors. Some studies have suggested that the actual usage rates are higher than
published ones because of underreporting [00009].
Use in women
An extensive literature has documented the physiological and psychological effects of
anabolic-androgenic steroids (AAS) in men, but little has been written about the effects of
these drugs in women. However, substantial numbers of women in the US abuse AAS. For
example, data from the 1993 National Household survey suggest that 145,000 American
women have abused AAS at some time in their lives (95 % confidence interval 64,000 to
830,000). Studies of high school populations have produced much higher estimates, with
0.5-3.2 percent of high school girls reporting that they had used AAS at least once. These
findings imply that AAS use among women, once restricted only to a small number of elite
athletes, may now be evolving into a commoner public health problem in the US. Although
numerous studies have documented the psychiatric and physiological effects of anabolic-
androgenic steroids (AAS) in males, virtually no studies have investigated the effects of illicit
AAS use in women. It was a performed psychiatric and medical evaluations of 75 dedicated
women athletes, recruited by advertisement primarily from gymnasiums in the Boston, Mass.,
area. Twenty-five (33 %) of the women reported current or past AAS use. Users were more
muscular than nonusers and reported use of many other “ergogenic” (performance-
enhancing) drugs in addition to AAS. Some described a frank syndrome of ergogenic
polysubstance dependence, often with significant morbidity. Fourteen (56 %) of the users
reported hypomanic symptoms during AAS use and 10 (40 %) reported depressive
symptoms during AAS withdrawal, but none met full DSM-IV criteria for a hypomanic or
major depressive episode. Nineteen (76 %) users reported at least one adverse medical
effect associated with AAS use. Perhaps the most interesting findings were several unusual
psychiatric syndromes reported by both the AAS users and nonusers. These included rigid
dietary practices (which we have termed “eating disorder, bodybuilder type”), nontraditional
gender roles and chronic dissatisfaction and preoccupation with their physiques (a syndrome
which we have termed “muscle dysmorphia”). It was concluded that dedicated women
athletes exhibit not only AAS abuse, but use of many other ergogenic drugs, sometimes
associated with significant morbidity. In addition, these athletes frequently display several
psychiatric syndromes which have not previously been well described [00010].
Self-perceived weight and anabolic steroid misuse among US adolescent boys

Anabolic steroid misuse is a growing concern among adolescent boys, and chronic misuse is
associated with multisystemic health consequences. However, little is known about weight
related predictors of anabolic steroid misuse. It was examined the prediction of lifetime
anabolic steroid misuse as a function of self-perceived weight status among US adolescent
boys. Analysis was undertaken using the 2013 Youth Risk Behavior Survey, a nationally
representative data set sampling public and private high school students throughout the
United States. Data from a total of 6,000 US adolescent boys were used in the present study.
The prevalence of ever misusing anabolic androgenic steroids was 12.6 percent among boys
who viewed themselves as very underweight, 11.9 percent for boys who viewed themselves
as very overweight, compared with 3.8 percent for boys who viewed themselves as about the
right weight. Compared to boys who viewed themselves as about the right weight, boys who
386
self-perceived themselves as very underweight (adjusted odds ratio 6.9, 95 % confidence
interval: 2.7 to 17.7) and very overweight (adjusted odds ratio 3.8, 95 % confidence interval:
1.8 to 7.7) were significantly associated with increased risk of anabolic androgenic steroid
misuse. Thus, large effect size estimates were revealed, suggesting that anabolic androgenic
steroid misuse is not solely a function of boys desiring increased mass; boys who desire
leanness are also likely to misuse anabolic androgenic steroids. Future prevention efforts
should target not only boys who view themselves as underweight but also those who
perceive themselves as overweight [150018].
Age- and activity dependence

To test the comparative value of strain theory and problem behavior theory as explanations
of adolescent anabolic steroid use, this study examined gender-specific relationships among
steroid use, physical activity, and other problem behaviors. Based on the United States
Centers for Disease Control and Prevention's 1997 Youth Risk Behavior Survey, a nationally
representative sample of over 16,000 U.S. public and private high school students, binge
drinking, cocaine use, fighting, and sexual risk-taking were associated with higher odds of
lifetime steroid use. In gender-specific analyses, steroid use was strongly associated with
female fighting and smokeless tobacco use as well as male sexual risk. Neither athletic
participation nor strength conditioning predicted odds of steroid use after controlling for
problem behaviors, nor did steroid-using athletes report more frequent use than steroid-using
nonathletes. The study's limitations and policy implications were noted. These data suggest
that other problem behaviors such as substance use, fighting, and sexual risk are better
predictors of adolescent steroid use than physical activity. Interventions to prevent steroid
use should not be limited to male participants in organized sports programs, but should also
target adolescents identified as at risk for other problem behaviors [05015].
In the US it has been demonstrated that 94 percent of the 1,955 adult AAS users began after
the age of 18 years with an overwhelming number being whites in their late 20s-30s with a
slightly above-average socioeconomic status. These men were self-reported perfectionists
and highly goal-oriented [14427].
Project EAT
A study was performed to explore the prevalence and correlates of using steroids for the
purpose of gaining muscle among adolescent males and females. More specifically, the
study objectives were to: (a) assess the prevalence of anabolic steroid use in a large
population-based sample of adolescent males and females; and (b) identify personal, socio-
environmental, and behavioral factors related to the health of adolescents that use anabolic
steroids. One study population included 4746 middle and high school students from St.
Paul/Minneapolis public schools who completed surveys and anthropometric measurements
as part of a population-based study of eating patterns and weight concerns among teenagers
(Project EAT: Eating Among Teens). Steroid use and a range of socio-environmental,
personal, and behavioral factors hypothesized to be correlates of steroid use were assessed.
Associations were examined separately among males and females through comparisons of
means (unadjusted and adjusted for grade-level and race) for continuous variables, and
percentages, and odds ratios for dichotomized variables. Steroid use was more common in
males (5.4 %, vs 2.9 % of females), non-Caucasians (especially Hmong, a subset of the
Asian-American population), and in middle school students (as compared to high school). In
males, steroid use was associated with poorer self-esteem and higher rates of depressed
mood and attempted suicide, poorer knowledge and attitudes about health, greater
participation in sports that emphasize weight and shape, greater parental concern about
weight, and higher rates of disordered eating and substance use. Among females, steroid
use was less consistent in its associations with other variables, although overall, a similar
pattern of results emerged. It was concluded that anabolic steroid use in adolescence is
387
associated with poorer health-related attitudes and behaviors and exposure to socio-
environmental influences encouraging weight preoccupation. Attention needs to be directed
toward youth who may be at increased risk for steroid use within clinical and community-
based settings [02005].
Prevalence of use
Although AAS use is widespread in Western countries, the United States appears to have
the largest absolute number of AAS users. This is not surprising because the United States
is the most populous country with substantial AAS use, and likely the first country in which
AAS use began to spread from elite athletics to the general population. A recent study based
on data from American surveys of school and youth populations used mathematical models
to generate estimates of the lifetime prevalence of AAS use in the United States (this value
should technically be called the cumulative incidence, although the term lifetime prevalence
is generally used in studies of substance abuse and other psychiatric disorders). Important to
note, this study took into account the fact that anonymous surveys of American high school
students almost always overestimate the prevalence of AAS use because students
erroneously answer that they have used steroids when in fact they have used corticosteroids,
rather than actual AASs, or have used over-the-counter supplements that the students
incorrectly believe are steroids. After adjusting for this source of bias and applying the
mathematical models, the analysis produced an estimate that 2.9 to 4.0 Americans have
used an AAS at some time in their lives. The AAS users at greatest risk for adverse effects
are likely those who develop AAS dependence and accumulate many years of AAS
exposure. Therefore, this same study sought to estimate the number of Americans who had
experienced AAS dependence. To do so, the investigators combined the data from 10
studies that collectively diagnosed AAS dependence in 1248 AAS users; we also included a
recently published paper that tabulates these studies [14426].
Applying a random-effects model to these 10 studies, the analysis yielded an estimate that
33 percent (95 % conference interval, 25 %-40 %) of AAS users develop AAS dependence.
Applying this proportion to the above estimates of the overall American AAS-using
population, it follows that in the United States alone, about 1 million men have experienced
AAS dependence at some time. As noted in the analysis, virtually all of these AAS-
dependent individuals are likely to be male, because only 2 of the 363 cases of AAS
dependence found in the 10 pooled studies described above were female. Thus, the lifetime
prevalence of AAS dependence in American men is likely in the same general range as that
of HIV infection or of type 1 diabetes, both of which afflict fewer than 1 million American men
[14426].
The use of PEDs is not limited to the United States. High rates have been consistently
documented in Scandinavia, Brazil, and British Commonwealth countries and more recently
in continental Europe. By contrast, AAS use is rare in East Asian countries such as China,
Korea, and Japan, perhaps because these cultures place less emphasis on male
muscularity, as explained in recent reports [14426].
Origin of AAS in the United States

AASs are DEA schedule III drugs of the Controlled Substances Act. The primary sources for
illicit AASs in the United States are manufacturing facilities in Mexico. Traffickers from the
United States enter Mexico and purchase AASs at pharmacies in Baja, California, where
AASs are available by prescription. They subsequently smuggle the AASs back in to the
United States. Other sources of illicit AAS in the United States include eRussia, Poland,
Hungary, Spain, Italy, Greece, Canada, and the Netherlands as well as some clandestine
labs in the United States. The diversion of legitimate pharmaceutic preparations provides a
much smaller portion (i.e.10-15 %) of illicit AASs compared with other illicit sources.
388
Information on the administration and procurement of AASs is widely available on the
Internet [13003].
Australia
There is evidence to suggest that the prevalence of anabolic-androgenic steroids (AAS) is

higher among young people than the general population. The purpose of one study was to
examine the proportion of students who reported lifetime and past-year AAS use, explore
other drug use among those who reported AAS use, and investigate demographic correlates
of AAS use. Data was taken from a cross-sectional survey of a representative sample of
Australian secondary students. A stratified two-stage probability sampling methodology was
employed and schools were randomly sampled from each Australian State and Territory. A
total of 376 schools participated in the survey. Lifetime AAS use was reported by 2 percent of
12-17-year-old students; use was more common among 12-15-year olds then 16-17-year
olds. Regardless of age, being male, speaking a language other than English at home, not
be at school on the previous school day, and rating own scholastic ability as below average
were all associated with a greater likelihood of using AAS in their lifetime and in the past
year. Those who reported AAS use also reported the use of a range of other substances,
suggesting that AAS use may be part of a broader experimentation with substances.
Interventions towards these groups regarding AAS may best be placed within a larger
substance use intervention rather than being AAS-specific. In light of the low levels of AAS
use among this group, more detailed research into AAS use among adolescent sporting
groups may be warranted [11017].
One study aimed to investigate the prevalence of illicit drug use among elite Australian
athletes with a focus upon cannabis, ecstasy, meth/amphetamine, cocaine, GHB and
ketamine; explore perceptions concerning the extent of drug use among this group; ascertain
opinions regarding specific drugs of concern; and investigate predictors of recent drug use.
Data were taken from surveys with 974 elite athletes. One-third of the sample had been
offered or had the opportunity to use illicit drugs in the past year; despite this, the self-
reported prevalence of all six drugs under investigation was lower than that reported by the
general population. Sixteen percent of athletes believed that there was a drug of concern in
their sport, with ecstasy, cocaine and alcohol being nominated. Knowing other athletes who
use illicit drugs, being offered or having the opportunity to use drugs and identifying as a “full-
time athlete” were significant predictors of recent drug use. The study found that one-third of
the athlete sample had been offered or had the opportunity to use illicit drugs in the past
year; despite this, there was low self-reported drug use. Despite media discussion regarding
alcohol use in sport, alcohol was nominated as a drug of concern only by a small proportion
of athletes, and further research investigating this issue may be warranted [11018].
Brazil
One study aimed to determine through a questionnaire applied to interviewers, the current or
past use of anabolic androgenic steroids (AAS), as well as other hormones, and other
medicines, food supplement and illicit drugs among strength training apprentices in the city of
Porto Alegre, RS. It was interviewed 288 subjects draw from a sample of 13 gyms. The
prevalence of current and past use of AAS was about 11 percent (32/288), other hormones 5
percent (16/288) and other medicine 4 percent (12/288). The most used AAS were
nandrolone and stanozolol; the other hormones were gonadotropin, triiodothyronine (T3).
The most frequent side-effects were behavioral such as humor oscillation, irritability and
hostility, and endocrine disturbances such as acne and increased or decreased libido. When
analyzed together with other hormones in a variable named "hormonal agents" (AH), AAS
389
presented a statistical difference among genders considering that the most frequent use of
hormones occurred among men and those who consume food supplements. The comparison
of these findings to other national and international results is difficult due to the
epidemiological design. Even if it is considered, the observed prevalence suggests that
preventive attitudes as well as special care in the orientation and education of this population
must be taken [07036].
The use of anabolic-androgenic steroids (AAS) is increasing among practitioners of

recreational physical activity. The aim of this research was to evaluate the prevalence of AAS
in practitioners of recreational physical activity in Brazil. After systematic review of four
databases, 14 articles were included. The results indicate that the prevalence of AAS varied
between 2 and 32 percent, according to the region analyzed and the sample characteristics
[14436]
Cameroon
To investigate use and awareness of lawful and unlawful substances by amateur footballers
in Yaounde, Cameroon, a total of 1116 amateur footballers (1037 male and 79 female) out of
1500 contacted participated in this study. They were divided into three groups: elite players
(n=314); local players (n=723); female players (n=79). They answered a questionnaire of 30
items grouped under six main topics: identification of players; use of lawful substances
subject to certain restrictions on the International Olympic Committee (IOC) list; use of IOC
banned substances; doping behaviour; awareness of doping; food supplements. Use by the
footballers of a banned substance (cocaine) and substances subject to certain restrictions
(alcoholic drinks, methylated spirits, and banga (marijuana)). Doping behaviour: use by the
footballers of substances with similar effects to some IOC banned substances but not listed
as such: tobacco, liboga, wie-wie (narcotic), bilibili (locally made alcohol drink). A large intake
of vitamin C (food supplements) in all three groups. In contrast, the footballers' knowledge of
doping was vague [03017].
Ghana
One study is a pioneering exploration of nonmedical anabolic-androgenic steroid (AAS) use

among Ghanaian high school students. Of 2683 students contacted, 2597 (1412 females)
participated in a survey (response rate 97 %). Participants (age range 11-35 years) provided
information on demographics, sports participation, and nonmedical AAS use. The overall
lifetime prevalence of use was 3.8 percent (males 4.9 %, females 3.1 %). Moreover, 18.5
percent reported having an acquaintance that has used AAS while 6.0 percent of the sample
had previously been offered AAS. However, none of the AAS users provided a valid name of
the AAS they had used. Use and intent to use AAS was also significantly higher among
males, teenagers (versus over 19-year-olds), athletes (versus recreational sportspeople, and
nonathletes), and participants in ball games (versus other sports). Female gender, parental
absence, religiosity, and participation in jogging had significant positive association with AAS
use attitudes whereas participation in martial arts, and swimming had significant negative
association with AAS use attitudes. It was concluded that the high prevalence of use and
intent to use AAS among Ghanaian high school students should be of concern to authorities
[14718].
Ghanaian high school students

One study is a pioneering exploration of nonmedical anabolic-androgenic steroid (AAS) use
among Ghanaian high school students. Of 2683 students contacted, 2597 (1412 females)
participated in a survey (response rate 96.8 %). Participants (age range 11-35 years,
390
M=17.2) provided information on demographics, sports participation, and nonmedical AAS
use. The overall lifetime prevalence of use was 3.8 percent (males 4.9 %, females 3.1 %).
Moreover, 18.5 percent reported having an acquaintance that has used AAS while 6.0
percent of the sample had previously been offered AAS. However, none of the AAS users
provided a valid name of the AAS they had used. Use and intent to use AAS was also
significantly higher among males, teenagers (versus over 19-year-olds), athletes (versus
recreational sportspeople, and nonathletes), and participants in ball games (versus other
sports). Female gender, parental absence, religiosity, and participation in jogging had
significant positive association with AAS use attitudes whereas participation in martial arts,
and swimming had significant negative association with AAS use attitudes. It was concluded
that the high prevalence of use and intent to use AAS among Ghanaian high school students
should be of concern to authorities [150021].
Ivory coast
It was conducted a survey of doping among soccer players in Côte d'Ivoire with a
representative sample of 150 soccer players who filled out an anonymous questionnaire. The
aim of this survey was to get a clearer picture of doping in Ivorian soccer in order to suggest
preventive actions against doping. The results of this study showed that doping was known
by the Ivorian soccer players; about 19 percent admitted to the use of doping substances, 42
percent recognised that they felt tempted by doping, while 38 percent knew another soccer
player who had already used a doping substance. Government and sports organisations
should recognize the importance of education and information in the antidoping campaign
and agree on effective preventive as well as repressive strategies [02007].
Different sports
Prevalence of use depending on type of sports
Prior research shows that college athletes have higher rates of substance use, especially
alcohol, than do college students who are not involved in athletics. To augment the literature,
the author sought to determine which sports/teams are at the greatest risk for substance use.
The author used data from the 1999 Harvard School of Public Health College Alcohol Study,
a national survey of college and university students in the United States. Findings indicated
that male hockey and female soccer athletes were the most likely to report substance use
and that male basketball and cross-country/track athletes reported lower levels of substance
use. There is variation in substance use on the basis of sport/team affiliation, and future
researchers should examine why certain groups of athletes have higher rates of substance
use [07037].
Correlation to selected socio-demographic, health-related, and sports-related predictors
Conducted researches recognize various risk factors, as well as protective factors against
doping behaviour in different sports i.e. sports disciplines or activities. The main goal of one
research was to identify the correlation between selected socio-demographic, health-related,
and sports-related predictors with doping factors in three different types of sports, which are
(1) highly energetic demanding sports (weightlifting), (2) highly technical demanding sports
(racquet sports), and (3) highly tactical demanding sports (sailing). The research consisted of
three separate studies, each one of them researching one of the sports. The sample of
subjects included altogether 293 athletes, senior level competitors (older than 18 years of
age). In total, the sample comprised three homogenous sub-samples, as follows: athletes in
highly energetic demanding sports (weightlifters and power lifters; n=27), athletes in highly
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technical demanding sports (table tennis, tennis and badminton players; n=188), and athletes
in highly tactical demanding sports (sailing; n=78). The first study involved weightlifters where
it should be pointed out the existence of high doping behaviour In this study, religiousness
was interpreted as the most significant protective factor against doping behaviour, while
sports factors are not found to be significantly related to doping. The study involving racquet
sport athletes suggests a high risk of doping behaviour among those athletes who observe
doping behaviour in their sport. It was noticed low levels of athletes' trust in their coaches'
and physicians' opinions on doping issues. This is an issue which should be researched in
the future, because the underlying cause has not been studied as yet. Briefly, it seems that
either the athletes are not convinced of their coaches'/physicians' expertise regarding doping
issues, and/or they do not believe in their good intentions. It is particularly important, as the
previous research has shown that with the increased trust in coaches and physicians, the
chance that an athlete will use doping decreases. As expected, it is characteristic for sailing
that it has a low likelihood of potential doping behaviour, although the consumption of dietary
supplements is high. Substance abuse in sports spreads beyond those that enhance athletic
performance. All of these issues should be studied in more detail in the future and, if
appropriately validated, incorporated into anti-doping intervention programs [13031].
Professional ballet dancers
One study investigated substance use and misuse among 16 female and 9 male Croatian
ballet professionals in 2008 using an original questionnaire. It was analyzed social, personal,
activity- and training-related, and educational factors, and criteria such as: binge alcohol
drinking, cigarette smoking, appetite suppressant consumption, analgesic use, and actual
and potential "doping" habits. Frequency tables and rank-order correlation were calculated.
More than one third of the male dancers reported binge drinking, while 20 percent of the
females smoked more than a box of cigarettes per day. Almost 25 percent of these dancers
would use "doping" if it will ensure successful ballet performance, regardless of negative
health consequences. In males, the risk of potential "doping" behavior increased with age. In
females, education level was negatively related to cigarette smoking, but positively correlated
to potential "doping" habits and behavior. In both genders, religiousness was the factor
negatively related to potential "doping" behavior and belief that "doping" exists in
professional ballet. Results suggest that there is evident need for more specific medical
and/or psychological services in professional ballet [10318].
Track and field
To document the use of prescribed medication and nutritional supplements in female and
male junior, youth, and adult track and field athletes depending on their sports discipline
analysis of 3 887 doping control forms undertaken during 12 International Association of
Athletics Federations World Championships and 1 out-of-competitions season in track and
field was performed. There were 6 523 nutritional supplements (1.7 per athlete) and 3 237
medications (0.8 per athlete) reported. Nonsteroidal anti-inflammatory drugs (NSAIDs; 0.27
per athlete, n=884), respiratory drugs (0.21 per athlete, n=682), and alternative analgesics
(0.13, n=423) were used most frequently. Medication use increased with age (0.33 to 0.87
per athlete) and decreased with increasing duration of the event (from sprints to endurance
events; 1.0 to 0.63 per athlete). African and Asian track and field athletes reported using
significantly fewer supplements (0.85 vs 1.93 per athlete) and medications (0.41 vs 0.96 per
athlete) than athletes from other continents. The final ranking in the championships was
unrelated to the quantity of reported medications or supplements taken. Compared with
middle-distance and long-distance runners, athletes in power and sprint disciplines reported
using more NSAIDs, creatine, and amino acids, and fewer antimicrobial agents. It was
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concluded that the use of NSAIDs in track and field is less than that reported for team-sport
events. However, nutritional supplements are used more than twice as often as they are in
soccer and other multisport events; this inadvertently increases the risk of positive results of
doping tests [10018].
Football
Big sports events like the 2008 European Football Championship are a challenge for anti-
doping activities, particularly when the sports event is hosted by two different countries and
there are two laboratories accredited by the World Anti-Doping Agency. This challenges the
logistics of sample collection as well as the chemical analyses, which must be carried out
timeously. In the paper it was discussed the handling of whereabouts information for each
athlete and the therapeutic use exemption system, experiences in sample collection and
transportation of blood and urine samples, and the results of the chemical analysis in two
different accredited laboratories. An overview of the analytical results of blood profiling and
growth hormone testing in comparison with the distribution of the normal population was also
presented [09033].
English professional football

To examine several issues related to drug use in English professional football. More
particularly the project sought to gather data on: players' use of permitted supplements
(mineral and vitamin pills and creatine); whether they sought advice, and if so from whom,
about their use of supplements; their experience of and attitudes towards drug testing; their
views on the extent of the use of banned performance enhancing and recreational drugs in
football; and their personal knowledge of players who used such drugs. With the cooperation
of the Professional Footballers Association (PFA), reply paid postal questionnaires were
delivered to the home addresses of all 2863 members of the PFA. A total of 706
questionnaires were returned, a response rate of just under 25 percent. Many players use
supplements, although almost one in five players does so without seeking qualified
professional advice from anyone within the club. Blood tests are rarely used to monitor the
health of players. One third of players had not been tested for drugs within the preceding two
years, and 60 percent felt that they were unlikely to be tested in the next year. The use of
performance enhancing drugs appears to be rare, although recreational drugs are commonly
used by professional footballers: 6 percent of respondents indicated that they personally
knew players who used performance enhancing drugs, and 45 percent of players knew
players who used recreational drugs. Thus, there is a need to ensure that footballers are
given appropriate advice about the use of supplements in order to minimize the risk of using
supplements that may be contaminated with banned substances. Footballers are tested for
drugs less often than many other elite athletes. This needs to be addressed. The relatively
high level of recreational drug use is not reflected in the number of positive tests. This
suggests that many players who use recreational drugs avoid detection. It also raises doubts
about the ability of the drug testing programme to detect the use of performance enhancing
drugs [05005].
Tennis
The sport of tennis has had its own experiences regarding nutritional supplements and
doping. Bohdan Ulihrach and Greg Rusedski are rare examples of athletes who tested
positive in a doping case, but were exonerated because they might have used contaminated
supplements. The complexity in these cases lies in the fact that the source of their positive
tests might have been supplied by the testing authority itself: the Association of Tennis
Professionals (ATP). The tribunals, special enquiries, and task forces that studied these
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cases all named the nutritional supplements provided by the ATP organisation as the most
likely source of the nandrolone metabolite. However, many minerals and vitamins that were
available on the ATP tour have actually been tested, and the true source has never been
confirmed. Because there were more (anonymous) cases within the ATP that were all linked,
as demonstrated by an analytical anomaly noticeable in the mass spectrogram of the urine
analysis, all of these tennis players have been cleared. Such exoneration by an anti‐doping
tribunal is extremely rare [07025].
Table tennis
Substance use and misuse was studied in athletes competing at the Slovenian Nationals
2008-2009 (responding rate was 100 percent; 50 males and 29 females; aged 18 years or
older). The anonymous questionnaire for studying the use and corresponding educational,
socio-demographic, and sport factors was used. More than 90 percent of all the athletes
included in our study do not rely on coaches' and/or physicians' opinion regarding nutritional
supplements and doping. Chi-square test revealed higher prevalence of binge drinking,
cigarette smoking, and potential doping behavior in males. In both genders, with the
advancement of the sport status, the probability for potential doping behavior increases
[10022].
Combined racket sports
Furthermore, it is apparent from annual lab statistics that the doping-testing programmes
concentrate the analyses on Olympic rather than in Non-Olympic sports, and on sports which
are already associated with doping (e.g. physically demanding sports). For example, in 2009,
26,593 urine and blood samples from track and field athletes yielded 398 total findings of
suspicious substances. At the same time, 467 tests of curling athletes resulted in only 14
total findings. The higher absolute number of adverse or atypical analytical findings in “highly
physically demanding” track and field athletics than in “less-demanding” curling seems
unsurprising (398 vs14). But, the surprising pattern is evident for aquatics (13,995 total
samples; 156 total findings, or 0.65 % of samples) in comparison to shooting (24/2,630; 0.91
% of samples) or archery (14/976; 1.44 % of samples). Doping in Olympic racket sports was
found to range from 0.17 to 0.94 percent in the following order: badminton: 2/1,175, tennis:
17/3,945 and table tennis: 10/1,066. Racket sport games are characterised by a handheld
racket that is used to propel a missile between two (or four) players with the purpose of
placing the missile in such a position that one player is unable to return it successfully. These
sports are also characterised by an area of play that has a specified size, within which the
missile must be contained, and by the presence of a net that the missile must pass above on
each play. The unique sizes and shapes of the area of play, the height of the net and the
type of missile and racket used give character to each variant of the game. Racket sports are
unique due to the fact that players can modify the physiological demands of the game by
controlling the rest intervals between rallies, games and sets. Average oxygen consumption
for single-match duration badminton is reported to be 39.6 ± 5.7 ml/kg/min (73 % VO 2max);
oxygen consumption for table tennis is 26 ± 4 ml/kg/min (47 % of VO 2max), and consumption
for tennis is 29 ± 6 ml/kg/min (51 % of VO2max). Of course, average match duration must also
be considered. In the 2006 badminton World Championship in Madrid, the average match
duration was 33:35 minutes. At the Olympic Games in Beijing, the average table tennis
match lasted for 27:31 minutes. The average duration of tennis matches depends on the type
of court but typically ranges from 120 to 180 minutes. For example, at Wimbledon in 2005,
the average duration of tennis matches was 137 minutes, whereas the average match in the
Australian Open that same year lasted for 154 minutes. A major determinant of the outcome
of a game is an individual's physical fitness, which can be influenced by SUM. Although SUM
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is regularly investigated in sports as a whole, SUM is rarely studied in racket sports. Apart
from studies dealing with sports and physical activity in youth and related SUM issues in
which racket sports (tennis mostly) were not studied systematically, there are only a few
papers addressing SUM in Olympic racket sports. Briefly, Kondric it was reported on SUM
habits in Slovenian table tennis players. Also, Maquirriain it was analysed offences to the
Doping Code committed by tennis players between 2003 and 2009. When studying SUM
problems in sports, previous investigations noted that SUM is gender-, sociodemographic-,
culture-, and sport-specific and, therefore should be studied accordingly. Apart from the fact
that SUM is rarely investigated among racket sports, we determined that these sports would
be particularly suitable for our study based on several factors. First, table tennis, tennis and
badminton are Olympic Sports that fall directly under WADA jurisdiction and anti-doping
legislation. Second, all three sports share similar competitive characteristics as they are all
individual sports with no physical contact between opponents. However, these sports are
also diverse in terms of physiological demands [11557].
It was studied a total of 188 participants divided into three groups: table tennis players
(n=78), badminton players (n=83), and tennis players (n=27). All players were 18 years of
age or older and had participated in at least one of the two most recent competitions at the
highest national level for their sport (e.g. Slovenian Nationals). The number of tennis players
is almost half the size of the other two groups because mature tennis players (+18) are
typically professionals and rarely compete in the Slovenian Nationals. SUM and its
corresponding educational, sociodemographic, and sport-specific factors were investigated
using a previously developed and validated questionnaire for studying SUM. Substance use
and misuse data consisted of questions on binge drinking (7-point scale from "I do not drink
alcohol" to "I binge a few times a week"), cigarette smoking (7-point scale from "not smoking"
to "2+packs daily"), consumption of drugs and oppiates (use of different drugs and opiates
were inquired after but subjects reported only marihuana and hashish use). Doping factors
were evaluated with questions concerning the athlete's opinions on doping practice in their
sport (4-point scale from "I do not think doping is used" to "Doping is often"), potential doping
habits (4-point scale from "I do not intend to use doping" to "I'll use it if assured it will help
me"), and trust in their coach regarding doping and trust in their physician regarding doping
(both nominal "yes-no" questions). Nutritional supplements were reported separately and
included the consumption of isotonics, proteins, carbohydrates, and recovery supplements.
Additionally, we asked athletes to indicate who advised them to use NS with the coach,
physician, friend, and self-decided as choices. Badminton players reported the highest level
of binge drinking. Statistically significant differences were found in cigarette use, but this was
mostly because of the high proportion of smoking cessation among badminton and tennis
players. No significant differences were found for doping factors, although 1 in 10 badminton
players said they would use doping if they were assured that it will improve their performance
without any negative health consequences. Data revealed that most female athletes do not
trust their own coaches regarding doping issues (mistrust in coaches ranges from 61 % in
badminton to 83 % in tennis), whereas their trust in their physicians' opinions on the same
issue was somewhat higher. Approximately 50 percent of females declared no use of
nutritional supplementation. Female racket sport athletes reported using vitamins, minerals
and isotonics almost exclusively. The reported use of other substances was very low among
tennis players who were mostly advised by coaches or medical professional to consume
nutritional supplements, whereas athletes in table tennis and badminton were not. In male
athletes, there was no statistically significant difference between players of different racket
sports in their perception of doping behaviours. One-third of the studied athletes thought that
doping is used in their sport. Sixty to 90 percent of the male athletes reported that they do
not trust coaches' or medics' opinions regarding doping issues and problems. A minority of
athletes (10 % in badminton, 15 % in table tennis and 24 % in tennis) indicated that they
would use doping if assured that it would help them achieve competitive results without any
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negative health consequences. However, 5 to 10 percent of the studied male athletes
declared that they might potentially dope regardless of the possible health hazard. Nutritional
supplement use was mostly frequently reported by tennis players, followed by badminton and
table tennis. More than half of the tennis and badminton players were formally advised by a
coach or medical professional to use nutritional. Players in this sample reported varied levels
of substance use with binge drinking and cannabinoids use reaching a concerning level with
40 and 30 percent binge drinking and 16 and 21 percent using cannabinoids, form males and
females respectively. This level of use is comparable with the national statistics as detailed
below. Tobacco use, in contrast, appears to be a male phenomenon. It was revealed an
overall trend among racket sport athletes showing that the most significant overlap between
self-reported use of these substances exist between binge drinking and opioid use, reaching
11 percent (males) and 7 percent (females). More precisely athletes who reported either
binge drinking or opioid use were more likely to also use the other. Although these activities
take place outside of the controlled sporting arena, the extent to which athletes reported
these activities is concerning. Almost half of the athletes in the sample reported NS use.
Interestingly, there was very little overlap between current NS use and willingness to use
prohibited substances. The majority of those who indicated that they would be willing to use
doping did not report current supplement use. Even more disturbing is the fact that more than
80 percent of the tennis and table tennis players and 60 percent of the badminton players
report not trusting physicians' opinions on doping issues. Additionally, sports physicians are
mostly focused on orthopaedic and locomotor injuries in sports and are rarely systematically
educated regarding nutritional supplementation and doping. Consequently, athletes do not
consider them to be reliable, leading to low levels of trust regarding the information they
provide on doping issues. These problems must not be overlooked because those who trust
physicians' and coaches' opinions on doping are less prone to doping behaviour in the future
[11557]
Biking
The human physiological system is stressed to its limits during endurance sports competition
events. It was described a whole body computational model for energy conversion during
bicycle racing. About 23 percent of the metabolic energy is used for muscle work, the rest is
converted to heat. It was calculated heat transfer by conduction and blood flow inside the
body, and heat transfer from the skin by radiation, convection and sweat evaporation,
resulting in temperature changes in 25 body compartments. It was simulated a mountain time
trial to Alpe d'Huez during the Tour de France. To approach the time realized by Lance
Armstrong in 2004, very high oxygen uptake must be sustained by the simulated cyclist.
Temperature was predicted to reach 39°C in the brain, and 39.7°C in leg muscle. In addition
to the macroscopic simulation, it was analysed the buffering of bursts of high adenosine
triphosphate hydrolysis by creatine kinase during cyclical muscle activity at the biochemical
pathway level. To investigate the low oxygen to carbohydrate ratio for the brain, which takes
up lactate during exercise, it was calculated the flux distribution in cerebral energy
metabolism. Computational modelling of the human body, describing heat exchange and
energy metabolism, makes simulation of endurance sports events feasible [11561].
Imagine a medicine that is expected to have very limited effects based upon knowledge of its
pharmacology and (patho)physiology and that is studied in the wrong population, with low-
quality studies that use a surrogate end-point that relates to the clinical end-point in a partial
manner at most. Such a medicine would surely not be recommended. The use of
recombinant human erythropoietin (rHuEPO) to enhance performance in cycling is very
common. A qualitative systematic review of the available literature was performed to
examine the evidence for the ergogenic properties of this drug, which is normally used to
treat anaemia in chronic renal failure patients. The results of this literature search show that
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there is no scientific basis from which to conclude that rHuEPO has performance-enhancing
properties in elite cyclists. The reported studies have many shortcomings regarding
translation of the results to professional cycling endurance performance. Additionally, the
possibly harmful side-effects have not been adequately researched for this population but
appear to be worrying, at least. The use of rHuEPO in cycling is rife but scientifically
unsupported by evidence, and its use in sports is medical malpractice. What its use would
have been, if the involved team physicians had been trained in clinical pharmacology and
had investigated this properly, remains a matter of speculation. A single well-controlled trial
in athletes in real-life circumstances would give a better indication of the real advantages and
risk factors of rHuEPO use, but it would be an oversimplification to suggest that this would
eradicate its use [13063].
The 1990-2010 period in professional cycling is labeled by some as the epo epidemic.
Surprisingly, performance enhancement by epo and blood doping is not that clear-cut for
endurance athletes, leading to the question whether doping indeed strongly influenced
cyclists' performances from the 1990s onwards. It was examined the records (1947-2008) of
the Tour de France, Giro d'Italia, and Vuelta a España (n=181) and assessed the time it took
riders to win the race. The findings revealed normally distributed performances and linear
and quartic relationships in victors' performances over time that correspond with Brewer's
(2002) sociohistorical analysis of professional cycling suggesting that effects of the epo
epidemic on professional cyclists'achievements may be overestimated [11562].
Professional cycling has suffered from a number of doping scandals. The sport's governing
bodies have responded by implementing an aggressive new antidoping program known as
the biological passport. Cycling's biological passport marks a departure from traditional
antidoping efforts, which have focused on directly detecting prohibited substances in a
cyclist's system. Instead, the biological passport tracks biological variables in a cyclist's blood
and urine over time, monitoring for fluctuations that are thought to indirectly reveal the effects
of doping. Although this method of indirect detection is promising, it also raises serious legal
and scientific concerns. Since its introduction, the cycling community has debated the
reliability of indirect biological-passport evidence and the clarity, consistency, and
transparency of its use in proving doping violations. Such uncertainty undermines the
legitimacy of finding cyclists guilty of doping based on this indirect evidence alone.
Antidoping authorities should address these important concerns before continuing to pursue
doping sanctions against cyclists solely on the basis of their biological passports [11563].
To understand why pharmacological enhancements should never be allowed in cycling, you

need to understand that all spectator sports thrive by selling simple stories to their fans. The
cycling story is that, with great talent and after years of training, the best riders ride faster
than the others at the very limits of natural human endurance. In the Tour de France, this
story has been told and retold for 100 years – over stages, tours and careers. It describes
the overall winner, the best hill climber and even the failed solo breakaway. How could
cycling's story survive if pharmacological enhancements were allowed? Even if the time
comes when botulinum toxin injections are available from vending machines, doping should
never be allowed in cycling [07038].
Using a psychosociological approach, the purpose of one study was to identify and
understand the use of doping substances by young elite cyclists. Semi-structured interviews
were conducted with young cyclists who were hoping to find a professional team and cyclists
who had recently become professional. All of the young cyclists interviewed took nutritional
supplements and believed that they improved their performance, which has been shown by
other scholars to be a risk factor for doping. These cyclists believed that doping at the
professional level in cycling was acceptable but did not approve of it at the amateur level.
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They were attracted to doping; they were open to using doping substances themselves if it
was the key to continuing their cycling career, but only after they became professional. Team
staff, doctors, parents and friends helped to create a "clean" environment that prevented the
young cyclists from doping before becoming professional. The more experienced cyclists,
who doped or used to dope, transmitted the culture of doping to the young cyclists, teaching
them doping methods and which substances to use. This study could help to improve
prevention and help to detect doping, as it is clear that doping behaviors begin at the
amateur level [10019].
After recent scandals in cycling involving doping, it has been asserted that top-level cycling is
impossible without pharmacological support. An important prerequisite for successfully
completing the Tour de France is maintaining energy balance. To compensate for the daily
caloric expenditure of 23-25 MJ, conventional food must be supplemented with liquid food.
Quick administration of liquid carbohydrates is essential for optimal recovery of glycogen
stores in the liver and skeletal muscle. Androgenic anabolic steroids are a frequently used
form of doping. In endurance sports, these drugs have not been shown to affect endurance
performance, and there is little evidence to suggest that they enhance recovery. Although
epoetin use can increase maximal oxygen uptake, its effects on maximal power output are
less pronounced than what is generally assumed. A relationship between haemoglobin
concentration and sport performance has not been proved. It has been found that growth
hormone rather has a negative than a positive influence on the sport performance. The
doping problem is due in part to superstition, hearsay and insufficient knowledge among the
athlete's support personnel, which frequently leads to medical malpractice in sport. Education
and quality control for sport professionals, including sports physicians, may help to control
the doping problem [06029].
In 2006, a couple of professional cycling teams initiated their own testing programs. The
objective of one study was to describe fluctuations in commonly measured blood parameters
among top-level riders. From 2006 to 2007, a total of 374 blood samples and 287 urine
samples were obtained from 28 elite, male cyclists. Blood was analyzed for hematocrit (Hct),
hemoglobin concentration ([Hb]) and % reticulocytes. Seventy-six percent of all samples
were collected out-of-competition (OOC). From December 2006 to September 2007, the
average Hct and [Hb] decreased by 4.3 percent point and 1.3 g/dL, respectively. After the
end of the competitive season, the values increased back to baseline levels. During the Tour
de France, the [Hb] decreased by 11.5 percent, with individual decreases ranging from 7.0 to
20.6 percent. Hct and [Hb] values were lower in-competition (40.9 % and 14.1 g/dL)
compared to out-of-competition (43.2 % and 15.0 g/dL) and pre-competition (43.5 % and
14.9 g/dL). The results suggest that when interpreting blood sample results in an anti-doping
context, the sample timing (OOC, pre- or in-competition) and time of year should be kept in
mind [08033].
The protection of the health of athletes is one of the three criteria taken into account when
registering a substance in the World Anti-Doping Agency prohibited list. Nevertheless, in
elite-level cycling, banned substance use is widespread. The present research adopted a
psychological approach to examine how or whether perceived health risks influence elite-
level cyclists' decisions to use banned substances. Sixteen semi-structured interviews were
conducted with cyclists hoping to join a professional team (n=6), neo-professional cyclists
(n=2), and former professional cyclists (n=8). Although an evolution was observed in the
organization of doping and perceptions of doping over the last decade, the perceived health
hazards did not influence, most of the time, decisions to use banned substances among the
sample of cyclists. There was a systematization of exogenous substance use in the cycling
environment and a trivialization of the side effects of the banned substances. Finally,
younger cyclists were not concerned about the long-term health consequences of banned
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substances; they were more focused on the short-term performance-enhancing benefits.
There is a need to implement more effective preventive programs to change athletes'
attitudes toward doping and its health risks [11015].
In 1998, the Festina scandal at the Tour de France provided the first proof of widespread
doping in professional cycling. This doping scandal marked the end of team-organized
doping in professional cycling and ushered in a new period marked by the increasing
implementation of anti-doping measures. One article evaluates the impact of the anti-doping
rules and tests instituted since the Festina scandal. It was adopted a psychosocial approach
to analyze the organization of doping and the development of doping attitudes and practices
in high-level cycling. Sixteen cyclists were interviewed, of which eight were young, current
cyclists and eight were former cyclists who became professionals before the Festina scandal.
The results show that although the fight against doping in the last decade has reduced
doping use in high-level cycling, anti-doping measures have also had unexpected effects.
The fight against doping in cycling is not over [13064].
Indirect evidence of effects

Since doping improves athletic performance, anti-doping policies should have the opposite
effect. One analysis examined whether changes in the speed of major cycling races reflect
recent anti-doping efforts. Average speeds of 5th place finishers of the Tour de France, Giro
d'Italia, and Vuelta a España cycling races were obtained for the period 1990-2009. Between
1990 and 2004, the average speed had been significantly increasing by 0.16 km/h per year.
In a downturn, since 2004, the average speed has significantly decreased by 0.22 km/h per
year. The slowing down of professional cycling races is compatible with the hypothesis that
recent anti-doping efforts in professional cycling have curbed the use of performance-
enhancing substances [10020].
Tour de France 2006

The saga of the 2006 Tour de France is disappointing. Cycling fans have always thought that
the greatest cycling competition in the world, next in terms of audience and media
involvement to the Olympics and the football Word Cup, was an endurance race in which
cycling performance and racing ability would have been the main requisites for wearing the
yellow jersey in Paris. However, recent developments have profoundly altered this scenario,
transforming the Tour into an elimination race. Just 24 h ahead of the prologue start in
Strasbourg, nine riders from five teams were ruled out of the race, suspected of doping by an
international probe based on blood transfusions involving more than 200 athletes from
different sporting disciplines. This huge doping scandal, conventionally known as the “affair
Fuentes”, began in an ordinary apartment in Madrid, where the Spanish police discovered
the covert structure of an international performance‐enhancement business; they seized
more than 200 blood bags, along with doping records and several other doping substances.
The investigators were able to match code names of athletes with their highly detailed doping
records. In the prosecution, the American rider Floyd Landis, who wore the 2006 yellow
jersey in Paris, tested positive for abnormal concentrations of testosterone after winning
stage 17 and is now set to lose his title. If this were not enough, the runner‐up Oscar Pereiro
of Spain, who would probably have been awarded the yellow jersey, is under investigation for
testing positive for the banned substance salbutamol! Metabolites of this drug, often
prescribed for asthma, were discovered by the French antidoping body, AFLD, in Pereiro's
urine sample after stages 14 and 16. Although the authority of the AFLD is limited to French
soil only, Pereiro may not be allowed to take part in the 2007 Tour and could be stripped of
second place in the 2006 race until he has been definitely cleared of this allegation. These
disappointing events warrant further consideration. Firstly, the unexpectedly high number of
cyclists still involved in doping cases clearly indicates that the strict control strategies
adopted by sports federations and antidoping organisations will probably not modify this
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trend. It must be assumed that the athlete's desire to win, along with the vision of glory and
money, will always overcome the risk of being found guilty. Different and multifaceted
strategies are needed, which should be based on preventing rather than identifying or
criminalising doping. Once the young athlete becomes familiar with unfair practices, it is
difficult to modify this attitude. It is clear that controls, suspensions and civil and penal
sanctions are not reliable deterrents [07030].
Floyd Landis
Another question is: are antidoping controls absolutely reliable? This is probably the foremost
question that should be answered. In the specific case of the 2006 Tour de France, former
winner Floyd Landis has denied ever taking performance‐enhancing drugs. His key defence
is the contention that the National Laboratory for Doping Detection in Paris made technical
and analytical errors. In testosterone cases, the first level of testing is to measure the ratio of
testosterone to epitestosterone (T/E ratio). A ratio higher than 4:1 triggers a further test
based on the measurement of trace isotopes of carbon in the urine sample. The documents
Landis has posted show that the laboratory measured the T/E ratio in his urine sample at
least three times, producing three different results: 4.9:1, 5.1:1 and 11.4:1. A variety of
additional reasons were offered for the failed test: one was that he drank whisky and beer on
the night before the incriminating stage. Additional explanations were dehydration,
anti‐inflammatory injections for hip pain, and natural metabolism. The third crucial issue is
the hullabaloo when an athlete tests positive. Doping cases are not supposed to be made
public until they are resolved, but most become public through the media once a positive “A”
test is confirmed. There is a tendency for there to be access to confidential information on
previous tests of athletes before a definitive result, either positive or negative, is released by
the competent sport authority and antidoping organisation. Enormous emphasis was given to
the exclusion of the nine riders before the official start of the competition in Strasbourg. On
some occasions, these athletes have been judged by the media in advance of the sport
authorities and declared guilty on the basis of contradictory evidence, causing enormous
human and financial problems and ultimately jeopardising their careers. None of these
athletes has yet been charged. On the contrary, some have been completely cleared and are
free to race. Landis criticised officials from the UCI and WADA for announcing the results of
his test without analysing the second sample, as normally takes place in the anti-doping
procedure. He also claimed that the test was not conducted anonymously, saying he had
evidence to prove that laboratory staff had access to the names of the cyclists whose
samples were being tested. In the Pereiro case, it appears that the UCI had allowed him to
use salbutamol under the therapeutic use exemptions scheme. However, AFLD claim that
Pereiro's team did not complete the relevant medical forms. Regardless of the definitive
conclusion of the cases, all these riders have been designated as doping athletes by the
media, with potential detrimental economic and moral consequences. This is unacceptable,
considering that a definitive test result may be unavailable for several months after the
opening of the case [07030].
Germany
Doctors and politicians in Germany are demanding stricter laws for sports medicine after
three doctors were discovered to have given performance enhancing drugs to professional
cyclists. Two of the three doctors, from Freiburg University Hospital, were suspended in
2007 by the university when they admitted doping professional cyclists. In separate
statements, Lothar Heinrich and Andreas Schmid said that they gave the blood cell
stimulating hormone erythropoietin to the cycling team of the German telephone company
Deutsche Telekom, now T-Mobile. The confessions were made after several cyclists had
recently publicly admitted to taking drugs for performance and accused the doctors of
involvement. The incident had spread to amateur ranks a few days later when another doctor
from the Freiburg sports medicine department who had worked several times for German
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Olympic teams (not only cyclists) admitted giving performance boosting testosterone to riders
as far back as 1980. The third doctor, Georg Huber, was suspended by both German cycling
authorities and the University of Freiburg. Two former cyclists had triggered his resignation,
naming him in a newspaper story and claiming that doping in amateur German cycling was
widespread long before team Telekom. The German Sports Medicine Association decided
that all its members who look after athletes, professional and lay, must sign a statement
distancing themselves from any doping. In Germany 11 000 doctors are qualified in sports
medicine [07039].
Bodybuilding
Bodybuilding is a sport in which competitors are judged on muscular appearance. One case
study tracked a drug-free male bodybuilder (age 26-27 years) for the 6 months before and
after a competition. The aim of one study was to provide the most comprehensive
physiological profile of bodybuilding competition preparation and recovery ever compiled.
Cardiovascular parameters, body composition, strength, aerobic capacity, critical power,
mood state, resting energy expenditure, and hormonal and other blood parameters were
evaluated. Heart rate decreased from 53 to 27 beats/min during preparation and increased to
46 beats/min within 1 month after competition. Brachial blood pressure dropped from 132/69
to 104/56 mmHg during preparation and returned to 116/64 mmHg at 6 months after
competition. Percent body fat declined from 15 to 5 percent during preparation and returned
to 15 months during recovery. Strength decreased during preparation and did not fully
recover during 6 months of recovery. Testosterone declined from 9.22 to 2.27 ng/mL during
preparation and returned back to the baseline level, 9.91 ng/mL, after competition. Total
mood disturbance increased from 6 to 43 units during preparation and recovered to 4 units 6
mo after competition. The case study provides a thorough documentation of the physiological
changes that occurred during natural bodybuilding competition and recovery [13062].
Dancesport
DanceSport is the competitive form of ballroom dancing, and even though it has more
participants worldwide than ballet and modern dance, there is less peer-reviewed research.
A review was conducted to identify all relevant literature to help researchers and clinicians
gain an enhanced understanding of dancesport. Eight databases were searched, with 34
articles found in topics including participation motives, psychology, exercise physiology,
fitness training, injuries and injury prevention, biomechanics, menstrual dysfunction, and
substance use. The results indicate that researchers have been inconsistently recording and
reporting anthropometric and dancesport data; for example, 31 studies separated
participants by gender, 21 included the competition classification of dancers, 19 reported
which style of dancesport participants competed in, and 13 described the participants as a
dance couple. Common injuries affected the neck, shoulder, spine, knee, lower leg, and foot.
Dancesport is in the very heavy to extremely heavy category in energy expenditure (mean
heart rate: male 175.2 ± 10.7, female 178.6 ± 8.6 bpm) and utilizes both aerobic and
anaerobic energy systems. Alpha-beta and heart rate variability intervention techniques are
reported to successfully enhance performance in dancers. Dancesport participants also
appear less likely to smoke cigarettes, but have little knowledge about anti-doping rules.
During events, professionals danced farther (30 m) and faster (0.3 m/sec) than junior
dancers. Female competitors were more likely to be eumenorrheic. Dancesport is a
physically and mentally demanding competitive sport, but there is a need to standardize
measurements in future studies to allow comparison [13065].
Recreational sports
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Substance abuse has become increasingly widespread among athletes at sub competitive
and recreational level, due in part to the lack of controls in form of doping tests. Hypertension
and the many other side effects related to the illicit use of prescription drugs pose a
substantial but often underestimated threat to public health. The symptoms are recognized
late and are then mostly repressed or misjudged. Since the abuse is concealed to the doctor
when help is finally sought, it results in extensive and expensive tests that can seldom lead
to an effective treatment. Two case reports were presented to elaborate on this issue
[10023].
Prevalence of use by fitness centre members
Studies on the use of performance enhancing drugs (PED) in fitness centres rely
predominately on conventional survey methods using direct questioning. However, research
indicates that direct questioning of sensitive information is characterized by under-reporting.
The aim of one study was to contrast direct questioning of different types of PED use by
Dutch fitness centre members with results obtained with the Randomized Response
Technique (RRT). Questionnaires were conducted among members of fitness centres. PED
were classified into the following categories: anabolic steroids, prohormones, substances to
counteract side-effects, growth hormone and/or insulin, stimulants (to reduce weight), and
miscellaneous substances. A total of 718 athletes from 92 fitness centres completed the
questionnaire. The conventional method resulted in prevalences varying between 0 and 0.4
percent for the different types of PED with an overall prevalence of 0.4 percent. RRT resulted
in prevalences varying between 0.8 and 4.8 percent for the different types of PED with an
overall prevalence of 8.2 percent. The overall prevalence of the two survey methods differed
significantly. The current study showed that the conventional survey method using direct
questioning led to an underestimation of the prevalence. Based on the RRT results, the
percentage of users of PED among members of fitness centres is approximately 8.2 percent.
Stimulants to lose weight had the highest prevalence, even higher than anabolic steroids.
The key task for future preventive health work is to not only focus on anabolic steroid use,
but also include interventions focusing on the use of stimulants to lose weight [13032].
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THEORETICAL ASPECTS ON DOPING-TESTING
Overview
It is difficult, virtually by definition, to identify the “undetectables” that are perhaps being used
by elite athletes. It is important not to simply assume that “the cheaters are always a step
ahead of the testers” . . . It is also important not to assume that any banned drug an athlete
does take is necessarily going to improve his or her performance, not to mention the
question of whether this black-market product is what the athlete thinks it is [00002].
Testing results show that cheating athletes alter their drug abuse behaviors to avoid
detection. For example, when a urinary test was developed for recombinant EPO, athletes
changed doses and routes of administration to shorten the detection window. This is
confirmed by information obtained from athlete blogs and government investigations. Thus,
continued research into innovative test methods and strategies is necessary to deter drug
abuse. Perhaps more importantly, the scientific contributions from antidoping research go
beyond the field itself to affect other scientific disciplines, such as analytical chemistry,
endocrinology, genetics, laboratory medicine, pharmacology, physiology, and sports
medicine. More collaborative research with experts in these fields should enhance the rate of
discovery of innovative approaches to solve doping problems. It is critical to recognize that
new tests and methods must be fit for purpose. The tests and methods developed must
make the transition into routine testing use. Thus, antidoping research is yet another field
where translational research is a key component to applying new technology to solve
problems [12006].
Doping in competitive sport is a peculiar phenomenon. The need for performance

enhancement is emerging from the desire to maximise or even expand human capacities,
and by doping to gain competitive edge in a situation where athletes’ performances are
judged on two levels simultaneously: athletes compete against the opponents in situations
where typically only one can win and are also automatically entered into a quest for breaking
records which opens up the competitive arena including all from the past. From the array of
substances with performance enhancing properties, a wide range represents fully acceptable
means, whilst a defined set is prohibited by some authorities. In general terms, and for the
purpose of this paper, the term “doping” refers to the latter category. From the system’s point
of view, the current detection-based anti-doping policy does not automatically eradicate the
use of prohibited substances, but rather presents a barrier with a quantifiable risk of being
caught. It is easy to see that such a system leads to two primary strategies employed by the
athletes [13016]:
- compliance, driven by respect for the rules, desire to compete clean or fear of being
caught
- circumvention, i.e. outwitting the system by using not-yet-known or undetectable
substances, masking or simply betting on chances of not being selected for
testing
In order to design effective anti-doping measures, gaining insight into the driving forces
behind doping behaviour is vital. Whilst the doping decision is very complex involving moral,
economical and health considerations, theoretically this complexity can be distilled into a
simple decision situation where pros and cons are weighed against each other in the context
of unknown but assumed choices of the opponents. Based on the assumption that
eradicating doping from sport requires a significant change in this decisional balance; by
403
formulating and formally solving multi-player doping games, we aim to make a contribution to
developing a better understanding of doping decisions [13016].
Theories on laboratory testing
A well-planned testing strategy for anti-doping organizations is a key element in order to

obtain representative samples and a correct biomatrix for efficient doping control, but the
contribution of anti-doping laboratories is also fundamental to the overall success. Analytical
methods may be needed to reveal potential masking attempts and fraud which could take
place outside the sample collection session (e.g. haemodilution) to complement carefully
supervised sample collection procedures. As pre-analytical processes, laboratories verify the
authorized origin, evaluate the transport time and conditions, and also verify the identity and
integrity of the biological samples at the time of receipt. In the actual analytical processes, fit-
for purpose methods and instrumentation are used for the detection of prohibited substances
which are specified by WADA. The majority of the prohibited substances are exogenous, i.e.
they are not naturally present in the body. In these cases, qualitative identification of the
substance and/or its metabolite is sufficient for establishing an anti-doping rule violation. In
the prohibited list, however, there are substances such as testosterone, erythropoietin
(EPO), growth hormone (GH), and insulin-like growth factor 1 (IGF-1), which are available as
pharmaceutical products for clinical purposes, but which are also produced endogenously. In
these cases the analytical methods and result interpretation should be capable of
discriminating between exogenous source and clinical or pathological conditions.
Implementation of gas chromatography-combustion-isotope ration mass spectrometry (GC-
C-IRMS) has enabled the analysis of endogenous anabolic steroids and their misuse,
whereas methods based on electrophoretic techniques (IEF-PAGE and SDS-PAGE) and
immunoassays have provided tools to detect large biomolecules, the concentration of which
is still often below the sensitivity limits of mass spectrometric approaches. Research projects
within these special topics require active and devoted scientists, and also provide significant
improvements for routine analysis in the form of enhanced detection times and by facilitating
the result evaluation [13078].
In general terms, technical improvements, for example enhanced chromatographic and mass
spectrometric resolution, higher sensitivity, scan-to-scan polarity switching, and more
powerful data analysis allow for faster, more sensitive, and more reliable screening
strategies. According to the prevailing International Standard for Laboratories (ISL), the
doping control samples can be re-analyzed within a time period of up to eight years following
the reporting of the original result. This is particularly interesting when new instrument
technologies are introduced in the analysis routine for the purpose of monitoring new drug-
derivatives or new chemical entities emerging onto market, and also when new long-term
excreted metabolites are reported for already known prohibited substances. Although the
flexibility of the ISL increases the risk of an adverse analytical finding, it also requires
adequate sample storage condition and knowledge on the long-term effects on various
analytes. The list of prohibited substances is updated annually; active research and up-to-
date analytical processes are mandatory in order to reveal the high-risk emerging
compounds and to target the appropriate sample matrix. The laboratories' assignment is to
search for and to adapt the applicable parts of modern methodologies to the anti-doping
context. In the success of these research projects and with efficient implementation of new
techniques, international and interdisciplinary co-operation plays a major role. Similar to any
branch of applied sciences, in doping control the science is not for science's sake only. With
respect to routine analysis and service to the customers, these improvements should be also
practical, prompt, flexible, and cost-effective [13078].
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Homo economicus: pay-offs and sanctions
Lay explanation for doping rests on the assumption that for those who engage in doping
practices “winning is everything”, hence they use prohibited methods to ensure this outcome.
This approach assumes that the choice is purely rational-economical and responds to
externally imposed incentives and deterrents. As long as the perceived advantages from
using doping constitute a far better scenario than any scenario with no doping, factoring in
the risk of being detected and its consequences, the only logical action is to dope. Contrary
to the detection-sanction based deterrence methods, economical models recognise the
importance of the prize structure, considering both benefits and costs. Prize structures can
also be manipulated so the monitoring cost is kept low. These models suggest that
eradicating doping would require changes in the external factors, such as increased dis-utility
(including the chance of being detected and its consequences), decreased utility (reduced
pay-off) or some combination of the two. In reality, it is unlikely that such change will be
effectively implemented. Based on a review of economical models of doping, posit that rank
order contests such as sport competitions with highly skewed prize structures inevitably lead
to undesirable practices (i.e. doping) players may employ in order to enhance their chances
to finish in positions with high pay-off. A follow-up study with empirical data from thirteen
different athletic events reinforced the assumption that increase in competition increases
doping, and consequently lead to a quest for sophisticated detection and a requirement for
equitable sanctions. The nature of doping makes policing difficult and leads to an imperfect
but costly monitoring system, where externally imposed sanctions may inadvertently motivate
doping use by indicating that doping is widespread, hence the need for harsh sanctions
[13016].
Aim of anti-doping
The aims of the World Anti-Doping Programme and the Code are to care for the athlete’s
fundamental right to participate in doping-free sport and thus promote health, fairness and
equality for athletes worldwide, and to guarantee harmonized, coordinated and effective anti-
doping programmes at the international and national level relating to the detection,
deterrence and prevention of doping [13017].
The Goldman dilemma
Discussions of doping often report Goldman's sensational results that half of the elite athletes
asked would take a drug that guaranteed sporting success which would also result in their
death in 5 years' time. There has never been any effort to assess the properties of the
“Goldman dilemma” or replicate the results in the post World Anti-Doping Agency context.
This research evaluated the dilemma with contemporary elite athletes. Participants at an
elite-level track and field meet in North America were segregated into an interview or online
response. After basic demographics, participants were presented with three variant
“Goldman dilemmas” counter-balanced for presentation order. Only 2 out of 212 samples
(119 men, 93 women, mean age 20.89) reported that they would take the Faustian bargain
offered by the original Goldman dilemma. However, if there were no consequences to the
(illegal) drug use, then 25/212 indicated that they would take the substance (no death
condition). Legality also changes the acceptance rate to 13/212 even with death as a
consequence. Regression modelling showed that no other variable was significant (gender,
competitive level, type of sport) and there was no statistical difference between the interview
and online collection method. It was concluded that Goldman's results did not match this
sample. A subset of athletes is willing to dope and another subset is willing to sacrifice their
life to achieve success, although to a much lesser degree than that observed by Goldman. A
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larger scale online survey is now viable to answer important questions such as variation
across sports [13018].
Sports drug testing – an analyst's perspective
Sport plays a major role in the lives of many people, both for active participation and as
entertainment. Sport is now a huge nationally and internationally based industry. The desire
to win has led some athletes to resort to the use of performance enhancing drugs. With huge
financial rewards now available in some sports the pressure to excel has grown. Some have
argued that drug use should be given free rein, however most people are of the view that it is
athletic prowess that should be applauded not the efficacy of various performance enhancing
drugs. Apart from the obvious aspects of equality and fair play, the use of drugs is associated
with significant health risks. In the 1960's the use of stimulants in sports such as cycling led
to the death of at least one cyclist. Since 1968 the International Olympic Committee (IOC)
has required all Olympic Games' host cities to provide laboratory facilities for the analysis
and detection of performance enhancing drugs. There are now 29 IOC accredited
laboratories throughout the world that routinely test samples from athletes for the presence of
such drugs. The purpose of this tutorial review is to give an overview of drug testing
procedures, including those that were used at the last summer Olympic Games in Sydney
2000, and the incorporation of the latest developments in analytical chemistry technology in
the drug testing process. More recently, developments in biotechnology mean that the use of
whole new classes of drugs are banned in sport, often requiring new methodologies and
techniques for their analysis. The contest between those who wish to cheat and those who
wish to maintain fair play in sport is an ongoing one [03029].
Besides anabolic steroids, the most common performance-enhancing hormones are

erythropoietin (EPO), insulin, GH, and gonadotropins, mostly indistinguishable from
endogenous hormones and with very short half-life. This makes virtually impossible to
demonstrate their use by measuring their concentration in the blood or urine. A possible
approach to the problem may lie in in-direct demonstration through detection of the biological
effects of these substances. The finding of an increased hematocrit level is suspicious but
not clearly demonstrative of EPO abuse. Very high levels of circulating EPO could be
associated with a strong suspicion of doping, when associated to other abnormal
parameters, such as Ht, sTFRr, EPO, RDW. The presence of antibodies against the
polysaccharide fraction of lateral chains of EPO has been observed only in patients treated
with rhEPO. Owing to the pulsatile pattern of GH, particularly during physical exercise,
pathologically high values may be found in normal subjects. Therefore, as in the case of
EPO, evidence of GH abuse can be gathered only indirectly by detecting the biological
effects of its administration. In training subjects GH treatment increased GH, IGF-I, IGFBP-3
and ALS, and decreased IGBP-2. After cessation of treatment IGF-I, IGFBP-3 and ALS
approached basal values between 49 and 96 h. Also the bone parameters PICP ICIP, PIUP
and osteocalcin increased significantly. Four days after cessation of treatment, levels of PIIIP
and ICTP were still abnormally elevated. In conclusion, increases in IGF-I, IGFBP-3, ALS,
PIIIP and ICTP are all indicative of recent GH abuse or of acromegaly [03030].
Forensic intelligence in anti-doping
Today's approach to anti-doping is mostly centered on the judicial process, despite pursuing
a further goal in the detection, reduction, solving and/or prevention of doping. Similarly to
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decision-making in the area of law enforcement feeding on Forensic Intelligence, anti-doping
might significantly benefit from a more extensive gathering of knowledge. Forensic
Intelligence might bring a broader logical dimension to the interpretation of data on doping
activities for a more future-oriented and comprehensive approach instead of the traditional
case-based and reactive process. Information coming from a variety of sources related to
doping, whether directly or potentially, would feed an organized memory to provide real time
intelligence on the size, seriousness and evolution of the phenomenon. Due to the
complexity of doping, integrating analytical chemical results and longitudinal monitoring of
biomarkers with physiological, epidemiological, sociological or circumstantial information
might provide a logical framework enabling fit for purpose decision-making. Therefore, Anti-
Doping Intelligence might prove efficient at providing a more proactive response to any
potential or emerging doping phenomenon or to address existing problems with innovative
actions or/and policies. This approach might prove useful to detect, neutralize, disrupt and/or
prevent organized doping or the trafficking of doping agents, as well as helping to refine the
targeting of athletes or teams. In addition, such an intelligence-led methodology would serve
to address doping offenses in the absence of adverse analytical chemical evidence [13079].
Non-analytic testing
Collection of nonanalytical evidence has become more important. Tips and substantial
assistance from athletes have resulted in a number of antidoping sanctions. Cooperation with
law enforcement has provided antidoping authorities with not only the Bay Area Laboratory
Cooperative investigation but also materials that athletes have tried to bring across borders
and evidence of illegal Internet purchases. These, along with “suspicious” (negative) test
results, have been used as evidence in cases to prove use under the WADA Code as
opposed to direct detection of the prohibited substance. In the 2015 version of the WADA
Code, there is increased enforcement potential to support sanctioning of doping enablers.
Deterrence benefits from any method of increasing the certainty of being caught [14727].
Colored illicite tablets
The necessity of specific, confirmatory tests in the identification of seized illicit products was
highlighted by the analysis of eighteen heart shaped, blue tablets confiscated by Police at a
street control in the North East of Italy. The tablets responded as amphetamines to a
preliminary color test (Marquis); a subsequent, confirmatory assay by gas chromatography-
mass spectrometry revealed the presence of two anabolic androgen steroids (AAS),
methandienone and methyltestosterone, in concentration of 1.7 and 1.5 mg respectively per
tablet; no trace of amphetamine-like or nitrogen containing compounds was found. The
observed orange coloration was due to the reaction of concentrated sulphuric acid, contained
in the Marquis reagent, with the delta(4) C-3 keto group of steroids. The two AAS, banned
under the world antidoping code, are not considered as psychoactive drugs of abuse in most
countries, although their trafficking may entangle severe public health concerns [13083].
Human mitochondrial DNA analysis
In a doping control case, a urine sample was tested positive for nandrolon. It was asked by
the athlete to perform DNA investigations on the questioned urine sample and compare
these to a fresh blood sample taken from the athlete in order to detect or rule out
manipulation and/or switching of the samples. The urine sample had been collected nine
months prior to the investigation and had been stored at 4 degrees C. In a first approach,
nuclear DNA systems were investigated that failed with the exception of the Amelogenin
system. Due to the high copy number of mitochondrial DNA molecules and the robustness of
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the mitochondrial genome, we investigated the HVR I and HVR II regions of mitochondrial
DNA and obtained reproducible and clear sequencing results for both the blood and the urine
samples. Due to the identical sequences, it could not be excluded that the blood sample and
the urine sample were from the same individual or an individual having the same maternal
lineage [02017].
Determining the authenticity of athlete urine in doping control
DNA analysis
The integrity of urine samples collected from athletes for doping control is essential. The
authenticity of samples may be contested, leading to the need for a robust sample
identification method. DNA typing using short tandem repeats (STR) can be used for
identification purposes, but its application to cellular DNA in urine has so far been limited.
Here, a reliable and accurate method is reported for the successful identification of urine
samples, using reduced final extraction volumes and the STR multiplex kit, Promega®
PowerPlex ESI 17, with capillary electrophoretic characterisation of the alleles. Full DNA
profiles were obtained for all samples (n = 20) stored for less than 2 days at 4 °C. The effect
of different storage conditions on yield of cellular DNA and probability of obtaining a full
profile were also investigated. Storage for 21 days at 4 °C resulted in allelic drop-out in some
samples, but the random match probabilities obtained demonstrate the high power of
discrimination achieved through targeting a large number of STRs. The best solution for
long-term storage was centrifugation and removal of supernatant prior to freezing at -20 °C.
The method is robust enough for incorporation into current anti-doping protocols, and was
successfully applied to 44 athlete samples for anti-doping testing with 100% concordant
typing [150116].
Theories on doping in sports
Human behavior occurs within a system, and as such, so do behaviors in performance-

related domains (e.g. athletics, academics). Doping is a performance enhancement behavior
that can be problematic because of the negative physical and psychological effects
associated with the use of some substances and the common argument that doping is unfair.
However, doping continues and may be increasing. Because a firm theoretical or empirical
understanding of doping does not exist, one article proposed a conceptual, comprehensive,
and innovative systemic model of doping behavior. The model is built from relevant
empiricism supporting the idea that contemporary doping behavior is a function of systemic
transactions between historical doping practices, the present environment, current antidoping
interventions, one's genetic makeup, developmental milestones, social factors, and
epigenetics [11424].
The fight against doping is a challenging task. Owing to the complexity of the doping
phenomenon, simultaneous consideration of physiological, medical, pharmacological,
psychological, ethical and systemic factors is required in order to be successful in this
endeavor. The need for effective deterrence policy is underscored by the fact that the
problem of performance enhancements has spread beyond the elite athlete population. It is
well documented that groups other than competitive athletes are at risk of using doping
agents, especially steroids. Furthermore, medical enhancement of non-sport performance
(i.e. quality of life, appearance) appears to be widely acceptable among non-athlete
population. For effective deterrence methods, individual, systemic and situational factors that
make an athlete or athlete group more susceptible to doping than others should be fully
investigated. Traditional behavioral models assume that the behavior in question is the
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ultimate end. However, growing evidence suggests that in doping situations, the doping
behavior is not the end but a means to an end, which is gaining competitive advantage.
Therefore, models of doping should include and anti-doping policies should consider
attitudes or orientations toward the specific target end, in addition to the attitude toward the
tool itself. Data were collected by questionnaires containing a battery of psychological tests
among competitive US male college athletes (n=199). Of the 199 athletes, 15 (8 %) reported
having personal experience with doping and an additional 9 (5 %) claimed to have used
substances classified as doping for medical reasons. The same figures for current use of
performance enhancing substances were lower: 5 (3 %) and 1 (1 %), respectively. Outcome
measures included sport orientation (win and goal orientation and competitiveness), doping
attitude, beliefs and self-reported past or current use of doping. A structural equation model
was developed based on the strength of relationships between these outcome measures.
Whilst the doping model showed satisfactory fit, the results suggested that athletes' win and
goal orientation and competitiveness do not play a statistically significant role in doping
behavior, but win orientation has an effect on doping attitude. The structural equation
modeling (SEM) analysis provided empirical evidence that sport orientation and doping
behavior is not directly related. It was concluded that the considerable proportion of doping
behavior unexplained by the model suggests that other factors play an influential role in
athletes' decisions regarding prohibited methods. Future research, followed by policy
development, should incorporate these factors to capture the complexity of the doping
phenomenon and to identify points for effective anti-doping interventions. Sport governing
bodies and anti-doping organisations need to recognize that using performance
enhancements may be more of a rational, outcome optimizing behavior than deviance and
consider offering acceptable alternative performance-enhancing methods to doping [07004].
Despite the increased anti-doping effort, the relative number of adverse analytical findings
has not decreased considerably in the past four years (written 2007). The appropriateness of
education as a deterrent is questionable as it has been shown that doping specific
knowledge is higher among doping users than among their non-user counterparts. While
prevention, complemented with detection, will be likely to be the main approach to the doping
problem, the ultimate goal for sport governing bodies should be creating policies for a truly
effective deterrence. Setting detection aside, there is still a fundamental distinction between
prevention and deterrence. It is suggested that prevention (and detection) create an
environment where the chances of detection and punishment for using doping are
uncomfortably high, hence keep athletes away from employing such means, regardless of
their motives. On the other hand, value-based deterrence in its true, perhaps Utopian sense,
is associated with the creation of an environment where athletes never feel motivated to use
illegal means for performance enhancement [07004].
Doping detection
Historically, the anti-doping movement has been based on detection and prevention, with the
initial emphasis on detection. Organisational structures and standard operating procedures
have been in place to ensure compliance with the anti-doping regulations. Detection relies on
testing, which has been increasingly problematic in high performance sport. It has been
argued persuasively that making testing effective as a deterrence method, either the volume
of tests conducted or the sanctions imposed have to be increased significantly, potentially to
the level that is practically not feasible. The new technologies in both the development of
undetectable methods and the detection of the new methods have led to rapidly escalating
costs, bearing in mind that tests are currently not even available for all banned substances
and methods. If the trend continues, costs of effective testing will soon became a prohibiting
factor. Athletes, as they progess in their sports career, are gradually drawn into the vicious
circle of the constant desire to enhance performance. In this process, some athletes may
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become more susceptible to doping than others, depending on the combination of their
personality and the situation. Therefore, both the individual and systemic factors contributing
to doping behavior should be fully investigated in order to underpin effective, targeted anti-
doping intervention. In support of the argument against detection from a psychological
perspective, it was provided empirical evidence for the failure of detection based deterrence
showing that in a hypothetical situation, athletes first consider their moral beliefs, followed by
the fear of negative health consequences and legal sanctions associated with the use
performance-enhancing drugs. The effect of the threat of legal sanctions practically
diminished when moral beliefs and health concerns were added to the behavioral model,
directing policy makers to alternative deterrence methods. Additionally, many speculate that
with gene doping on the horizon of competitve sport, detection based regulation will soon be
seriously undermined [07004].
Understanding the basics of testing for banned substances
Whenever athletes willfully or accidentally ingest performance-enhancing drugs or other

banned substances (such as drugs of abuse), markers of those drugs can be detected in
biological samples (e.g. biofluids: urine, saliva, blood); in the case of some drugs, that
evidence can be apparent for many weeks following the last exposure to the drug. In addition
to the willful use of prohibited drugs, athletes can accidentally ingest banned substances in
contaminated dietary supplements or foods and inadvertently fail a drug test that could mean
the end of an athletic career and the loss of a good reputation. The proliferation of
performance-enhancing drugs and methods has required a corresponding increase in the
analytical tools and methods required to identify the presence of banned substances in
biofluids. Even though extraordinary steps have been taken by organizations such as the
World Anti-Doping Agency to limit the use of prohibited substances and methods by athletes
willing to cheat, it is apparent that some athletes continue to avoid detection by using
alternative doping regimens or taking advantage of the limitations in testing methodologies.
One article reviewed the testing standards and analytical techniques underlying the
procedures used to identify banned substances in biological samples, setting the stage for
future summaries of the testing required to establish the use of steroids, stimulants, diuretics,
and other prohibited substances [150117].
Doping prevention
The WADA and national sport governing bodies have added preventive measures to their
detection programs. Examples for anti-doping prevention include: WADA's Athlete Outreach
Program (launched in 2001) targeting top performing athletes at major sporting events, the
Anti-Doping Development Program (started in 2004), which aims to help countries and
organizations to set up quality doping control, and the Educational Programme, which is a
major tool of the WADA in an attempt to create a doping free culture by providing education
to all stakeholders about the dangers of doping and its consequences. Congruently, the 100
percent me programme of UK Sport aims to promote positive attitudes and values of those
who successfully competed drug-free and to provide accurate and relevant information on
anti-doping. The 100 percent me is an educational program with three distinct but related
strands. Outreach programme provides a framework for delivering accurate information and
giving advice on anti-doping issues to athletes, athlete support personnel, and parents
across the UK via sports events, workshops, training sessions and conferences. The
accreditation programme allows interested individuals to gain knowledge in anti-doping and
became a “100 % me” tutor. The 100 percent me is also a brand promoting the image of the
“clean athlete”' based on values of personal responsibility, choices, fairness and honesty.
This image is linked to the Ambassador programme where successful drug-free athletes
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committed to anti-doping use the 100 percent me platform to promote drug-free sport among
their fellow athlete The Education Model Guidelines (EMG) are in place to help National
Governing Bodies (NGBs) develop their own programmes using the 100 percent me
framework. The UK model is one of the existing anti-doping national programmes. In the US,
the U.S. Anti-Doping Agency (USADA) is responsible for similar testing and education
programmes, and in place to eliminate conflict of interest of NGBs testing and sanctioning
their own athletes. The Australian Sports Anti-Doping Authority (ASADA) has also launched
a comprehensive the ASADA Education Service Charter in 2007. The Charter places an
emphasis on developing athletes' and support personnels' understanding of the physical and
psychological risks of doping to ensure that athletes and support personnel are aware of their
rights and responsibilities [07004].
Explaining the doping behavior
Whether it is a realistic goal or not, effective deterrence is hindered as long as doping

behavior is poorly understood. Before any serious consideration is given to deterrence
methods, factors that make an athlete or athlete group more inclined to doping than others
must be fully investigated. The WADA has only just started to channel funds to social science
doping research to develop better understanding and consequently, more effective
deterrence programs. Aiming to add to the body of knowledge on one possible cause of
doping behavior (i.e. individual dispositions and attitudes) is congruent with the current
priorities of the WADA Social Science Research Programme [07004].
Both the eminent literature and the official global sport organisational stance suggest that
athletes' attitudes are responsible for the deviant behavior of doping. Being overly
competitive or exceedingly win-orientated is often used as a lay explanation for doping.
Although gender, cultural and competitive level differences among athletes have been
scrutinized since the late '80s the relationship between these factors and doping behavior
has not been empirically tested, except in one project. In one study the classic “heory of
planned behavior” (TPB) model provided a theoretical framework for a study among Italian
adolescents, where attitude was found to be the strongest predictor for behavioral intention.
The TPB model held across different levels of sport involvement and gender. Alternative
theoretical models of doping have been developed attempting to explain the complex nature
of doping. The models are based on existing general models from either health science or
criminology but their application to the doping situation has not been empirically validated.
The first among the few used the “Health Belief Model” to develop a theoretical drug control
model. Although it was not explicitly stated, the model also incorporates some kind of
economic rationality when it considers the balance between deterrence and incentives and
availability and affordability of performance enhancing substances. According to the model,
athletes' doping behavior is the ultimate function of this cost/benefit ratio, personality and
morality, legitimacy of sanctioning organisation, social context (reference group) and attitude
toward doping. The “Drugs in Sport Deterrence Model” also considered costs and benefits
but used these concepts in a broader sense. Their model is based on “Deterrence Theory”
used in criminology and costs and benefits include material and social consequences, as
well as individual effects, such as health concerns, guilt or even satisfaction from sport
achievement. Situational factors (i.e. prevalence perception, professional status, type of
drug, experience with testing, etc.) were also thought to have an effect on the final decision
regarding doping use. The common element of all three models is that subjective norms play
a seemingly important role in doping behavior. As it is evidenced in a recent, WADA Social
Science research funded extensive literature review, published research into doping attitude
is dominantly descriptive and with a few exceptions, it falls short on theoretical underpinning
or on establishing causal relationships between attitudes and behavior. The major
achievement of the existing doping models is that they draw attention to the complexity of the
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doping problem. Many of them touched upon attitudes and many other perhaps important
factors contribution to doping but their claims have not been supported with empirical
evidence [07004].
The traditional one-step behavioral models assume that the behavior in question is the
ultimate end and considers antecedents, such as beliefs, attitudes, subjective norms and
perceived behavioral control regarding the particular behavior. Research into athletes'
motivation and reasons for doping use reveal an important factor that has been prominent in
game theory models but overlooked in the existing doping behavior models: that doping
behavior is not the ultimate end but rather a means to an end. It can be argued whether the
ultimate end is winning or achieving a specific sport related goal (i.e. breaking a record); and
it may vary from athlete to athlete. Nevertheless, if doping is a tool to achieve an end-goal,
then models of doping should include attitudes or orientations toward the specific target end,
in addition to attitudes toward the tool itself [07004].
In consideration of the structural model of doping, the existing literature, more specifically the
Theory of Reasoned Action (TRA), the Theory of Planned Behaviour (TPB), previous
structural equation models of attitude and behavior and previous doping models were
consulted. The Theory of Reasoned Action established a linear sequence of cognition
(beliefs), affects (attitude), conation (behavioural intention) and behaviour. Later, the model
has been criticized for the underlying and unrealistic assumption of absolute behavioural
control, hence perceived behavioural control was added and the model expanded into the
TPB. Models have been empirically tested and refined by showing interaction between the
predictors and by questioning the generality of the model. An earlier model suggests an
important notion, namely multiple factors influencing the behavior. The notion of multiple
factors is, of course, not new. In 1977, it was already mentioned multiple-act criterion, where
attitudes toward a target (i.e. doping in general) is linked with observed heterogeneous
behaviors (i.e. supporting the anti-doping movement but using doping at the same time).
Influencing factors can be learned experiences (past behavior), perceived control,
personality, cost/benefit ratio and most importantly: goals. Also, it was focused on past
behavior (in general) whereas new doping models consider personality, availability, free
choice of actions and situational factors as well as perceived control over behavior and free
choice. Curiously, a situation where multiple attitudes are influencing a single behavior has
not been considered in doping attitude-behavior modeling. If doping behavior is considered
as a means to an end, attitude toward the end point should be taken into account. Support
for this assumption can be found in the literature for at least two decades [07004].
Others have suggested that doping may be used to achieve one or more of many goals,
including reaching unattainable goals, breaking off the plateau, or even to signal group
membership; or mark transition from being recreational to professional athlete. Contrary to
the argument that the crucial element of doping is the intent to gain unfair advantage at the
expense of other competitors, athletes do not necessarily see using doping as unfair or
advantageous. Doping may be employed as a useful tool to improve performance to the level
that is, or perceived to be necessary to have a reasonable chance for winning. When
athletes assume that their competitors follow the same logic, the motivation for doping use is
often reduced to the desire to level the playing field and ensure equal chances. These
rational decisions regarding doping behavior could easily be against the general attitude
toward doping, which is suppressed by other, stronger driving forces such as the desire to
win, goal orientation or competitiveness [07004].
One of the most important features of the present study was the finding that sport orientation
is not strongly related to doping behavior, or to doping attitude. The only exception was win
orientation, which showed a significant relationship with doping attitude. Thus the importance
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of winning may have influenced what athletes think about doping, but it does not necessarily
manifest in their behavior. From the path coefficients, it was clear that athletes' desire to win,
to achieve their personal goals or their competitive nature is not necessarily related to their
decision regarding use of prohibited performance enhancements. None of the measures,
except expressed belief, had a significant path to behavior. Apparently, athletes using
prohibited means of performance enhancements do not have to be overly competitive or win-
orientated. They do not have to endorse such pharmaceutical agents, or agree with the use
of such substances in order to actually use them. Many athletes claimed that they would
prefer not to use drugs and would not do it if they were certain that the competition was drug-
free. The paranoia about other competitors using performance enhancement is a
reappearing theme in these papers. In addition, it has been noted that athletes often feel an
external pressure to win, most often in the form of warning about exceptionally good
opponents. Hence, using doping agents may be more of a rational, outcome optimizing
behavior than deviance. If this is the case, sport governing bodies may do well if in addition
to placing a ban on certain performance enhancing substances and methods, they provide
athletes with acceptable alternatives. The small negative (but not significant) relationship
between goal orientation and doping behavior was a logical connection because among the
three sport orientation measures, goal orientation reflects an orientation to personal
standards, regardless of the situation. The other two measures, desire to win and
competitiveness reflect a tendency to enter and strive for success in a sport situation. Using
banned performance enhancements in most athletes' view was expected to be against their
standards as sportsmen. However, at the same time doping is often viewed as a means to
an end; a 'tool' that is bad but necessary to ensure success in competition. Therefore, a
positive relationship was expected. Of the two measures studied, competitiveness had a
small but insignificant, positive path to doping behavior, whilst winning practically showed no
relationship at all. On the other hand, the only statistically significant relationship with sport
orientation measures and other factors was between win orientation and doping attitude.
Sport orientation and attitude appear to be similar constructs and distinctly different from
behavior. Athletes may think that doping is needed or not needed for winning but when it
comes to actual behavior, it might be influenced by other factors more than attitude or
orientation. This is not to say that personality, attitude, values should be discarded in order to
make room for other factors. As probably no two individuals would react identically to the
same combination of environmental factors, it is fair to assume that contextual contingencies
are mediated through the combination of individual factors. Adherence to norms is a
particularly difficult question. Decisions regarding doping use are influenced by at least two
possibly competing norms:
- the general social norms, such as fair play, condemnation of cheating

- the special norms held by the athletes' immediate subcultures
One possible explanation for the strong path between belief and behavior is justification.
Those athletes who use doping or wish to use such performance enhancements would prefer
to do so without social stigmatization. Such a view is in keeping with previous research
where athletes expressed their view of doping as a necessary means to a desired end and
whilst they acknowledge rule breaking behavior, they do not consider themselves cheaters or
more cheating than any other [07004].
The results of this research have both theoretical and practical implications. At the theoretical
level, the findings of the paper are a step toward a comprehensive doping model. It highlights
the need for the inclusion of other influencing factors and makes suggestions for future
model testing. At the practical level, understanding the driving forces behind doping and how
athletes wish to deal with these factors must be at the center of informed deterrence policies.
Athletes are, by nature, highly motivated and achievement oriented individuals and have
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grown to appreciate methods for performance enhancement (training, nutrition,
physiotherapy, equipment, etc.). The distinction between acceptable and prohibited methods
must be made clear and convincing. To be effective, authorities must be able to
- justify the doping ban in general

- use evidence-based selection of substances and methods included into the prohibited
list
- use the same criteria for all substances and methods
- communicate such decisions to all stakeholders
Suggesting anti-doping education and perhaps changes in attitudes to doping is a rather

futile approach if the other influencing factors are kept constant. A value-based deterrence
requires changes at all levels and in all stakeholders. Large scale research aiming to
understanding the driving forces behind doping behavior and gaining knowledge of effective
deterrent factors is much needed and should be extended beyond the athlete population to
include coaches, managers and officials. Sport governing bodies and anti-doping
organisations are in the unique position to endorse and foster such research. International
and national anti-doping organisations should make targeted funding opportunities for
doping-related research aiming at increasing knowledge regarding both the doping behavior
and alternative acceptable means of performance enhancement. Constant improvement of
performance is, after all, the core characteristic of competitive sport [07004].
Multiple drug use
Because the use of anabolic steroids remains illicit, detailed knowledge of their side effects is
unknown. Reported side effects emanate from case reports, retrospective studies, and
comparisons drawn from prospective studies of steroid therapy in patients with a variety of
wasting syndromes. Further confusing the specific side effects of a given anabolic steroid is
the common practice that many athletes take multiple drugs simultaneously and in multiple
administration routes. Additionally, physiologic differences between patients with wasting
syndromes and athletes limit our ability to extrapolate the results of controlled studies to
anabolic steroid use in sports. One other compounding factor in determining which side
effects arise from anabolic steroid use is that users also ingest other illicit drugs including
cocaine, marijuana, alcohol, and tobacco. Doctors and training staff must be aware of the
potential harms in order to both recognize and more importantly educate patients about
these possibly life-threatening adverse effects [06031].
Athletes often alternate or combine multiple forms of AAS in an effort to maximize benefits
while minimizing side effects. Users often take an average of five different drugs to achieve
supraphysiologic anabolic levels exceeding 40 to 100 times the normal physiologic hormonal
effects. This use of a combination of products is commonly referred to as stacking, and it is
this stacking that makes it difficult to pinpoint the direct effect of different agents. The effects
of AAS tend to follow a linear doseresponse curve for both anabolic and androgenic actions,
although at high doses there appears to be a plateau to their physiologic effects. The
supraphysiologic doses used by athletes far exceed the saturation point of the androgenic
receptors. Therefore, there must be additional mechanisms to explain why supraphysiologic
doses of steroids seem to enhance strength. There are three different physiologic
mechanisms through which AAS may exert their effects [06031]:
- First, AAS improve the body’s utilization of ingested protein, which favorably alters
nitrogen balance. They mainly stimulate protein synthesis by turning on gene
transcription after binding to androgenic receptors at the cellular level. Androgens
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exert their effects on a number of varied tissues including bone, adipose tissue,
skeletal muscle, brain, prostate, liver, and kidney, as well as reproductive tissues.
Therefore, a more complex understanding at the cellular level is needed to explain
the variety of effects, knowing that their actions are mediated by only one androgen
receptor. These receptors appear to be identical in muscle and various other organs,
but their absolute number and affinity for various types of AAS potentially explain the
variety of different effects in multiple organ systems and from various AAS products.
Some effects of testosterone are mediated through conversion to other bioactive
compounds including dihydrotestosterone and estradiol
- A second effect of steroids is to displace glucocorticoids from binding to their
receptors, thus exerting an anticatabolic effect. Because glucocorticoids usually
depress protein synthesis, this antagonistic effect explains muscle mass gain and
also the utility of these drugs in wasting syndromes
- A third postulated effect of steroids is that they confer a psychologic benefit to
athletes wishing to gain strength. AAS users describe a euphoria or high following
ingestion that allows them to work harder during workouts and recover more rapidly,
which allows them both to intensify their training and become more aggressive in
competition
The types and patterns of performance-enhancing drug use
AAS are the most commonly used PEDs, with testosterone, boldenone, and trenbolone
being the most frequently detected drugs among illicit PEDs users in the US. Although
boldenone is a veterinary steroid not approved for human use, this fact has not diminished its
popularity among illicit AAS users. In the small subgroup of PED users who are elite athletes,
WADA most commonly detects testosterone, stanozolol, and nandrolone; and the highest
prevalence of positive tests occur in bodybuilding, power lifting, weightlifting, boxing, and
kickboxing. PEDs users often combine multiple drugs, including classical drugs of abuse
such as opiates. The Monitoring the Future survey question states: “Steroids, or anabolic
steroids, are sometimes prescribed by doctors to treat certain conditions. Some athletes, and
others, have used them to try to increase muscle development. On how many occasions (if
any) have you taken steroids on your own – that is, without a doctor telling you to take
them?” The limitations of these data include the potential for false positives from a
respondent’s lack of understanding of the question, as well as the potential underestimation
of the problem since AAS users do not begin using steroids until they reach the early 20’s.
Most AAS-users engage in high-intensity exercise to maximize anabolic gains. The
combined use of AAS and opiates enables the user to continue training despite muscle and
joint pain. Inevitably, some individuals develop opioid dependence. In particular, nalbuphine
hydrochloride (Nubain®) is popular among weightlifters, and is associated with other
substance abuse. It has been suggested that AAS could act as a gateway drug to opioid
dependence. In another study of 223 men entering a drug treatment program, AAS use was
considerably higher (25 %) among opioid users, compared withmenusing other drugs (5 %).
In yet another study, 50 percent of dependent AAS users met Diagnostic and Statistical
Manual of Mental Disorders-IV criteria for a lifetime history of opioid abuse or dependence,
as compared to eight nondependent AAS users (19 %) and five nonusers (7 %). In one case
report of a man with AAS dependence, naloxone precipitated symptoms suggestive of opiate
withdrawal, even though the man denied using opiates. AAS may also interact with heroin in
accidental drug overdose. Recent studies increasingly suggest that the use of AAS and other
PEDs often occurs in conjunction with use of multiple classical drugs of abuse. PED users
are increasingly encountered in needle-exchange programs, where they may sometimes
represent most the clientele [14017].
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Combination of anabolic steroids and other substances
AAS use has also been linked to alcohol use in humans and rats. Chronic AAS use may
make rats susceptible for alcohol intake. Steroid-induced alterations in opioid peptides in the
brain reward system may explain the increased sensitivity to alcohol. Other studies have
observed an imbalance in dopaminergic pathways in nucleus accumbens, a brain area
involved in reward, leading to speculation that the alterations in the actual peptidergic and
monoaminergic systems promote the rewarding effects of ethanol, thereby increasing alcohol
intake. Additional studies have reported increased sensitivity to cocaine and amphetamine in
rats exposed to high doses of AAS. Thus, AAS may induce effects on the brain reward
system that may render individuals susceptible to other drugs of abuse. Athletes and
nonathlete weightlifters that use AAS commonly combine different steroids (“stacking”) in
cycles of increasing and decreasing concentrations (“pyramiding”). Most stacks will include
both androgens and nonsteroidal drugs. The latter are typically chosen to provide further
anabolic effects (hGH, IGF-I, insulin), to counteract negative side effects of AAS (aromatase
inhibitors, estrogen receptor antagonists), to enhance fat and water loss (diuretics, thyroid
hormones, alpha2 adrenergic receptor agonists), to reactivate endogenous testosterone
production at the end of a cycle (gonadotropins), and to reduce the risk of detection
(diuretics, probenecid) [14017].
Side effects of these nonsteroidal drugs include headache, nausea, nervousness, diarrhea,
perspiration, hot flushes, and bone pain. Athletes may add epitestosterone to normalize their
testosterone/epitestosterone ratios, thus avoiding testosterone-use detection. Researchers
have not adequately investigated interactions of AAS with nonsteroidal drugs [14017].
Association of performance-enhancing drug use with other high-risk behaviors
Athletes and nonathlete weightlifters that use PEDs often engage in other high-risk health
behaviors. In addition to the risks associated with concomitant use of other drugs such as
alcohol and opiates with AAS, users of high doses of AAS may be more susceptible to rage,
antisocial and violent behaviors, and suicidality. Sharing of needles and other paraphernalia
and unprotected sex may increase the risk of infections such as hepatitis and HIV. The use
of PEDs, especially in conjunction with analgesics or stimulants, may allow athletes to
engage in extremely high-intensity exercise, increasing the risk of musculoskeletal injuries
[14017].
Recombinant proteins
Advances in recombinant DNA technology have created one of the most powerful weapons
in the current doping arsenal: recombinant proteins [Sweeney HL. Gene doping. Sci Am
2004; 291: 62-9; Unal M, Ozer Unal D. Gene doping in sports. Sports Med 2004; 34: 357-62].
Recombinant erythropoietin (EPO) and human growth hormone (hGH) are currently being
abused but are fortunately detectable either directly by employing isoelectric focusing and
immunoassays or indirectly by assessing changes in selected hematopoietic parameters.
The detection is technically demanding due to the extent of similarity between the
recombinant proteins and their endogenous counterparts. Another issue facing detection
efforts is the speed and conditions at which blood samples are collected and analyzed in a
sports setting. Recently, gene doping, which stemmed out of legitimate gene therapy trials,
has emerged as the next level of doping. Erythropoietin (EPO), human growth hormone
(hGH), insulin-like growth factor-1 (IGF-1), peroxisome proliferator-activated receptor-delta
(PPAR delta), and myostatin inhibitor genes have been identified as primary targets for
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doping. Sports clinical scientists today are racing against the clock because assuring the
continued integrity of sports competition depends on their ability to outpace the efforts of
dopers by developing new detection strategies [05007].
Co-operation in drug testing
There are many areas of common interest between anti-doping laboratories and those
working in the clinical, legal and forensic fields. In addition to methodological similarities,
there are aspects of the findings in sport drug testing that overlap with other fields in such a
way that sport drug testing and clinical, legal or forensic work may benefit from mutual
interaction. Three recent examples are presented from the author's experience. Case report
1 concerns the clinical relevance of hCG findings in sport drug testing as potential indicators
of the presence of a (testicular) tumour in athletes. Case report 2 refers to difficulties that
accredited laboratories can encounter due to differences between national legal systems and
the administrative regulation systems of sport authorities. The example involves a network of
blood collection for further autologous transfusion. Case report 3 relates to additional
forensic-type investigations needed to interpret a situation where intoxication of a whole
delegation was responsible for apparent doping cases. Clinical, legal and forensic fields must
recognize the added value that some results and developments coming from anti-doping
laboratories may have. At the same time anti-doping analysts should be aware of new
issues, methodologies and problems appearing in related fields [10438].
Methodology for investigation of doping in the society
To date, there are estimates for the percentage of unknown cases of doping and illicit drug
use in fitness sports, but not for elite sports. This can be attributed to the problem of
implementing questionnaires and surveys to get reliable epidemiological estimates of deviant
or illicit behaviour. All athletes questioned were subject to doping controls as members or
junior members of the national teams. In order to estimate the prevalence of doping and illicit
drug abuse, the athletes were either issued an anonymous standardized questionnaire (SQ;
n=1394) or were interviewed using randomized response technique (RRT; n=480). It was
used a two-sided z-test to compare the standardized questionnaire and RRT results with the
respective official German NADA data on the prevalence of doping. Official doping tests only
reveal 0.81 percent (n=25,437; 95 % confidence interval 0.70 to 0.92 %) of positive test
results, while according to RRT 6.8 percent (n=480; 95 % confidence interval 2.7 to 10.9 %)
of our athletes confessed to having practiced doping. Standardized questionnaire and
randomized response technique both revealed a prevalence of about 7 percent for illicit drug
use, but SQ failed to indicate a realistic prevalence of doping (0.20 %; 95 % confidence
interval 0.02-0.74 %). It was thus demonstrate for the first time that data from official doping
tests underestimate the true prevalence of doping in elite sports by more than a factor of
eight. The results indicate that implementing randomized response technique before and
after anti-doping measures could be a promising method for evaluating the effectiveness of
anti-doping programs [09017].
Understanding athletes' attitudes and behavioural intentions towards performance

enhancement is critical to informing anti-doping intervention strategies. Capturing the
complexity of these attitudes beyond verbal declarations requires indirect methods. One pilot
study was aimed at developing and validating a method to assess implicit doping attitudes
using an Implicit Associations Test (IAT) approach. The conventional IAT evaluation task
(categorising ”good” and ”bad” words) was combined with a novel ”doping” versus ”nutrition
417
supplements” category pair to create a performance-enhancement related IAT protocol (PE-
IAT). The difference between average response times to ”good-doping” and ”bad-doping”
combinations represents an estimate of implicit attitude towards doping in relation to
nutritional supplements. 111 sports and exercise science undergraduates completed the PE-
IAT, the Performance Enhancement Attitude Scale (PEAS) and answered questions
regarding their beliefs about doping. Longer response times were observed in the mixed
category discrimination trials where categories ”good” and ”doping” shared the same
response key (compared to ”bad-doping” combination on the same key) indicating a less
favourable evaluation of doping substances. The PE-IAT measure did not correlate
significantly with the declared doping attitudes, indicating a predictable partial dissociation.
Action-oriented self-report expressed stronger associations with PE-IAT: participants who
declared they would consider using doping showed significantly less implicit negativity
towards banned substances. Similarly, those who reported more lenient explicit attitudes
towards doping or expressly supported legalizing it, showed less implicit negativity towards
doping in the sample, although neither observed differences reached statistical significance.
Known-group validation strategy yielded mixed results: while competitive sport participants
scored significantly lower than non-competitive ones on the PEAS, the two groups did not
differ on PE-IAT. It was concluded that the results suggest a potential of the PE-IAT method
to capture undeclared attitudes to doping and predict behaviour, which can support targeted
anti-doping intervention and related research [08019].
n=1394) or were interviewed using randomized response technique (RRT; n=480). It was
used a two-sided z-test to compare the standardized questionnaire and RRT results with the
respective official German NADA data on the prevalence of doping. Official doping tests only
reveal 0.81 percent (n=25,437; 95 % confidence interval 0.70 to 0.92 %) of positive test
results, while according to RRT 6.8 percent (n=480; 95 % confidence interval 2.7 to 10.9 %)
of our athletes confessed to having practiced doping. Standardized questionnaire and
randomized response technique both revealed a prevalence of about 7 percent for illicit drug
use, but SQ failed to indicate a realistic prevalence of doping (0.20 %; 95 % confidence
interval 0.02-0.74 %). It was thus demonstrate for the first time that data from official doping
tests underestimate the true prevalence of doping in elite sports by more than a factor of
eight. The results indicate that implementing randomized response technique before and
after anti-doping measures could be a promising method for evaluating the effectiveness of
anti-doping programs [09401].
Current antidoping policy, essentially a costly repressive zero-tolerance approach in elite

sport, will continue to be hampered by the limits of technology. False negatives and false
positives are inherent possibilities with testing technology and current protocols do not
adequately address the problem of biological and pre-analytical variability, which both may
lead to unreliable test results. This uncertainty is acceptable in the field of therapeutic
medicine but problematic in sport because athletes can never be considered truly clean,
whereas false accusations should be avoided at all cost. Pragmatically, the introduction of
additional analyses to the already huge armamentarium of antidoping tests is questionable,
both in ethical and economical terms, especially when the diagnostic efficiency of the new
tests is not proven [08045].
Enforcement of the antidoping-code is performed by doping controls. For this purpose, blood
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and urine samples of athletes are collected and analysed. In 2006 approximately 200,000
samples were analysed worldwide, with 1.96 percent being tested positive [08046].
Drug-testing practices are based on separation of a sample's constituents by

chromatography and detection of target compounds by mass spectrometry, one of the work-
horses in anti-doping research. Identification is reported as positive when the test and
reference sample signals agree within a particular tolerance window. However, the size of
this window is not constructed with an acceptable risk of false positives in mind. Rather, fixed
decision criteria hold, regardless of the quality of the laboratory or the signal properties of the
target compound. However, a laboratory that produces relatively precise results should
deploy stricter criteria. Likewise, target compounds should be differentiated so that
information in their signals can be respected [08047].
Always deploying the same rigid criteria leads to a probability of false positives that depends
on the particular laboratory and target compound in an undefined way. This situation is
frustrating because the statistical solution - flexible criteria that account for various
complications - was already published and thoroughly tested five years before these rigid
criteria were introduced. So, is it ignorance of the literature or failure to understand the
analytical problem at hand that underlies the ongoing usage of these arbitrary decision rules?
Laboratories, as well as their clients and (re-)accrediting organizations, should start reflecting
on their accountability with regard to this avoidable malpractice [08047].
Already, in the run-up to the Olympic Games, vast amounts of time, money and media
coverage have been spent on sports doping. Several doping experts have contended that
tests aren't sensitive enough and let dozens of cheaters slip through the cracks, but only
some athletes are facing sanctions. For example upon testing positive for clenbuterol, US
swimmer Jessica Hardy was held back from the 2008 Olympic team and faces a two-year
ban from the sport. China has already banned several athletes, some of them for life, on
doping charges. Indeed, many world-class athletes will find their life's accomplishments and
ambitions, their integrity and their reputations hinging on urine or blood tests [08048].
One factor at play in many cases that involve statistical reasoning, is what's known as the
prosecutor's fallacy. At its simplest level, it concludes guilt on the basis of an observation that
would be extremely rare if the person were innocent. Consider a blood test that perfectly
matches a suspect to the perpetrator of a crime. Say, for example, the matching profile
occurs in just 1 out of every 1,000 people. A naive prosecutor might try to convince a jury
that the odds of guilt are 999:1, that is, the probability of guilt is 0.999. The correct way to
determine odds comes from Bayes rule and is equal to 999 times P/(1-P) where P is the
“prior probability'“of guilt. Prior probability can be difficult to assess, but could range from very
small to very large based on corroborating evidence implicating the suspect. The
prosecutor's claim that the odds are 999:1 implies a prior probability of guilt equal to 0.5 (in
which case P and 1-P cancel). Such a high value of P is possible, but it would require
substantial evidence. Suppose there is no evidence against the suspect other than the blood
test: he was implicated only because he was from the city where the crime occurred. If the
city's population is one million then P is 1/1,000,000 and the odds of his guilt are 1001:1
against, which corresponds to a probability of guilt of less than 0.001 [08048].
Therefore it must be asked if, when an athlete tests positive, he or she without doubts is
guilty of doping? The international Court of Arbitration for Sport upheld doping charges
against cyclist Floyd Landis, stripping him of his title as winner of the 2006 Tour de France
and suspending him from competition for two years. The court agreed with the majority
opinion of a divided three-member American Arbitration Association (AAA) panel and
essentially placed a stamp of approval on a laboratory test indicating that Landis had taken
419
synthetic testosterone. Despite that there might be be inherent flaws in the testing practices
of doping laboratories. Close scrutiny of quantitative evidence used in Landis's case show for
example it to be non-informative. This says nothing about Landis's guilt or innocence. It
rather reveals that the evidence and inferential procedures used to judge guilt in such cases
don't address the question correctly [08048].
The prosecutor's fallacy is at play in doping cases. For example, Landis's positive test result
seemed to be a rare event, but just how rare? In doping cases the odds are dictated by the
relative likelihood of a positive test assuming the subject was doping (“sensitivity“) against a
positive result assuming no doping (which is one minus “specificity“). Sensitivity and
specificity are crucial measures that must be estimated with reasonable accuracy before any
conclusion of doping can be made. The studies necessary to obtain good estimates are not
easy to do. They require known samples, both positive and negative for doping, tested by
blinded technicians who use the same procedures under the same conditions present in
actual sporting events. Most often in doping testing, such studies have not been adequately
done, leaving the criterion for calling a test positive unvalidated [08048].
Urine samples from cyclists competing in the 2006 Tour de France were analysed at the
French national anti-doping laboratory in Châtenay-Malabry. This is one of 34 laboratories
accredited by the World Anti-Doping Agency to receive and analyse test samples from
athletes. The laboratory flagged Landis's urine sample following race stage 17, which he
won, because it showed a high ratio of testosterone to epitestosterone. Based on the initial
screening test, the laboratory conducted gas chromatography with mass spectrometry, and
isotope ratio mass spectrometry on androgen metabolites in Landis's sample. Such
laboratory tests involve a series of highly sophisticated processes that are used to identify
the likelihood of abnormal levels of plant-based androgen metabolites (from dietary or
pharmaceutical sources) in a urine sample. The goal is to differentiate from endogenous
androgen metabolites normally found in urine. Mass spectrometry requires careful sample
handling, advanced technician training and precise instrument calibration. The process is
unlikely to be error-free. Each of the various steps in handling, labelling and storing an
athlete's sample represents opportunity for error. The criteria used to discriminate a positive
from a negative result are set by the World Anti-Doping Agency. However, there is no way of
knowing which cases are truly positive and which are truly negative. It is proper to establish
threshold values such as these, but only to define a hypothesis; a positive test criterion
requires further investigation on known samples. However, the method used to establish the
criterion for discriminating one group from another has rarely been published, and tests have
not been performed to establish sensitivity and specificity. Without further validation in
independent experiments, testing is subject to extreme biases [08048].
Landis seemed to have an unusual test result. Because he was among the leaders he
provided 8 pairs of urine samples (of the total of approximately 126 sample-pairs in the 2006
Tour de France). So there were 8 opportunities for a true positive - and 8 opportunities for a
false positive. If he never doped and assuming a specificity of 95 percent, the probability of
all 8 samples being labelled “negative” is the eighth power of 0.95, or 0.66. Therefore,
Landis's false-positive rate for the race as a whole would be about 34 percent. Even a very
high specificity of 99 percent would mean a false-positive rate of about 8 percent. The single-
test specificity would have to be increased to much greater than 99 percent to have an
acceptable false-positive rate [08048].
More important than the number of samples from one individual is the total number of
samples tested. With 126 samples, assuming 99 percent specificity, the false-positive rate is
72 percent. So, an apparently unusual test result may not be unusual at all when viewed
from the perspective of multiple tests. This is well understood by statisticians, who routinely
420
adjust for multiple testing. It is believe that test results much more unusual than the 99 th
percentile among non-dopers should be required before they can be labelled “positive“
[08048].
Other doping tests are subject to the same weak science as testosterone, including tests for
naturally occurring substances, and some that claim to detect the presence of a foreign
substance. Detecting a banned foreign substance in an athlete's blood or urine would seem
to be clear evidence of guilt. But as with testing for synthetic testosterone, such tests may
actually be measuring metabolites of the drug that are naturally occurring at variable levels.
Whether a substance can be measured directly or not, sports doping laboratories must
prospectively define and publicize a standard testing procedure, including unambiguous
criteria for concluding positivity, and they must validate that procedure in blinded
experiments. Moreover, these experiments should address factors such as substance used
(banned and not), dose of the substance, methods of delivery, timing of use relative to
testing, and heterogeneity of metabolism among individuals. To various degrees, these same
deficiencies exist elsewhere - including in some forensic laboratories. It was stated that all
scientists share responsibility for this [08048].
Against this discussion representatives of the World Anti-Doping Agency (WADA) argued
that all WADA's accredited laboratories, including the French national laboratory must meet
standards set by the International Standard for Laboratories (ISL) in validation methods, staff
competency and chain of custody, for example. Compliance is assessed independently by
bodies of the International Laboratory Accreditation Cooperation. It should also be mentioned
that the majority of the substances reported by the anti-doping laboratories are exogenous
substances not naturally present in human beings. The development of testing procedures
for endogenous substances includes samples from normal reference populations and from
subjects administered with the substance under investigation, so that test-sample status and
positivity criteria can be established. To determine cut-offs for the ratio of testosterone to
epitestosterone (T/E), tens of thousands of athlete samples were analysed to establish
reference values. To detect exogenous administration of endogenous substances (such as
pharmaceutical testosterone) by isotope-ratio mass spectrometry (IRMS), validation is based
on a combination of hundreds of known positive and negative samples analysed by many
WADA anti-doping laboratories operating under the scrutiny of the ISL and of the
International Organization for Standardization (such as ISO 17025) [08049].
Each sample test includes positive and negative quality-control samples to assess the
possibility of a false result. If these samples fail, the test must be repeated. An adverse
analytical finding is not reported unless the quality criteria are met. All the laboratories
participate in at least four rounds of blind and one double-blind proficiency test per year; the
results of each round determine the accreditation status of the laboratory. False positives
mean immediate revocation of accreditation [08049].
Also, mass-spectrometry identification of exogenous substances relies on at least three

diagnostic ions to avoid any interference or misidentification. For immunoassays, antibodies
in the initial testing and confirmation procedures must have different antigen-epitope
specificity. For analytes that are too small to have two independent antigenic epitopes, two
different purification methods or two different analytical methods are used [08049].
Testosterone abuse is conventionally assessed by the urinary testosterone/epitestosterone

(T/E) ratio, levels above 4.0 being considered suspicious. A deletion polymorphism in the
gene coding for UGT2B17 is strongly associated with reduced testosterone glucuronide (TG)
levels in urine. Many of the individuals devoid of the gene would not reach a T/E ratio of 4.0
after testosterone intake. Future test programs will most likely shift from population based- to
421
individual-based T/E cut-off ratios using Bayesian inference. A longitudinal analysis is
dependent on an individual's true negative baseline T/E ratio. The aim of one study was to
investigate whether it is possible to increase the sensitivity and specificity of the T/E test by
addition of UGT2B17 genotype information in a Bayesian framework. A single intramuscular
dose of 500 mg testosterone enanthate was given to 55 healthy male volunteers with either
two, one or no allele (ins/ins, ins/del or del/del) of the UGT2B17 gene. Urinary excretion of
TG and the T/E ratio was measured during 15 days. The Bayesian analysis was conducted
to calculate the individual T/E cut-off ratio. When adding the genotype information, the
program returned lower individual cut-off ratios in all del/del subjects increasing the
sensitivity of the test considerably. It will be difficult, if not impossible, to discriminate
between a true negative baseline T/E value and a false negative one without knowledge of
the UGT2B17 genotype. UGT2B17 genotype information is crucial, both to decide which
initial cut-off ratio to use for an individual, and for increasing the sensitivity of the Bayesian
analysis [08050].
Alternative matrices
Human routine doping control samples currently include the matrices urine, serum, and
whole blood for the various different test menus allowing the conduction of targeted as well
as non-targeted analyses. Despite the broad analytical picture provided by these specimens,
complementary options such as oral fluid, sweat, and dried blood spots (DBS) are
continuously assessed to probe for added value enabled by these alternative matrices. It has
been comprehensively reviewed the potential role of oral fluid in sports drug testing, outlining
the apparent advantages (e.g. fast and non-invasive/non-intrusive sample collection, analysis
of intact drug, correlation of blood and oral fluid concentrations particularly helpful with drugs
prohibited in-competition only) and current limitations (drug stability, limited volume, short
detection windows, considerable knowledge gaps concerning drug disposition, contamination
issues, etc). Especially under consideration of the few controlled drug administration studies
currently available with respect to oral fluid analysis, a potential use of this matrix for selected
compounds banned in-competition only was concluded [14715].
With the continuously growing complexity of the options presumably enhancing athletic
performance, research in anti-doping science is becoming extremely multifaceted. Besides
the efforts made to expand test methods and to include more/new prohibited substances and
methods of doping into routine sports drug testing programs, an increasing number of studies
have been dedicated to complementary goals. These include predominantly the identification
of alternative doping control matrices as well as the fine-tuning of existing approaches to
reduce the burdens imposed on both the testing and the tested party and to improve the
sensitivity for ‘traditional’ doping agents. As a result of the latter, findings for stanozolol and
dehydrochloromethyltestosterone for instance increased considerably from approximately
290 in 2012 to over 540 in 2013. To accommodate the enormous number of legal highs,
designer stimulants, and synthetic cannabinoids, a substantial number of publications has
been recorded also in an anti-doping context although the larger misuse of these substances
by elite athletes has not been observed in laboratory analyses or obtained by other means of
anti-doping investigations. In contrast, the issue of (adulterated) nutritional supplements has
seemingly affected anti-doping statistics, where the drug with arguably modest stimulating
properties methylhexaneamine has resulted in 169 findings in 2013. Overall, the scientific
efforts undertaken by numerous international research groups have allowed for significant
accomplishments in the international fight against doping; much information has been
generated on doping agents and their detection as well as “traps” that athletes might fall into
inadvertently. For both scenarios, scientific data are invaluable and indispensable [14715].
422
A support vector machine
Due to their performance enhancing properties, use of anabolic steroids (e.g. testosterone,
nandrolone, etc.) is banned in elite sports. Therefore, doping control laboratories accredited
by the World Anti-Doping Agency (WADA) screen among others for these prohibited
substances in urine. It is particularly challenging to detect misuse with naturally occurring
anabolic steroids such as testosterone (T), which is a popular ergogenic agent in sports and
society. To screen for misuse with these compounds, drug testing laboratories monitor the
urinary concentrations of endogenous steroid metabolites and their ratios, which constitute
the steroid profile and compare them with reference ranges to detect unnaturally high values.
However, the interpretation of the steroid profile is difficult due to large inter-individual
variances, various confounding factors and different endogenous steroids marketed that
influence the steroid profile in various ways. A support vector machine (SVM) algorithm was
developed to statistically evaluate urinary steroid profiles composed of an extended range of
steroid profile metabolites. This model makes the interpretation of the analytical data in the
quest for deviating steroid profiles feasible and shows its versatility towards different kinds of
misused endogenous steroids. The SVM model outperforms the current biomarkers with
respect to detection sensitivity and accuracy, particularly when it is coupled to individual data
as stored in the Athlete Biological Passport [13019].
Performance profiling
In recent years, antidoping strategies underwent a significant development, from purely

biochemical analyses and the detection of substances in urine samples to a biological
approach, using blood samples, longitudinal monitoring, and probabilistic techniques.
Nowadays, the appropriate timing of testing and the targeting of the athletes to be tested with
antidoping tests is a major issue. A new strategy to improve the targeting of suspicious
athletes might be the longitudinal monitoring of individual performances. By these means,
suspect athletes might be identified, as doping will not only alter their blood or steroid
profiles, but ultimately boost their performance, as well. Through the proposed approach, the
effectiveness in the fight against doping might be improved considerably [09002].
Controls at random
Substance abuse, particularly among young people, does not seem to have the tendency to
decrease. The knowledges on this phenomenon are manifold and they validly compete to
address the actions of contrast. Nevertheless, it would seem profit to be able to have further
informations, to place side by side to those already existing, with the aim to improve the
surveillance of the phenomenon. For this purpose, in one paper it was proposed a monitoring
model based on the results of random controls on road, carried out by the Police (or by the
Hospital) in relationship to the road safety. The representativeness of the data that we could
get this way appears elevated as a high percentage of the population own the driver's
licence. As it is shown, these controls could be both individual and related to drivers' pools of
biological samples. This last approach would seem to be more practicable since problems
relating to the drivers' privacy would be avoided [09003].
Errors in drug testing
423
n=1394) or were interviewed using randomized response technique (RRT; n=480). Official
doping tests only reveal 0.81 percent (n=25,437; 95 % confidence interval 0.70 to 0.92 %) of
positive test results, while according to RRT 6.8 percent (n=480; 95 % confidence interval
2.7-10.9 %) of our athletes confessed to having practiced doping. SQ and RRT both revealed
a prevalence of about 7 percent for illicit drug use, but SQ failed to indicate a realistic
prevalence of doping. It was demonstrate for the first time that data from official doping tests
underestimate the true prevalence of doping in elite sports by more than a factor of eight
[10011].
Incongruity of data
Antidoping testing is currently exclusively based on hematochemical analysis performed in

specialized laboratories accredited by WADA (World Anti-Doping Agency). Many of the
analytical methods used for the determination of the parameters considered, such as
hematological parameters (hemoglobin, hematocrit and reticulocytes), proteins (soluble
transferrin receptor and hepcidin) and hormones (erythropoietin and growth hormone) are
often affected by lack of clear standardization and harmonization. The observed incongruity
of the data deriving from different laboratories often results in the risk of false positive results
in athletes [10012].
Gather data to reveal true extent of doping in sport
How many elite athletes take performance enhancing drugs? Sporting bodies say that it is a
very small minority. But a documentary broadcast in Germany suggested a much higher
figure. Several Russian athletes claimed that nearly all of their colleagues dope, and with the
knowledge of officials. The World Anti-Doping Agency (WADA) immediately launched an
investigation, which is expected to report this year. Science helps to keep sport clean by
developing tests to screen athletes for banned substances. Bodies such as WADA and the
US Anti-Doping Agency (USADA) say that they are doing all they can to deter doping. But
they have so far neglected to carry out a simple scientific analysis of how widespread the
problem is. Or if they have, they have not published the results. This makes it impossible for
the rest of us to assess whether anti-doping policies are working. Drug testing in sport, as
currently implemented, might catch the occasional cheat and could deter others, but these
results do little to help design an anti-doping strategy, and to independently assess whether it
works. For that, we need to know whether the number of athletes doping is going up or
down. And to do that, we need a reliable measure of what proportion of athletes dope. The
problem – and the best way to manage it – is very different if 1 percent of athletes dope than
if 50 percent of them do. Although the stated goal of anti-doping agencies is to prevent
prohibited drug use, they simply do not gather the data to enable evaluation of how effective
their policies are. This is despite sporting bodies across the world spending an estimated
USD 350 million on drug testing each year. Estimating the number of elite athletes who dope
is straightforward, and perfectly suited to the tools of science. Determining this number is
much easier than other efforts by scientists to quantify unknowns, such as estimates of the
number of planets in the Galaxy or whales in the sea. In probability-speak, it is a ball and urn
problem: how do we determine how many black balls there are in an urn that contains
1,000 white and black balls if we can sample only a small number? To assess the prevalence
of sports doping, such an analysis needs two things: a reliable estimate of the total
population of elite athletes and a proper randomized testing protocol. The first is readily
available. For instance, at the London 2012 Summer Olympics, nearly 11,000 athletes
424
participated from more than 200 countries. Each country conducted Olympic trials with its
own pool of registered, domestic competitors seeking to qualify for the games. For the
second requirement, because screening every athlete over a year is impractical, anti-doping
agencies could carry out randomized tests designed to support estimates of the prevalence
of doping alongside existing testing programmes at a marginal cost [151001].
Contextual modulation of androgen effects on agonistic interactions

Seasonal changes in steroid hormones are known to have a major impact on social behavior,
but often are quite sensitive to environmental context. In the bi-directionally sex changing
fish, Lythrypnus dalli, stable haremic groups exhibit baseline levels of interaction. Status
instability follows immediately after male removal, causing transiently elevated agonistic
interactions and increase in brain and systemic levels of a potent fish androgen, 11-
ketotestosterone (KT). Coupling KT implants with a socially inhibitory environment for
protogynous sex change induces rapid transition to male morphology, but no significant
change in social behavior and status, which could result from systemically administered
steroids not effectively penetrating into brain or other tissues. Here, it was first determined
the degree to which exogenously administered steroids affect the steroid load within tissues.
Second, it was examined whether coupling a social environment permissive to sex change
would influence KT effects on agonistic behavior. It was implanted cholesterol (Chol, control)
or KT in the dominant individual (alpha) undergoing sex change (on d0) and determined the
effects on behavior and the degree to which administered steroids altered the steroid load
within tissues. During the period of social instability, there were rapid (within 2 h), but
transient effects of KT on agonistic behavior in alphas, and secondary effects on betas. On
d3 and d5, all KT, but no Chol, treated females had male typical genital papillae. Despite
elevated brain and systemic KT 5 days after implant, overall rates of aggressive behavior
remained unaffected. These data highlight the importance of social context in mediating
complex hormone-behavior relationships [14033].
Theoretical testing
Currently a large range of pure substance reference materials are available for calibration of
doping-control methods. These materials enable traceability to the International System of
Units (SI) for the results generated by World Anti-Doping Agency (WADA)-accredited
laboratories. Only a small number of prohibited substances have threshold limits for which
quantification is highly important. For these analytes only the highest quality reference
materials that are available should be used. Many prohibited substances have no threshold
limits and reference materials provide essential identity confirmation. For these reference
materials the correct identity is critical and the methods used to assess identity in these
cases should be critically evaluated. There is still a lack of certified matrix reference materials
to support many aspects of doping analysis. However, in key areas a range of urine matrix
materials have been produced for substances with threshold limits, for example 19-
norandrosterone and testosterone/epitestosterone (T/E) ratio. These matrix-certified
reference materials (CRMs) are an excellent independent means of checking method
recovery and bias and will typically be used in method validation and then regularly as
quality-control checks. They can be particularly important in the analysis of samples close to
threshold limits, in which measurement accuracy becomes critical. Some reference materials
for isotope ratio mass spectrometry (IRMS) analysis are available and a matrix material
certified for steroid delta values is currently under production. In other new areas, for
example the Athlete Biological Passport, peptide hormone testing, designer steroids, and
gene doping, reference material needs still need to be thoroughly assessed and prioritized
[11029].
425
Efforts for drug free sport include developing a better understanding of the behavioural
determinants that underline doping with an increased interest in developing anti-doping
prevention and intervention programmes. Empirical testing of both is dominated by self-
report questionnaires, which is the most widely used method in psychological assessments
and sociology polls. Disturbingly, the potential distorting effect of socially desirable
responding (SD) is seldom considered in doping research, or dismissed based on weak
correlation between some SD measure and the variables of interest. The aim of one report
was to draw attention to the potential distorting effect of SD and to the limitation of using
correlation analysis between a SD measure and the individual measures. Models of doping
opinion as a potentially contentious issue was tested using structural equation modeling
technique (SEM) with and without the SD variable, on a dataset of 278 athletes, assessing
the SD effect both at the indicator and construct levels, as well as testing SD as an
independent variable affecting expressed doping opinion. Participants were categorised by
their SD score into high- and low SD groups. Based on low correlation coefficients observed
in the overall sample, SD effect on the indicator variables could be disregarded. Regression
weights between predictors and the outcome variable varied between groups with high and
low SD but despite the practically non-existing relationship between SD and predictors in the
low SD group, both groups showed improved model fit with SD, independently. The results of
this study clearly demonstrate the presence of SD effect and the inadequacy of the
commonly used pairwise correlation to assess social desirability at model level. In the
absence of direct observation of the target behaviour (i.e. doping use), evaluation of the
effectiveness of future anti-doping campaign, along with empirical testing of refined doping
behavioural models, will likely to continue to rely on self-reported information. Over and
above controlling the effect of socially desirable responding in research that makes
inferences based on self-reported information on social cognitive and behavioural measures,
it is recommended that SD effect is appropriately assessed during data analysis [11030].

alternative doping regimens or taking advantage of the limitations in testing methodologies
[150112].
Self-reporting
Social psychology research on doping and outcome based evaluation of primary anti-doping
prevention and intervention programmes have been dominated by self-reports. Having
confidence in the validity and reliability of such data is vital. Epidemiological and social
science research assessing social cognitions linked to doping behaviour has been
constrained by the almost exclusive use of self-report methodology. Anti-doping prevention
programmes are also evaluated via self-reported changes in attitudes and willingness to use
doping substances, anabolic steroids in particular. However, recent research has drawn
attention to a potential distorting effect of social desirability observed in self-reported social
426
cognitive measures related to doping. It has been shown, albeit on a small sample, that
taking self-reports at face value could lead to misleading conclusions about the social
cognitive processes that underlie doping behaviour. Whilst differences in explicit (self-
reported) social cognitive measures between user and non-user groups were observed in the
expected direction when groups were created from self-report, generally the reverse was
evidenced when the user status was based on hair analysis results (i.e. based on the
presence of at least one prohibited performance enhancing drug in hair). Implicit measures
were consistent with the grouping based on hair analysis. The outcome of this project
suggested that respondents may consistently manipulate their answers on all related
measures in order to maintain the image they wish to project, although the possibility that this
response bias might stem, at least partially, from self-deception (as opposed to strategic
responding for impression management) cannot be ruled out. The strikingly different patterns
in self-reports and implicit associations in the context of behavioural data inevitably lead to
the question of: Which data should we trust? The self-report methodology has endured a mix
of support and criticism in the past. Whilst a plethora of literature suggests that self-report
can yield a valid assessment of substance use behaviour, it has also drawn equally strong
criticism. Whilst these tests have provided adequate evidence of validity and reliability, other
studies using various biomarkers to validate self-reported behaviour data have put
convincing evidence forward for a considerable under-reporting of substance use. A
systematic review revealed that this bias is not limited to socially undesirable behaviours; it
also extends to simple measures such as height and weight with a tendency towards over-
and under-reporting, respectively. The effect of gender, race, age and contextual
contingencies such as drug type and seriousness of the offence on over- and under-reporting
substance use has also been investigated with race as the only factor so far demonstrating
an effect on admitting drug taking behaviour. One study utilised a mixed method design to
afford triangulation between explicit measures through self-reported questionnaire, implicit
associations using a computerised test for latency measures, and bioanalysis via hair
specimens. The sample of 82 athletes from 30 sports (52 % female, mean age: 21 years)
was split into quasi-experimental groups based on self-admitted previous experience with
prohibited performance enhancing drugs (PED) and the presence of at least one prohibited
PED in hair covering up to 6 months prior to data collection. Approximately 100 mg untreated
head hair, cut at scalp, was screened using ELISA kits for the presence of the most
commonly used anabolic steroids (stanozolol, nandrolone and boldenone). Positive samples
for anabolic steroids were confirmed and quantified using liquid chromatography-tandem
mass spectrometry (LC-MS/MS) methods. Erythropoietin (EPO) was detected using
quantifiable ELISA. Hair digestion and analytical methods using LC-MS/MS were developed
in house to increase sensitivity and to reduce the amount of hair required. Participants
responded to questionnaires assessing a range of social cognitive determinants of doping via
self-reports; and completed a modified version of the Brief Implicit Association Test (BIAT)
assessing implicit attitudes to doping relative to the acceptable nutritional supplements (NS).
Social projection regarding NS was used as control. PEDs were detected in hair samples
from 10 athletes (12 % prevalence), none of whom admitted doping use. Nutritional
supplement use was reported by 60 percent, whereas those having personal experience with
doping constituted 13 percent of the athletes. Admitted doping appears to be independent of
self-reports on nutritional supplements or social drug use. Eight hair samples were positive
for stanozolol and two for EPO, giving a 12 percent prevalence rate for prohibited
performance enhancing substance use in the sample. EPO levels were 13 picog/mg and
12.53 picog/mg for the two positives. Interestingly, more males admitted having experience
with doping than females (21 % vs 7 %, respectively) with a reversed pattern for positive hair
analysis (5 % vs 19 %, respectively). None of the athletes who returned positive hair samples
admitted doping use. Conversely, no self-admitted doping was confirmed by current hair
sample tests. Social projections for nutritional supplements, doping and social drug use
showed a positive, statistically significant but relatively weak relationship between fellow
427
athletes using prohibited performance enhancing drugs and nutritional supplements and
social drugs by the general population. Based on self-declared doping behavior, an
interaction effect between user group and gender was only found for social projection of
doping and nutritional supplements use by fellow athletes. No gender difference was
evidenced for any of the outcome variables except the pressure to use banned substances.
A statistically significant difference was found between admitted doping users and non-users
in explicit attitude and social projection of doping use, with a borderline significance for
pressure to use doping. As expected, those who admitted having personal experience with
doping exhibited more of a lenient attitude (shown by higher PEAS score) towards doping
and gave higher estimated proportions of doping users among athletes and reported higher
perceived pressure to use doping. Athletes who denied doping contrary to the evidence in
their hair samples exhibited less lenient attitudes toward doping. These results support the
previous observation that athletes who deny their doping use behaviour gave answers on
social cognitive measures that are consistent with a typical non-user. In other word, they are
“faking good” in a consistent manner. These results are not surprising, considering that the
outcome measures were exclusively based on self-declarations. The picture, however, has
changed for the implicit associations. Albeit the differences in latency or D-scores did not
reach statistical significance, the sample means suggest that performance on the BIAT was
revealing for deniers. That is, latency measures and D-scores set deniers apart from clean
and self-admitted users, but did not differentiate between the latter two groups. The majority
of the athletes (89 %) indicated that they would prefer to compete in a doping free
environment. The remaining 11 percent opted for a scenario in which both players use
doping. The fact that no athletes opted for a unilateral use of doping suggests that there is a
proportion of athletes who might be more motivated about enhancing performance in general
(including using prohibited means) than gaining competitive advantage against opponents.
Interestingly, 62 percent of all respondents believed that athletes use performance
enhancing substances in training and competition. Overall, discrepancies in the relationship
between declared doping-related opinion and implicit doping attitudes were observed
between the groups, with control measures remaining unaffected. The results suggest, with
considerable confidence, that the “denier” group is characterised by a pattern of dissociation
between explicit and implicit responding. This dissociation is, in fact, likely to be a cognitive
marker for this group, which may lead to a promising application of the combined explicit-
implicit cognitive protocol used in this study as a proxy for the less readily available
biochemical detection methods for large scale social science research on doping. Athletes'
views on doping were first and foremost influenced by the “legality” of the substance, then on
performance. In this, athletes interviewed considered prohibited performance enhancing
substances as cheating but acceptable enhancers as essential. The most important
contribution that the results can add to drug use research is the observed distinct patterns of
explicit and implicit responding among self-declared doping users and deniers which may
lead to significant advances in both detection and treatment interventions for these groups.
The findings question the validity of self-reports which may have significant implications in
interpreting previous and future doping research. A combination of self-report and implicit
cognitive measures seems to hold the strongest promise for future doping research. It is this
combination that is likely to produce, with attendant methodology refinements, robust
cognitive markers of denial. Objective verification using biomarkers or chemical analysis may
not be a feasible approach in all social science research. However, our results suggest that
triangulating results obtained on the same or related constructs but using different
methodologies could be a cost-effective avenue. Hence, further research into the methods of
combining self-report methodology, with indirect, implicit methods is warranted. Assuming
that social desirability has a root in contextual contingencies, research among different user
groups could be beneficial. Doping social science research, particularly quantitative
research, is seriously lacking in studies using samples drawn from athletes banned from
competition owing to doping offences and longitudinal research. Research in this field would
428
benefit from looking beyond doping and having a greater use of direct and indirect methods
from social psychology, particularly those used successfully in substance use and addiction
research. Incorporating implicit social cognition is one promising avenue for doping social
science research. Although it is still debated whether implicit social cognitions reveals
something about the individual or the individual's environment, implicit social cognition
research is among the thriving areas in social psychology. Doping research, owing to the
unique nature of doping (i.e. being positioned between illicit behaviour and functional use of
ergogenic aids) provides an excellent testing field for developing a better understanding of
the explicit and implicit social cognition and the environment. Questionnaire responses thus
showed a pattern consistent with self-reported doping use. Following this preliminary work,
the study provides further evidence that both self-reports on behaviour and social cognitive
measures could be affected by some form of response bias. This can question the validity of
self-reports, with reliability remaining unaffected. Triangulation of various assessment
methods is recommended [11031].
Testing as a way of decreasing intent for doping
To assess the effects of random drug and alcohol testing (DAT) among high school athletes
a 2-year prospective randomized controlled study was performed. It was a study of a single
cohort among five intervention high schools with a DAT policy and six schools with a
deferred policy, serially assessed by voluntary, confidential questionnaires. DAT school
athletes were at risk for random testing during the full academic year. Positive test results
were reported to parents or guardians, with mandatory counseling. Indices of illicit drug use,
with and without alcohol use, were assessed at the beginning and end of each school year
for the past month and prior year. Potential mediating variables were evaluated. Student-
athletes from intervention and control schools did not differ in past 1-month use of illicit drug
or a combination of drug and alcohol use at any of the four follow-up periods. At the end of
the initial school year and after 2 full school years, student-athletes at DAT schools reported
less drug use during the past year compared to athletes at the deferred policy schools.
Combining past year drug and alcohol use together, student-athletes at DAT schools
reported less use at the second and third follow-up assessments. Paradoxically, DAT
athletes across all assessments reported less athletic competence, less belief authorities
were opposed to drug use, and indicated greater risk-taking. At the final assessment, DAT
athletes believed less in testing benefits and less that testing was a reason not to use drugs.
It was concluded that DAT deterrent effects were evident for past month use during any of
four follow-up periods. Prior-year drug use was reduced in two of four follow-up self-reports,
and a combination of drug and alcohol use was reduced at two assessments as well. Overall,
drug testing was accompanied by an increase in some risk factors for future substance use.
More research is needed before DAT is considered an effective deterrent for school-based
athletes [07018].
Fatigue as a limit for test performance
Muscle physiologists often describe fatigue simply as a decline of muscle force and infer this
causes an athlete to slow down. In contrast, exercise scientists describe fatigue during sport
competition more holistically as an exercise-induced impairment of performance. The aim of
one review was to reconcile the different views by evaluating the many performance
symptoms/measures and mechanisms of fatigue. It was describe how fatigue is assessed
with muscle, exercise or competition performance measures. Muscle performance (single
muscle test measures) declines due to peripheral fatigue (reduced muscle cell force) and/or
central fatigue (reduced motor drive from the CNS). Peak muscle force seldom falls by >30
percent during sport but is often exacerbated during electrical stimulation and laboratory
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exercise tasks. Exercise performance (whole-body exercise test measures) reveals impaired
physical/technical abilities and subjective fatigue sensations. Exercise intensity is initially
sustained by recruitment of new motor units and help from synergistic muscles before it
declines. Technique/motor skill execution deviates as exercise proceeds to maintain
outcomes before they deteriorate, e.g. reduced accuracy or velocity. The sensation of fatigue
incorporates an elevated rating of perceived exertion (RPE) during submaximal tasks, due to
a combination of peripheral and higher CNS inputs. Competition performance (sport
symptoms) is affected more by decision-making and psychological aspects, since there are
opponents and a greater importance on the result. Laboratory based decision making is
generally faster or unimpaired. Motivation, self-efficacy and anxiety can change during
exercise to modify RPE and, hence, alter physical performance. Symptoms of fatigue during
racing, team-game or racquet sports are largely anecdotal, but sometimes assessed with
time-motion analysis. Fatigue during brief all-out racing is described biomechanically as a
decline of peak velocity, along with altered kinematic components. Longer sport events
involve pacing strategies, central and peripheral fatigue contributions and elevated RPE.
During match play, the work rate can decline late in a match (or tournament) and/or
transiently after intense exercise bursts. Repeated sprint ability, agility and leg strength
become slightly impaired. Technique outcomes, such as velocity and accuracy for throwing,
passing, hitting and kicking, can deteriorate. Physical and subjective changes are both less
severe in real rather than simulated sport activities. Little objective evidence exists to support
exercise-induced mental lapses during sport. A model depicting mind-body interactions
during sport competition shows that the RPE centre-motor cortex-working muscle sequence
drives overall performance levels and, hence, fatigue symptoms. The sporting outputs from
this sequence can be modulated by interactions with muscle afferent and circulatory
feedback, psychological and decision-making inputs. Importantly, compensatory processes
exist at many levels to protect against performance decrements. Small changes of putative
fatigue factors can also be protective. We show that individual fatigue factors including
diminished carbohydrate availability, elevated serotonin, hypoxia, acidosis, hyperkalaemia,
hyperthermia, dehydration and reactive oxygen species, each contribute to several fatigue
symptoms. Thus, multiple symptoms of fatigue can occur simultaneously and the underlying
mechanisms overlap and interact. Based on this understanding, it was reinforce the proposal
that fatigue is best described globally as an exercise-induced decline of performance as this
is inclusive of all viewpoints [11032].
Psychometric testing in doping control in sport
One of the fundamental challenges in anti-doping is identifying athletes who use, or are at
risk of using, prohibited performance enhancing substances. The growing trend to employ a
forensic approach to doping control aims to integrate information from social sciences (e.g.
psychology of doping) into organised intelligence to protect clean sport. Beyond the
foreseeable consequences of a positive identification as a doping user, this task is further
complicated by the discrepancy between what constitutes a doping offence in the World Anti-
Doping Code and operationalized in doping research. Whilst psychology plays an important
role in developing our understanding of doping behaviour in order to inform intervention and
prevention, its contribution to the array of doping diagnostic tools is still in its infancy. In both
research and forensic settings, we must acknowledge that (1) socially desirable responding
confounds self-reported psychometric test results and (2) that the cognitive complexity
surrounding test performance means that the response-time based measures and the lie
detector tests for revealing concealed life-events (e.g. doping use) are prone to produce false
or non-interpretable outcomes in field settings. Differences in social-cognitive characteristics
of doping behaviour that are tested at group level (doping users vs non-users) cannot be
430
extrapolated to individuals; nor these psychometric measures used for individual diagnostics.
In this paper, we present a position statement calling for policy guidance on appropriate use
of psychometric assessments in the pursuit of clean sport. It was argued that, to date, both
self-reported and response-time based psychometric tests for doping have been designed,
tested and validated to explore how athletes feel and think about doping in order to develop a
better understanding of doping behaviour, not to establish evidence for doping. A false
positive psychological profile for doping affects not only the individual clean athlete but also
their entourage, their organisation and sport itself. The proposed policy guidance aims to
protect the global athletic community against social, ethical and legal consequences from
potential misuse of psychological tests, including erroneous or incompetent applications as
forensic diagnostic tools in both practice and research [150113].
Implicit Association Test
Attitudes are among the strongest social cognitive predictors of human behavior. Direct (i.e.
self-report) assessment of socially sensitive attitudes can be distorted by social desirability
bias because the purpose of a given test often is easy to determine, and thus allows
participants to deliberately choose and alter their responses. The Implicit Association Test
(IAT) constitutes a class of reaction-time based indirect tests that aim to hide the true goal of
measurement better than do direct tests. It is presented typically as a lexical sorting task on a
computer, where two concepts (one target and one evaluative) are mapped on the same
response key of the keyboard. The task is easier and reaction times are faster when the two
concepts that share the same response key (e.g. flowers + like) are closely associated,
rather than when they are not associated (e.g. insects + like). The IAT methods have evolved
as one standard for indirect attitude testing in social cognition research. One of the IAT's
most important features is its postulated potential to control for the social desirability bias by
evading voluntary control and being rather robust toward deception attempts compared to
direct tests. Indeed, compared with questionnaires, IATs display higher predictive validity
when socially sensitive constructs are measured. As a more economic, but equally valid and
reliable variant, Brief IATs (BIAT) have received considerable scientific attention in the past
few years [150119].
Doping attitudes are among the strongest statistical predictors of doping behavior. Doping in
sports is a socially and legally sanctioned behavior. Therefore, people with rather permissive
doping attitudes are often motivated to disguise their real attitude and instead provide the
socially desired response, namely that they dislike doping. Whereas, data from doping
attitude questionnaires is often skewed and of very limited value for the prediction of doping
behavior, the doping BIAT has been found to be a valid predictor for positive biochemical
doping test results. In general, the IAT's robustness toward faking has been heavily studied
as of late. It was used the terms faking and deception synonymously in this article because
the former is more common in the cited social cognition research, whereas the latter is more
common in the neuroscientific research that was cited. So far, results have indicated that the
IAT can be deceived to some extent. However, most participants need to be instructed
regarding a successful faking strategy. For example, it was shown that participants could not
effectively conceal their positive attitude toward flowers unless they were told how to do so.
Only after having been provided with the explicit strategy to respond more slowly when the
concepts flower and like were mapped on the same response key did participants not reveal
their positive attitude. Recently, considerable research efforts also have been devoted to
autobiographical IAT (aIAT) faking. Here, the truthfulness of a previously established
autobiographic memory is evaluated using reaction-time based IAT methodology. Regarding
faking the aIAT, response slowing likewise has been demonstrated as an effective means to
fake this test. In addition, a study has demonstrated the possibility to fake the aIAT by
speeding up responses in the incongruent block. However, the aIAT differs from traditional
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IAT variants in that it focuses on autobiographical memories and not on social cognitive
predictors of behavior, such as attitudes [150119].
There are theoretical and practical reasons for why research on IAT faking has become
topical in the social cognition literature: Evidence showing that IATs can be faked has
challenged the theoretical claim that IAT scores really reflect implicit associations. These are
theorized to represent output from the impulsive system of the social information system and
should therefore be immune to faking. From a practical perspective, test-takers with high
motivation to disguise their true attitude will most likely begin to develop and apply deception
strategies. It is thus important to investigate possibilities to detect fake test results and
potential threats to test validity in general. Faking and deception, in general, have also been
studied using EEG, most often investigating guilty-knowledge paradigms. Overall, results
have suggested that there is no specific lie response in ERPs. Rather, ERP differences may
strongly reflect the involved cognitive processes. For example, investigated groups of
participants who were either instructed to commit a mock crime or not. When presenting
“guilty” participants with rare crime-relevant – compared to frequent crime-irrelevant – stimuli,
these participants showed an increased P300 compared to an “innocent” control group that
was shown rare information that was autobiographical, but not related to the mock crime.
This is in line with increased P300 amplitudes often found in oddball experiments for novel
stimuli, and for stimuli that are inconsistent with the established context or inconsistent with
participants' attitudes. However, it has also been found a decrease in P300 for deceptive
responses in a design where participants had to make an equal number of honest and
deceptive responses. It might be that the visual processing of the faking stimuli seems to
have been down-regulated, suggesting perceptual disengagement from critical target stimuli
as one mechanism of successful faking. Previous research has indicated that P300
amplitudes decrease as the cognitive resources needed by a secondary task increase. Thus,
in balanced faking designs, a decrease in the P300/LPP is related to the amount of
monitoring processes needed and cognitive control required [150119].
More recently, even earlier differences are reported when participants faked responses to
self-related vs. non-self-related information. Previously for the N1 and N2, an increased
negativity was found for faking. This could reflect the conflict between the automatic and the
response actually given, as an increased N2 is also found for responses to incongruent
prime-target pairs. In line with this, increased N1 and N2 were also found when participants
had to inhibit responses in NOGO tasks compared to equally frequent GO tasks. These
differences were linked to enhanced activity in right inferior frontal regions. In previous
deception studies, participants responded either truthfully or deceitfully to stimuli in yes/no
forced-choice formats. We aimed to apply these findings to reaction-time based tests. We
therefore investigated the cerebral correlates of faking an attitude test by enabling test takers
to alter their responses in a test where faking is difficult (i.e. when participants are not
informed of how this test can be faked), but whose socially sensitive content induces
participants to do so. In line with the experimental paradigm most often used in social
cognition research on IAT faking, participants were given an explicit faking strategy.
Response slowing on one's true attitude is the most commonly implemented strategy
although it also seems possible to cheat on at least some variants of the IAT via response
acceleration. As response slowing has been used more often in the literature, and as
response acceleration suffers from the problem that there is a natural lower limit on reaction
times – such that if participants really show full effort on baseline testing, they may not be
able to go much faster – response slowing was the faking instruction chosen in the present
study [150119].
Direct assessment of attitudes toward socially sensitive topics can be affected by deception
attempts. Reaction-time based indirect measures, such as the Implicit Association Test (IAT),
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are less susceptible to such biases. Neuroscientific evidence shows that deception can
evoke characteristic ERP differences. However, the cerebral processes involved in faking an
IAT are still unknown. It was randomly assigned 20 university students (15 females, 25 years
of age) to a counterbalanced repeated-measurements design, requesting them to complete a
Brief-IAT (BIAT) on attitudes toward doping without deception instruction, and with the
instruction to fake positive and negative doping attitudes. Cerebral activity during BIAT
completion was assessed using high-density EEG. Event-related potentials during faking
revealed enhanced frontal and reduced occipital negativity, starting around 150 ms after
stimulus presentation. Further, a decrease in the P300 and LPP components was observed.
Source analyses showed enhanced activity in the right inferior frontal gyrus between 150 and
200 ms during faking, thought to reflect the suppression of automatic responses. Further,
more activity was found for faking in the bilateral middle occipital gyri and the bilateral
temporoparietal junction. Results indicate that faking reaction-time based tests alter brain
processes from early stages of processing and reveal the cortical sources of the effects.
Analyzing the EEG helps to uncover response patterns in indirect attitude tests and broadens
our understanding of the neural processes involved in such faking. This knowledge might be
useful for uncovering faking in socially sensitive contexts, where attitudes are likely to be
concealed [150119].
The Implicit Association Test (IAT) aims to measure participants' automatic evaluation of an
attitude object and is useful especially for the measurement of attitudes related to socially
sensitive subjects, e.g. doping in sports. Assessment of attitudes on socially sensitive
subjects using traditional self-report measures is susceptible to faking. The Implicit
Association Test (IAT) aims to reflect test participants’ automatic evaluations of an attitude
object. It is a reaction time-based, computerized sorting task intended to conceal the true
measurement target, and thus potentially more resistant to faking. Research has shown that
IATs are especially useful in the assessment of attitudes related to socially sensitive subjects
(e.g. stereotyping or prejudice). Faking an IAT on the very first exposure appears to be
almost impossible, but evidence is accumulating that IATs can be faked to some extent when
participants are allowed to become familiar with the test format. All previous studies have
explicitly asked participants to try faking their IAT score. Successful faking has been
assessed using both within- and between-subject designs, and in some studies participants
were given explicit instructions on how to fake the test (e.g. slowing of responses in sensitive
trials and reacting as quickly as possible in others). In summary, extant studies indicate that
successful IAT faking increases when participants are given repeated opportunities to fake
the test, and that provision of an effective faking strategy is an even more effective aid to
faking. Instructing participants to fake test scores in order to assess the vulnerability of the
test to faking, or to learn how to detect faking attempts, is a method which has also been
used in other research areas e.g. in memory malingering. In these studies participants are
usually assigned to a faking or a non-faking condition by the experimenter. This approach
secures internal control on the participants’ group membership. Other studies from the same
research area however suggest that participants who have been instructed to fake poor
performance on memory tests tend to over-exaggerate their memory deficits. Taking our cue
from this line of research it is suggested that the same could happen when participants are
asked and instructed to fake an IAT score: results from instructed faking attempts (a behavior
that is ethically acceptable in this case) might differ from uninstructed, hidden faking
attempts. It is believed that this difference might account for conflicting data on the IAT’s
vulnerability to faking as well as for ambiguous findings with respect to the IAT’s predictive
validity [150120].
Athletes’ attitudes to doping are subject to social pressure in a way that they are expected to
be strongly anti-doping. Direct assessments with attitude questionnaires are heavily biased
by socially desirable responding. Attitudes to doping are therefore a suitable substrate in
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which to investigate the IAT’s susceptibility to faking. In the following study we tested the
hypothesis that in an experimental condition in which an implicit incentive to fake was
present, participants would be capable of faking an attitude on a questionnaire, the
Performance Enhancement Attitude Scale (PEAS), but not on a response time-based
measure, the doping Brief IAT (BIAT). Several studies indicate that IAT scores can be faked
on instruction. But fully or semi-instructed research scenarios might not properly reflect what
happens in more realistic situations, when participants secretly decide to try faking the test.
The present study is the first to investigate IAT faking when there is only an implicit incentive
to do so. Sixty-five athletes (23 years ± 2; 25 women) were randomly assigned to an
incentive-to-fake condition or a control condition. Participants in the incentive-to-fake
condition were manipulated to believe that athletes with lenient doping attitudes would be
referred to a tedious 45-minute anti-doping program. Attitudes were measured with the
pictorial doping brief IAT (BIAT) and with the Performance Enhancement Attitude Scale
(PEAS). A one-way MANOVA revealed significant differences between conditions after the
manipulation in PEAS scores, but not in the doping BIAT. In the light of our hypothesis this
suggests that participants successfully faked an exceedingly negative attitude to doping
when completing the PEAS, but were unsuccessful in doing so on the reaction time-based
test. This study assessed BIAT faking in a setting that aimed to resemble a situation in which
participants want to hide their attempts to cheat. The two measures of attitude were
differentially affected by the implicit incentive. The findings provide evidence that the pictorial
doping BIAT is relatively robust against spontaneous and naïve faking attempts. (B)IATs
might be less prone to faking than implied by previous studies [150120].
Economical aspects
According to the framework legislation promulgated as part of the reform of finance laws in
France, quality is a mandatory feature of all governmental actions. In this context, this work
was conducted to assess the construction cost of a national health program designed to
promote physical and sports activities and prevent doping behaviors. This program was
considered to have the characteristic features of a successful governmental health
intervention. Four cost categories were evaluated: cost of the activity itself, transportation
costs, communication costs and promotion costs. It was found that the program costs for
2002-2007 were 100,000 euros, with 15 percent of the costs in the communication category.
This means that economic elements could be associated with factors of successful health
service interventions in order to help decision makers responsible for the public interest and
the consistency of public health actions [09012].
The overall economic costs of the fight against a doping allegation deserve consideration.
WADA is the international independent organisation created in 1999 to promote, coordinate
and monitor the fight against doping in sport in all its forms. Composed of and funded equally
by the sports movement and governments of the world, WADA received its first 2 years of
funding (USD18.3 million) from the Olympic Movement. Since 2002, according to its statutes,
WADA's funding is sourced equally from the Olympic Movement and the governments of the
world. The total budget for the year 2006 has been estimated at nearly USD 24 million, 60
percent for research, 15 percent for out of competition testing, 15 percent for education and
10 percent for contingencies. The budget of the Australian Sports Anti‐Doping Authority
(ASADA) for the period 1 July 2005 to 30 June 2006 was 13.681 million Australian dollars.
The total revenue from grants and other financial support of the US Anti‐Doping Agency
(USADA) reached USD 10.9 million in the year 2005. These are only part of the huge
economic resources currently devoted to the fight against doping worldwide, which are
predicted to reach or exceed USD 10 billion. Therefore, in practice, healthcare systems and
national governments worldwide are expected to devote to the fight against doping the same
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resources that the US government dedicates to prevention and treatment of diseases that
cause great morbidity, mortality and economic burden for individuals, families and the entire
population. Is this really necessary and morally acceptable? [07030].
Chemoinformatics-based classification of prohibited substances
Representative molecules from 10 classes of prohibited substances were taken from the
World Anti-Doping Agency (WADA) list, augmented by molecules from corresponding activity
classes found in the MDDR database. Together with some explicitly allowed compounds,
these formed a set of 5245 molecules. Five types of fingerprints were calculated for these
substances. The random forest classification method was used to predict membership of
each prohibited class on the basis of each type of fingerprint, using 5-fold cross-validation. It
was also used a k-nearest neighbors (kNN) approach, which worked well for the smallest
values of k. The most successful classifiers are based on Unity 2D fingerprints and give very
similar Matthews correlation coefficients of 0.836 (kNN) and 0.829 (random forest). The kNN
classifiers tend to give a higher recall of positives at the expense of lower precision. A naïve
Bayesian classifier, however, lies much further toward the extreme of high recall and low
precision. The results suggest that it will be possible to produce a reliable and quantitative
assignment of membership or otherwise of each class of prohibited substances. This should
aid the fight against the use of bioactive novel compounds as doping agents, while also
protecting athletes against unjust disqualification [06034].
Need of excretion studies
The list of prohibited anabolic steroids in sports has grown due to the addition of numerous
steroids that have been introduced on the market by non-pharmaceutical companies.
Moreover, several designer steroids, specifically developed to circumvent doping control,
have also been detected. Because anabolic steroids are most often intensively subjected to
phase I metabolism and seldom excreted unchanged, excretion studies need to be
performed in order to detect their misuse [06004].
Chemical and physical manipulations of doping tests
Detection and proof of doping control sample manipulation is a challenging task, and one of
the most efficient tools to identifyurine substitution is careful steroid profile evaluation. In
2009/2010, identical steroid profiles of supposedly eight different athletes (from different
teams and collection sites) were found and DNA analyses requested, demonstrating that all
eight urine specimens were provided from a single donor. This donor was eventually
identified as the doping control officer and none of the athletes was actively involved in the
sample manipulation. In another case of urine substitution, no natural endogenous steroid
was observed in steroid profile analyses, triggering further investigations into the composition
of the specimen. Based on findings of hordenine, trace amounts of alcohol, various
saccharides and intact proteins including Serpin-Z4, the liquid was identified as non-alcoholic
beer. This manipulation however entailed the suspension of the athlete [12017].
Manipulation of urine specimens provided by elite athletes for doping control purposes has
been reported several times in the past, and in most of these cases urine substitution was
eventually proven. Recent findings of suspected and substantiated manipulation have
outlined the complexity and diversity of tampering options, sample appearance alterations
435
resulting from non-manipulative influence, and the analytical challenges arising from these
scenarios. Using state-of-the-art mass spectrometric and immunological doping control and
forensic chemistry methodologies, four unusual findings were observed. One sports drug
testing specimen was found to contain an unusually high content of saccharides
accompanied by hordenine and Serpine-Z4, while no endogenous steroid (e.g. testosterone,
epitestosterone, androsterone and etiocholanolone) was detected. This specimen was
identified as non-alcoholic beer filled into the doping control sample container, constituting an
undisputed doping offense. A doping control sample of bright green color was received and
found to contain residues of methylene blue, which is not considered relevant for doping
controls as no masking or manipulative effect is known. In addition, the number of urine
samples of raspberry to crimson red coloration received at doping control laboratories has
constantly increased during the last years, attributed to the presence of hemoglobin or
betanin/isobetanin. Also here, no doping rule violation was given and an impact on routine
analytical results was not observed. Finally, a total of 8 sports drug testing samples collected
at different competition sites was shown to contain identical urine specimens as indicated by
steroid profile analysis and conclusively proven by DNA-STR (short tandem repeat) analysis.
Here, the athletes in question were not involved in the urine substitution act but the doping
control officer was convicted of sample manipulation [12088].
The case of seven urine samples collected for anti-doping purposes during a cycling stage
race with moderately elevated testosterone and epitestosterone ratio (T/E) is reported. The
very low probability of having all seven urine samples with such similar elevated T/E ratio
(from 3.2 to 4.7) was very suspicious. Different pattern classification tools were tested to
categorize the most similar steroid profiles, but none of the models enabled a clear
classification of the different urine samples. Subsequently, genetic profiling of all urine
samples was performed and demonstrated that three of the seven samples were collected
from the same cyclist. Finally, the International Federation confirmed DNA profiling results.
This suggests that urinary steroid data using several methodologies are not appropriate for
identification purposes and to an extent not unique to individuals [06033].
Designer drugs in Japan
In recent years, many analogs of narcotics have been widely distributed as easily available
psychotropic substances and have become a serious problem in Japan. To counter the
spread of these non-controlled substances, the Pharmaceutical Affairs Law in Japan was
amended in 2006 to establish a new category; Designated Substances in order to more
strictly control these substances. In April 2007, 31 compounds and 1 plant were first
controlled as Designated Substances. Before 2007, the major compounds distributed in the
Japanese illegal drug market were tryptamines, phenethylamines and piperazines. Alkyl
nitrites, such as isobutyl nitrite and isopentyl nitrite, were also widely distributed. After they
were listed as Narcotics or Designated Substances in 2007, these compounds, especially the
tryptamines, quickly disappeared from the market. In their place, cathinone derivatives have
been widely distributed, as well as different phenethylamines and piperazines. Additionally, in
recent years, new herbal products containing synthetic cannabinoids have appeared globally.
As at July 2012, 78 substances (including 1 plant; Salvia divinorum) were listed in the
category of Designated Substances. They were 13 tryptamines, 17 phenethylamines, 11
cathinones, 4 piperazines, 23 synthetic cannabinoids, 6 alkyl nitrites, 3 other compounds and
1 plant. In this review, we show our survey of the spread of new designer drugs in Japan,
focusing especially on synthetic cannabinoids and cathinone derivatives. Also, the
prevalence and legal status of these substances in other countries will be presented [13057].
436
Testing efficiency
In the early 2000s, United States track and field athlete Kelly White passed 17 drug tests
while on steroids (tetrahydrogestrinone, THG, testosterone), stimulants (modafinil), and EPO
before she was caught on modafinil, then confessed to having used the whole regimen. THG
was not found in her samples because laboratories were still blind to this designer steroid
(used only to beat the test). Testosterone use was not detected because she masked it by
taking epitestosterone as well; because her T/E never exceeded the cut-off, it never triggered
IRMS analysis, which would have detected exogenous testosterone. Modafinil was first
targeted and found by the French WADA-accredited laboratory; her EPO use was not
detected because sprinters' samples were not tested for EPO yet [07011].
Anti-doping research opportunities
The key areas of antidoping research include detection of compounds and methods for
increasing the absorption, transportation, and delivery of oxygen from the lung to the muscle;
detection of compounds or methods that increase the efficiency of conversion of oxygen to
intracellular energy; detection of compounds and methods that enhance muscle growth and
recovery; detection of genetic modifications applied to sport performance; identification of
additional matrices (oral fluid, dried blood spots, etc) to detect doping; and identification and
detection of masking techniques. The challenge of developing and validating methods for the
long list of prohibited substances and methods is daunting, requiring analytical skills, a
thorough understanding of drug metabolism and pharmacokinetics, and an appreciation of
human physiology and endocrinology. Antidoping science would benefit from the expertise of
scientists working on proteomics collaborating with scientists interested in changes in the red
blood cell during storage – hopefully resulting in a test for autologous blood transfusions.
Other key areas of research in the future will involve recombinant protein and glycoprotein
characterization and quantification using mass spectrometry and other techniques. The
potential for genetic modification for performance enhancement is of great concern, and
methods will need to be developed to detect gene doping. New methods of drug
administration may impact detection methods. Alternative testing matrices, such as oral fluid,
dried blood spots, and hair, may become more important [12006].
Economy
According to the WADA website, they have distributed over USD 50 million in research
funding since 2001. Between 2002 and 2010, USADA distributed an additional USD 9 million
in research funding. In 2008, the USADA, the U.S. Olympic Committee, the National Football
League, and Major League Baseball joined forces to create the Partnership for Clean
Competition (PCC) to support research in antidoping science. PCC has now funded 28
proposals totaling USD 6 million In addition to research proposals, PCC will continue to
establish collaborative working groups to focus the efforts of the best researchers on the
science of antidoping. While significant advances have been made in improving detection
strategies and methods, increased collaboration with the basic research community should
enhance the progress of drug abuse deterrence. Translation of those findings to routine
testing will also require funding and effort [12006].
Placebo
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While an increasing number of research is devoted to the understanding of placebo effects in
sports, athletes' experiences with and attitudes towards the use of placebo for performance
enhancement remain poorly understood. In this study, 79 elite athletes from different sports
were surveyed on five issues related to placebo use in sports. Results showed that 47
percent of the athletes have experienced placebo effects in the past. A majority of the
athletes (82 %) thought that placebos could affect their sports performances. A wider use of
placebos in sport settings was endorsed more by those who have experienced placebo
effects in the past than those who did not. Regardless of past experience with placebo, more
than half of the athletes (53 %) would accept an unknown but legitimate substance from the
coach, and 67 percent of them would not mind a placebo-linked deception if that was
effective. These findings confirm that most elite athletes believe in the power of placebos in
enhancing sports performance, and those having a positive past experience exhibit slightly
more favourable attitudes in contrast to those without such experiences [150056].
In the perpetual quest for better performance, athletes are using an increasingly diverse
range of ergogenic aids. Some are permitted; however, this "drug" use is often seen as an
ethically questionable behavior. A variety of research suggests that much of the impact of
such aids may be due to expectancy-the belief that the substance will aid performance. It
would be useful to demonstrate this to athletes considering such usage, especially as a pillar
of antidrug education. Accordingly, this investigation used sodium bicarbonate and placebo
additives in a double disassociation design, with athletes completing a series of 1,000-m time
trials. Results showed that believing one had taken the substance resulted in times almost as
fast as those associated with consuming the drug itself. In contrast, taking the drug without
knowledge yielded no significant performance increment. Results are discussed against the
backdrop of applying expectancy effects in high-performance sport, including dissuading
athletes from using illegal aids [07026].
One article described a study examining placebo effects associated with the administration of
a hypothetical ergogenic aid in sport. Forty-two team-sport athletes were randomly assigned
to 2 groups. All subjects completed 3 x 30-m baseline sprint trials after which they were
administered what was described to them as an ergogenic aid but was in fact 200 mg of
cornstarch in a gelatin capsule. Group 1 was provided with positive information about the
likely effects on performance of the substance, whereas Group 2 was provided with negative
information about the same substance. The sprint protocol was repeated 20 min later.
Although for Group 1 mean speed did not differ significantly between baseline and
experimental trials, a significant linear trend of greater speed with successive experimental
trials suggested that positive belief exerted a positive effect on performance. Group 2 ran 1.6
percent slower than at baseline (95 % confidence intervals 0.32 to 2.82 %), suggesting that
negative belief exerted a negative effect on performance. Collectively, data suggest that
subjects' belief in the efficacy or otherwise of a placebo treatment might significantly
influence findings in experimental research [07027].
The recent advances in the neurobiology of the placebo effect have shown that the
administration of a placebo (inert substance), along with verbal suggestions of clinical
benefit, activates different neurotransmitters in the brain, like endogenous opioids and
dopamine and is associated to neural changes at both the cortical and subcortical level
Powerful placebo responses can be obtained after pharmacological preconditioning, whereby
the repeated administration of a drug is replaced with an inert substance. For example, the
morphine-like effects of placebos after morphine preconditioning have been shown in the
context of pain management. Although these drug-like effects of placebos represent an
interesting phenomenon in the clinical setting, they also have implications that have been
ignored so far. One of these has to do with the use of drugs in sport competitions to boost
physical performance. Among performance-boosting drugs, morphine is known to be a
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powerful analgesic that increases tolerance to pain, thereby improving physical performance.
The importance of opioid-mediated placebo responses consists in the fact that they can be
exploited when one wants morphine-like effects without giving morphine. For example, in the
context of pain management, it has been shown that morphine administration for 2 days in a
row may induce robust placebo analgesic responses when morphine is replaced with a
placebo on the third day. This raises the important question whether two morphine
administrations separated several days or weeks from each other have similar powerful
effects on subsequent placebo responses. The neurobiological investigation of the placebo
effect has thus shown that placebos can activate the endogenous opioid systems in some
conditions. So far, the impact of this finding has been within the context of the clinical setting.
Here it was present an experiment that simulates a sport competition, a situation in which
opioids are considered to be illegal drugs. The subjects were healthy males who agreed to
participate in one of the experimental groups after they signed an informed consent form in
which the details of the experiment, including the drugs to be administered, were explained.
In particular, the subjects were told that either morphine or naloxone would be administered
at a given time, depending on the experimental group. None of them were training as a
competitive athlete, but all the subjects engaged in recreational fitness training. After
repeated administrations of morphine in the precompetition training phase, its replacement
with a placebo on the day of competition induced an opioid-mediated increase of pain
endurance and physical performance, although no illegal drug was administered. The
placebo analgesic responses were obtained after two morphine administrations that were
separated as long as 1 week from each other. These long time intervals indicate that the
pharmacological conditioning procedure has long-lasting effects and that opioid-mediated
placebo responses may have practical implications and applications. For example, in the
context of the present sport simulation, athletes can be preconditioned with morphine and
then a placebo can be given just before competition, thus avoiding administration of the
illegal drug on the competition day. However, these morphine-like effects of placebos raise
the important question whether opioid-mediated placebo responses are ethically acceptable
in sport competitions or whether they have to be considered a doping procedure in all
respects. The present study demonstrates that a pharmacological preconditioning, with
morphine given twice at intervals as long as 1 week, can induce robust placebo analgesic
responses when morphine is replaced with a placebo. It should also be noted that placebo
administration without previous morphine conditioning induced a small but significant
increase in pain endurance, which indicates smaller effects when a placebo is given for the
first time compared with its administration after pharmacological conditioning. The study
shows that long time lags between two consecutive administrations of morphine and the
administration of the placebo are not very different from short time lags, at least in the range
of days/weeks. This indicates that the pharmacological conditioning procedure has long-
lasting effects. In addition to the mechanisms of placebo responsiveness and the
preconditioning effects of morphine, this study raises important ethical questions: do opioid-
mediated placebo effects during competitions have to be considered a doping procedure?
Should we consider morphine conditioning in the training phase ethical and legal? This issue
is not easy to be resolved and will need both an ethical and legal discussion. Although one
must be aware that the experimental conditions of the present study do not represent a real
competitive event, but a pain challenge paradigm, the increase in pain endurance after the
placebo is real and robust and has key attributes relevant to situations encountered in sport
competitions. For example, our model of tonic ischemic arm pain represents a long-lasting
painful stimulation that is likely to be encountered in real long-lasting sport activities.
Therefore, if the conditioned subjects of this study engaged in a real sport activity, they would
tolerate pain for a longer time [07202].
Medical history of placebo
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Over the last 200 years, the placebo effect has cast a large and persuasive shadow over the
medical field. In that time it has been by turn; harmless charade, charlatan’s ruse, therapeutic
device, methodological tool, ethical dilemma, research theme and source of controversy.
Despite popular recognition, pervasive problems underlie conceptualisation of placebos and
placebo effects. With medicine now firmly entrenched in the age of evidence based practice,
there is a question as to whether it is time to leave the old placebo behind us. The idea of a
magical black box from which unexplained therapeutic effects spring up is archaic and also
unhelpful from a scientific point of view. If there really is an effect, surely we are best served
by directly investigating what is responsible for it. This knowledge can be used in the clinic to
improve treatment effectiveness and in research to inform study design. The view and role of
placebos have developed over time. Modern medical understanding of placebos dates back
to the late 1700s, and since then, placebo interventions appear to have been used fairly
commonly in medical practice alongside other treatments. Up until midway through the 20th
century, the prevailing opinion appears to have been that placebo interventions had no effect
on pathophysiology. Placebos were used only to bring comfort to the patient, “a camouflage
behind which to watch nature takes its course.” From the 1950s onwards, there was a shift
towards regarding placebos as having genuine effects of their own. This period also saw the
start of rapid growth in the use of placebos as a control intervention for testing the efficacy of
other treatments. Thus, as research generally, and randomised controlled trials (RCTs) in
particular, grew in stature and volume, placebo interventions also gained a more prominent
face in the medical literature [13029].
Effect of placebo on runnig results
To quantify the placebo effect magnitude on endurance running performance, in “real-world”

field-based head-to-head competition settings, of an injected placebo (“OxyRBX”) purporting
to have similar effects to recombinant human erythropoietin (r-HuEPO). Fifteen endurance-
trained club-level men (age 28), with personal best 10 km times of 39 ± 4 min completed the
randomised cross-over study design of 3 km races before and after 7-day “control” and
'placebo' phases. During the placebo phase participant’s self-administered subcutaneous
saline injections daily, believing it to be OxyRBX, with no intervention during the control
phase. At the start and end of each 7-day phase 3 km running performance was assessed.
Qualitative assessments of participants' perceptions and experiences were recorded
throughout and in semi-structured interviews on completion. Race time improved significantly
more in response to the placebo intervention (9.7 ± 2.0), than in response to control (1.8 ±1.9
s). In response to the placebo, participants reported reductions in physical effort, increased
potential motivation and improved recovery. Beliefs and congruence between positive
expectations of the effects of the placebo and perceptions of physical change during training
also appeared to impact on competitive performance. Compared to control, the injected
placebo improved 3 km race time by 1.2 percent. This change is of clear sporting relevance,
but is smaller than the performance improvement elicited by r-HuEPO administration. The
qualitative data suggest that placebo may have improved performance by both reducing
perception of effort and increasing potential motivation, in accord with the psychobiological
model for exercise performance, and that cognitive and non-cognitive processes appear to
have influenced placebo response [14705].
Placebo effect of carbohydrate feedings during a 40-km cycling time trial
The placebo effect, a favorable outcome from belief that one has received a beneficial
treatment, may be an important phenomenon in athletic performance. It was therefore
investigated the placebo effect of a carbohydrate supplement on endurance performance.
Forty-three competitive endurance cyclists (2 female, 41 male) performed two simulated 40-
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km time trials on an air-braked ergometer. In the first trial they ingested water to establish
baseline performance. For the second trial 6-8 d later they were randomized to two groups:
one group ingested 16 mL/kg of a drink containing 7.6 g/100 mL carbohydrate; the other
ingested an indistinguishable noncaloric placebo drink. Cyclists in each group were further
randomized to three subgroups according to whether they were told the drink contained
carbohydrate, placebo, or either (not told). Changes in mean power in the second trial were:
told carbohydrate, 4.3 + 4.8 percent; told placebo, 0.5 + 5.8 percent; and not told, -1.1 + 8.5
percent. The difference between the told-carbohydrate and told-placebo groups was 3.8
percent (95 % likely range 7.9 to -0.2 %). The change in performance in the not-told group
was more variable than that of the told groups by a factor of 1.6 (2.6 to 1.0). The real effect of
carbohydrate was a slight reduction in power of 0.3 percent (4.4 to -3.8 %). It was concluded
that the placebo effect of a potentially ergogenic treatment during unblinded laboratory time
trials lasting approximately 1 h is probably a small but worthwhile increase in endurance
power. Blinding subjects to the treatment increases individual differences in endurance effort,
which may reduce precision of performance outcomes in controlled trials [00022].
Powerless placebo
The view that placebos have a powerful therapeutic effect remained largely unchallenged
until the 1990s. Around this time, authors began to question the scientific basis upon which
the claims of large therapeutic placebo effects were made. Critically, it was pointed out that
longitudinal changes in a group of patients who have received a placebo intervention are not
only due to placebo effects. From the point of onset (or clinical contact), many conditions
improve with the passage of time independent of clinical management, due to the natural
history of the condition. Improvements over time are likely to be further enhanced by
regression to the mean which is a statistical artifact whereby scores that are high at one point
in time, i.e. at study entry, are always more likely to be lower at a later time. Thus, when a
group of patients receive a placebo intervention the changes afterwards may be due to
placebo effects plus changes due to natural history plus the effects of regression to the
mean. Experimental pain researchers pointed out the apparent discrepancy between the
very small placebo effect sizes reported in clinical studies and the much larger effects in
studies investigating placebo mechanisms [13029]
Lack of effect of flavor
One study investigated whether a change in beverage flavor during endurance cycling
improves subsequent performance. Eight trained male athletes (age 24 years) undertook 3
trials, with training and diet being controlled. Trials consisted of 120 min of steady-state (SS)
cycling at approximately 70 percent VO2peak, immediately followed by a 7-kJ/kg time trial (TT).
During exercise subjects were provided with fluids every 20 min. After 80 min of SS cycling
subjects either continued drinking the same-flavor sports drink or changed to an alternate
flavor-either an alternate-flavor sports drink (AFSD) or cola. All beverages were carbohydrate
and volume matched. Changing drink flavor caused no significant change in TT time. The
various flavors produced no treatment effects on heart rate, blood glucose, or rating of
perceived exertion throughout the SS exercise protocol. The influence of other taste
variables such as palatability, bitterness, or timing of flavor change on endurance-exercise
performance requires more rigorous investigation [07028].
Ethical issues
Along with the developing view of the role of placebos, the last half century has seen a shift
towards acceptance of the evidence-based medicine paradigm. These two factors have led
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to increasing recognition of ethical and legal concerns associated with the administration of
placebos in practice. The issues are difficult and consensus as to the appropriateness of
placebos in clinical practice has not been achieved. At the heart of the matter lies the
question of whether deliberate deception of the patient is acceptable in the course of their
treatment. Implicit in the definition of placebo effects is that they are psychologically
mediated. Whether the placebo is an ingested agent such as a sugar or vitamin pill, or a
procedural intervention such as non-penetrating acupuncture, sham electrotherapy or an
inert topical formulation, it must be physically inert. However, there follows the issue of
disentangling inert from non-inert psychological interventions. The problem is, if there is a
(psychological) mechanism, can the psychotherapeutic part of the intervention still be said to
be inert? Along a similar line, researchers have proposed the term “nocebo” to describe
undesirable effects of placebos. For example, studies have described immunosuppressive
effects of placebo interventions which may be undesirable (a nocebo) for one patient group,
for example general surgery patients but desirable (a placebo) for another group, for
example, organ transplant recipients. Further, an intervention assumed to have an effect at
one point in time could subsequently be shown to have no physiological effect and be used
as a placebo. In some ways, it is therefore not surprising a definition for the placebo effect
has proven so elusive. Conceivably, the same agent might be a placebo, a nocebo or an
active intervention depending on who gives it, who gets it or when it is given [13029].
Concluding remarks on placebo
The conception of placebos and placebo effects has developed significantly over the past
200 years in concert with changes in the nature and practice of healthcare as a whole. In the
past 20 years, questions have arisen as to the clinical significance of placebo effects and
perhaps more importantly, the logical basis of the concept itself. As things stand, enquiry into
placebo effects faces the nonsensical situation of attempting to explain an effect that has no
mechanism. Abandoning the placebo black box in favour of theory-directed research that
specifically targets and investigates the cause of therapeutic effects offers several
advantages. Effective techniques can be manipulated and incorporated in clinical practice to
produce better outcomes for patients, and better control interventions can be designed too
[13029].
Measurement uncertainty in anti-doping quantitative analysis
The standards of laboratory performance of the World Anti-Doping Agency (WADA)-

accredited laboratories are defined in the WADA International Standard for Laboratories and
its associated Technical Documents. These sets of rules aim to harmonize the production of
valid laboratory test results and evidentiary data as well as the reporting of laboratory
analytical findings. The determination of anti-doping rule violations in sport made on the
basis of analytical quantitative confirmatory analyses for the presence of prohibited threshold
substances, in particular, requires the application of specific compliance decision rules,
which are established in the WADA Technical Document on Decision Limits. In one article,
the use of measurement uncertainty information in the establishment of compliance Decision
Limits and in evaluating the performance of a laboratory's quantitative analytical procedures
over time and in relation to other laboratories through WADA's External Quality Assessment
Scheme program is reviewed and discussed. Furthermore, a perspective is provided on the
emerging challenges associated with the harmonization of the quantitative measurement of
large-molecular weight biomolecules [12053].
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Statistical aspects
The detection of growth hormone (GH) abuse by athletes raises statistical problems as well
as biochemical ones. It was outlined the statistical approaches to the various issues which
have arisen ; in particular, it considers the need to develop a test which detects GH abuse in
any elite athlete “beyond reasonable doubt“. The test needs to be robust enough to withstand
legal challenge, while minimising the risk of false accusation. Since GH is a naturally
occurring hormone whose concentration varies substantially, its abuse cannot be detected by
direct measurement. The methodology considered here made use of markers whose levels
are more stable but are influenced by GH. The statistical methods employed aimed to make
the best use of these markers, taking account of all factors contributing to errors in
measurement. There were two key steps in the statistical investigation undertaken to develop
the GH detection algorithm. The first was the requirement to identify GH-dependent
biomarkers which would identify GH doping reliably and robustly for a significant length of
time. The second was to calibrate the GH detection method in the elite athlete population, so
that the method would be applicable to all athletes, regardless of age, sex and ethnicity, and
regardless of whether they had recently sustained an injury. In practice, further work was
needed to ensure that the methodology met the WADA testing protocol rules, but also that
the proposed method can be used by any WADA accredited lab without placing any athlete
at an unfair disadvantage and ensuring a high level of confidence in any result produced
[09004].
The comparison among different modelling techniques, such as multiple linear regression,
partial least squares and artificial neural networks, has been performed in order to construct
and evaluate models for prediction of gas chromatographic relative retention times of
trimethylsilylated anabolic androgenic steroids. The performance of the quantitative
structure-retention relationship study, using the multiple linear regression and partial least
squares techniques, has been previously conducted. In the present study, artificial neural
networks models were constructed and used for the prediction of relative retention times of
anabolic androgenic steroids, while their efficiency is compared with that of the models
derived from the multiple linear regression and partial least squares techniques. For overall
ranking of the models, a novel procedure [Trends Anal Chem 2010; 29: 101-9] based on sum
of ranking differences was applied, which permits the best model to be selected. The
suggested models are considered useful for the estimation of relative retention times of
designer steroids for which no analytical data are available [12052].
False negatives
A 2011 article published in Clinical Chemistry covered the recent state of affairs and the
outlook for future efforts to combat the use of illicit or performance-enhancing drugs in sports
(i.e. doping). The interviewees in that article made several poignant admissions: that the rate
of detection for doping is <100 percent (and maybe far less), that deterrent measures need to
be stronger yet fair, and that future additions to the pharmacopoeia of doping agents will
seriously challenge our ability to provide reliable and comprehensive testing. One of the most
important principles of laboratory testing is the statistical concept implied by the Bayes
theorem: the likelihood of a finding being true is related to the pretest probability of truth
times the likelihood ratio associated with the test. Thus, in the case of a drug test, if it is
tremendously unlikely that the subject is using the drug, a positive drug test result is most
likely a false positive. Conversely, if a subject is highly likely to be using a drug, a negative
drug test result is most likely a false negative. Consider, for example, a hypothetical
screening population in which 90 percent of athletes use a banned substance. Bayesian
analysis tells us that, even if the test used for the banned substance is 95 percent sensitive
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and 95 percent specific for detection, more than 32 percent of the negative tests are false
negatives. Because antidoping testing is often designed to afford higher specificity at the cost
of lower sensitivity to lower the probability of false-positive results, and because athletes are
known to actively evade detection, the problem of false negatives could be worse, depending
on the prevalence of doping [14728].
High true prevalence
In the context of antidoping measures if the true prevalence of doping is very high, then the
results of testing are not terribly informative. The problem, however, is that no one knows the
true prevalence of doping, and it is difficult to measure. Compounding the problem, some
organizations have a vested interest in finding a low prevalence. Professional sports
associations worry about their public images, and the World Antidoping Agency (WADA) may
worry about justifying its existence. But mounting evidence (the recent admissions of cyclist
Lance Armstrong, the words of cyclist Floyd Landis, and the US government's Mitchell report
on baseball doping, to name a few sources) indicates that the pretest probability of doping, at
least in some sports, is very high. For example, in one study it was found that the prevalence
of doping resulting in increased hematocrit was at least 46-48 percent in one unnamed
country's track and field athletes. The methods used in that study generated conservative
estimates, however, and focused on only one type of doping (increasing hematocrit by
transfusion or erythropoietin abuse), so the cumulative prevalence of all types of doping in
this cohort is almost surely higher. The prevalence of doping is probably not 100 percent in
these athletes, but how high does it really need to get before we decide that testing, in
Bayesian terms, is futile? [14728].
Preanalytical variability
The use of illicit substances and methods contravenes the ethics of sports and may be
associated with side effects. Antidoping testing is an essential tool for preventing or limiting
the consequences of cheating in sports. As for conventional laboratory testing, major
emphasis has been placed on analytical quality, overlooking the inherent risks that may arise
from analysis of unsuitable doping samples. The adherence to scrupulous criteria for
collection, handling, transportation and storage of samples, especially blood and urine
samples, is essential. The leading preanalytical variables that influence doping sample
quality include biological variability, sample collection, venous stasis, spurious hemolysis and
presence of other interfering substances, sample manipulation and degradation, and
inappropriate conditions for transportation and storage. One article provided a personal
overview about the current challenges in preanalytical management of doping samples, as
well as potential solutions for preventing the negative impact of preanalytical variables on
sample quality and test results [13080].
Doping prevalence at drug testing
Doping prevalence can not be estimated by drug testing of athletes. This method is flawed:
as the autobiographies of some athletes attest, regular dopers have a track record of
avoiding testing positive. To estimate doping's true prevalence, two procedures that
circumvent inherent weaknesses in simple counts of positive test results are useful. First,
Bayesian inference methods can be used to compare two distributions of biological
parameters affected by doping: one distribution among sampled athletes and the other in a
suitable reference population, allowing the number of manipulated samples to be estimated.
A major advantage of this population-level analysis is that it can recognize abnormalities
even when dopers' values remain within the “normal” range. Second, specially tailored
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questionnaires allow athletes to give honest answers to doping questions under cover of
anonymity. The questionnaires are based on the randomized response technique and are
used by social scientists to study illegal and deviant behaviours. A combination of these
approaches estimates that 14-39 percent of elite athletes have intentionally doped. This
contrasts markedly with the 2 percent of samples designated as suspect in the World Anti-
Doping Agency's published statistics [150114].
Prevalence of doping use in elite sports: a review of numbers and methods

The prevalence of doping in elite sports is relevant for all those involved in sports, particularly
for evaluating anti-doping policy measures. Remarkably, few scientific articles have
addressed this subject so far, and the last review dates back to 1997. As a consequence, the
true prevalence of doping in elite sports is unknown. Even though it is virtually impossible to
uncover the exact prevalence of a prohibited activity such as doping, various methods are
available to uncover parts of this particular problem, which enables the circumvention (to a
certain degree) of the issues of truthfulness, definition problems and the limits of
pharmacological evidence. This review outlines the various methods that exist and presents
the scarce data available in this area. It is concluded that a combination of questionnaires
using the Randomised Response Technique and models of biological parameters is able to
provide the statistical possibilities to reveal accurate estimates of this often undisclosed
practice. Data gathered in this way yield an estimation of 14-39 percent of current adult elite
athletes who intentionally used doping. These period prevalences have been found in
specific sub-groups of elite athletes, and the available data suggest that the prevalence of
doping is considerably different between sub-groups with varying types of sport, levels and
nationalities. The above-mentioned figure of 14-39 percent is likely to be a more accurate
reflection of the prevalence of intentional doping in elite sports than that provided by doping
control test results (estimate of doping: 1-2 % annually) or questionnaire-based research
(estimations between 1 and 70 % depending on sport, level and exact definitions of intent
and doping). In the future, analytical science may play a more important role in this topic if it
may become feasible to detect very low concentrations of prohibited substances in sewage
systems downstream of major sporting events. However, it is clear that current doping
control test results show a distinct underestimation of true doping prevalence. It does not
seem feasible to distil better estimates of the prevalence of doping based on performance
indicators or ego documents because of the various existing effects that influence athletic
performance. Such information can only be used as extra information to augment the
accuracy of prevalence rates that have been found by using other techniques. True doping
prevalence studies have been scarce in elite sports so far. With the correct application of the
available scientific methods, preferably using harmonised definitions of the terms “doping”
and “elite sports”, more information on this topic may be gathered in a relatively short time.
This would assist anti-doping professionals in the future in order to evaluate the effects of
possible anti-doping measures, and better anti-doping policies would serve athletes who
compete without doping. The existing anti-doping measures seriously impact the lives of elite
athletes and their immediate entourage, which imposes a moral burden to evaluate these
measures in the best possible way [150115].
Wald test
One article derived the power curves for a Wald test that can be applied to randomized
response models when small prevalence rates must be assessed (e.g. detecting doping
behavior among elite athletes). These curves enable the assessment of the statistical power
that is associated with each model (e.g, Warner's model, crosswise model, unrelated
question model, forced-choice models, item count model, cheater detection model). This
power analysis can help in choosing the optimal model and sample size and in setting model
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parameters in survey studies. The general framework can be applied to all existing
randomized response model versions. The Appendix of the article contains worked-out
numerical examples to demonstrate the power analysis for each specific model. (PsycINFO
Database Record) [12051].
Bayesian statistics
Research on biological markers is a fast-growing field for assessing evidence in biomedical

toxicology. In forensic toxicology in particular, major goals are to develop and validate
measurements of endogenous substances that may reveal the presence of toxic substances,
drugs of abuse, and/or doping agents. The development and validation of biomarkers of
response are either based on the statistical description of endogenous substances measured
on a population or on a longitudinal evaluation of a series of repeated tests performed on the
same individual. Longitudinal studies are particularly interesting in forensic toxicology when
the biomarker has a significantly smaller intraindividual variability than interindividual
variability. This is the case for several biomarkers currently used in antidoping investigations,
such as indirect markers of blood doping and the testosterone over epitestosterone (T/E)
ratio for the detection of the abuse of testosterone and its precursors. These biomarkers are
all characterized by a small ratio of intra- to interindividual variation. In contrast to the
abundant number of statistical models that analyze serial biomarkers of disease, the
development of reliable methods for the detection of abnormal variations of a longitudinal
biomarker has remained astonishingly limited in forensic toxicology. Current methods employ
either population-derived limits – to detect “absolute” abnormal values of the biomarker – or
individual-based thresholds – to detect abnormal deviations relative to an individual baseline.
There is no reason why a test cannot combine formally population-based information with
individual-based data for better decision making. Failure to combine these two types of
information may lead to a low sensitivity/specificity relation of the biomarker. For example,
when a biological product such as the carbohydrate-deficient transferrine or a transminase
(alanine transaminase (ALAT) and/or aspartate transaminase (ASAT)) is used as an indirect
marker of chronic alcohol abuse no effective method integrates previous readings for better
decision making despite several measurements being available on the same individual. In
the antidoping world, at least four readings are currently required by the World Anti Doping
Agency (WADA) for the detection of abnormal variations of the T/E ratio, with no knowledge
of the rate of false positives. Note that a precise valuation of the specificity is important in
forensic toxicology, because a very low false-positive rate is demanded in order to prevent
the accusation of an innocent individual. Finally, with biomarkers of blood doping, there is
currently no procedure that has a specificity that does not depend on the number of test
results. It is believed that “at least six samples for the baseline reading and possibly
considerably more” are needed to derive cutoff thresholds that take into account the
intraindividual variability of the biomarker. It was developed a test that compares sequential
measurements of a biomarker against previous readings performed on the same individual.
A probability mass function expresses prior information on interindividual variations of
intraindividual parameters. Then, the model progressively integrates new readings to more
accurately quantify the characteristics of the individual. The idea is to use prior knowledge on
interindividual variations of intraindividual parameters, and to progressively integrate
previous readings to more accurately quantify the characteristics of the individual. Each new
measurement of the biomarker is compared to a critical range. Before the first measurement
on the individual, the critical range is derived from the population only. Then, this range
progressively adapts itself as the number of readings performed on the same subject
increases to finally characterize a particular individual only as the number of readings
becomes very large. The rate of false positives does not vary with the number of previous
test results. The specificity is independent of the number n of previous test results, with a
model that gradually evolves from population-derived limits when n = 0 to individual-based
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cutoff thresholds when n is large. We applied this model to detect abnormal values in an
athlete's steroid profile characterized by the testosterone over epitestosterone (T/E) marker.
A cross-validation procedure was used for the estimation of prior densities as well as model
validation. The heightened sensitivity/specificity relation obtained on a large data set shows
that longitudinal monitoring of an athlete's steroid profile may be used efficiently to detect the
abuse of testosterone and its precursors in sports. Mild assumptions make the model
interesting for other areas of forensic toxicology [06032].
Decision limit (CCalpha) and detection capability (CCbeta)
Initially in the Decision 2002/657/EC the criteria for the calculation of the decision limit
(CCalpha) and the detection capability (CCbeta) have been estimated as purely quantitative
(alpha-error is 1 % and beta-error is 5 %). In 2004, the European Commission has issued a
document to provide guidance for the interpretation of the 2002/657/EC. In this document it is
mentioned that also qualitative criteria should be fulfilled. Therefore, the calculated CCalpha
and CCbeta must be verified by using fortified samples. The method should be able to
detect/identify the target component in 50 percent of the cases at CCalpha and in 95 percent
of the cases at CCbeta. Analytical methods for the analysis of nitroimidazoles, nitrofurans
and corticosteroids with LC-MS/MS have been validated by fortifying blank samples below
and above the MRPL. CCalpha and CCbeta were calculated using the ISO 11843 approach.
In addition, the frequency of methodical compliance for the qualitative criteria was
determined at each concentration level. It was observed that at the calculated CCalpha and
CCbeta levels the qualitative criteria were not fulfilled. It was concluded that the detection
capability of the analytical method should be calculated by using decreasing fortification
levels at and below the MRPL. A protocol validating methods for banned substances by
limiting the number of samples is presented and the qualitative criteria for the assessment of
CCalpha and CCbeta were verified based on the same set of data without the need of
performing additional validation experiments [06035].
Prediction of future doping
The World Anti-Doping Agency (WADA) publishes the Prohibited List, a manually compiled
international standard of substances and methods prohibited in-competition, out-of-
competition and in particular sports. It would be ideal to be able to identify all substances that
have one or more performance-enhancing pharmacological actions in an automated, fast
and cost effective way. Here, it was used experimental data derived from the ChEMBL
database (7,000,000 activity records for 1,300,000 compounds) to build a database model
that takes into account both structure and experimental information, and use this database to
predict both on-target and off-target interactions between these molecules and targets
relevant to doping in sport. The ChEMBL database was screened and eight well populated
categories of activities (Ki, Kd, EC50, ED50, activity, potency, inhibition and IC50) were used
for a rule-based filtering process to define the labels "active" or "inactive". The "active"
compounds for each of the ChEMBL families were thereby defined and these populated our
bioactivity-based filtered families. A structure-based clustering step was subsequently
performed in order to split families with more than one distinct chemical scaffold. This
produced refined families, whose members share both a common chemical scaffold and
bioactivity against a common target in ChEMBL. It was thus used the Parzen-Rosenblatt
machine learning approach to test whether compounds in ChEMBL can be correctly
predicted to belong to their appropriate refined families. Validation tests using the refined
families gave a significant increase in predictivity compared with the filtered or with the
original families. Out of 61,660 queries in an Monte Carlo cross-validation, belonging to
447
19,639 refined families, 41,300 (67 %) had the parent family as the top prediction and 53,797
(87 %) had the parent family in the top four hits. Having thus validated our approach, we
used it to identify the protein targets associated with the WADA prohibited classes. For
compounds where we do not have experimental data, we use their computed patterns of
interaction with protein targets to make predictions of bioactivity. It was hoped that other
groups will test these predictions experimentally in the future [13081].
448
ATHLETE BIOLOGICAL PASSPORT (ABP)
Overviews and background
Blood doping in all its facets is still a major issue in sports and much effort has been invested
also in order to improve existing doping control strategies as well as to probe for
complementary methodologies. Recent confessions of doped athletes have once more
underlined the extent of blood manipulation, by ESAs and/or blood transfusions and the
doping control analytical challenges are inherent to the nature of the manipulative actions,
i.e. use of low- to microscale dosages of ESAs combined with small units of blood
transfusions. Various approaches converging mainly to “indirect” detection methods (such as
the Athlete Biological Passport, ABP) and assays providing supporting evidence have been
pursued as means to tackle the abusive transfusion of autologous blood, while flow
cytometry has proved capable of uncovering homologous blood transfusions in a “direct”
manner in the past. In order to apply the ABP, numerous aspects need to be thoroughly
assessed such as the stability of the ABP parameters under extended storage conditions as
recently done. The stability of the most crucial variables, i.e. haemoglobin concentration and
percentage of reticulocytes, under the defined pre-analytical conditions has been established
to be at least 36 h, requiring rapid and cooled transport of doping control blood samples to an
accredited laboratory. Extending the storage time to 168 h at +4°C, +6°C, and +12°C as
done in this study demonstrated that these parameters were not significantly altered if the
temperature was kept at 4-6°C, hence allowing to rely on analytical data up to seven days
after blood collection when adequately cooled. Physiologically, the percentage of
reticulocytes can vary depending on factors such as seasonal stress (training, competition,
and recovery), sport discipline, diseases, etc. However, intra-individually, the parameter has
proved extraordinary informative and has thus become a major pillar of the ABP. In order to
support the significance of values measured from individuals and the comparison of analyses
conducted at different laboratories and/or on different analytical systems of the same
instrument type, a study to improve the between-instrument comparability by optimizing the
calibration was conducted. By means of a stabilized whole blood matrix used as a calibrant,
mean values of authentic samples were within 0.1 percent among the test instruments, thus
allowing an improvement in the commonly observed bias between systems still operating
within the manufacturers’ specifications. The robustness and efficiency of the ABP has
resulted in numerous undisputed AAFs in the past as summarized for the International
Cycling Union (UCI) in 2012. However, the “inventiveness” of cheating athletes and their
entourage must be kept in mind, and other factors potentially influencing, for example, the
percentage of reticulocytes have been investigated such as the injection of the granulocyte
colony-stimulating factor (G-CSF), for which anecdotal evidence existed as to its abuse in
elite sport. In a controlled study, the repeated administration of therapeutic dosages of G-
CSF (10 microg/kg/day) over a period of 5 days resulted in a statistically significant increase
of %reticulocytes while all volume-dependent parameters such as red blood cell count,
haematocrit and haemoglobin were found decreased [14009].
In 2011, WADA re-established a Haematological Expert Group to further refine and develop
the athlete biological passport. The goal was to evaluate analytical elements and possible
confounding factors with a rigorous scientific approach. For example, it is clear that
fluctuations in plasma volume will have an influence on a number of the measured
haematological variables (biomarkers). During exercise, plasma volume will contract before it
settles back to normal levels in about 2 h postexercise. Therefore the Technical Document
on Blood Sample Collection Requirements for the ABP4 mandates that blood collection does
not occur within 2 h of training or competition to maintain the validity of the sample [14015].
449
As of the beginning of 2014, 41 Anti-Doping Organisations run haematological ABP
programmes; these are in different phases of implementation. This has resulted in more
effective targeting for testing (e.g. EPO tests) which contributed to the doubling of positive
analytical findings for EPO in the first years of the programme. From the implementation of
the ABP to the end of 2013, more than 40 cases resulted in direct sanctions (ADRVs) without
the benefit of an adverse analytical finding. These have been reported by both National Anti-
Doping Organisations and IFs. WADA continues to develop and improve the ABP with input
from experts and stakeholders. The principles and practice of an ABP should be a model of a
modern, integrated antidoping programme. This combines traditional and new analytical
approaches along with non-analytical methods such as information from intelligence. The
world of sport and antidoping continues to evolve with increasing challenges. The access
and supply of prohibited substances has increased dramatically in the past decades and is
now readily and rapidly available via the Internet. There are newer substances including
peptides and designer drugs that are harder to detect. The potential for significant financial
gains in sport has encouraged the involvement of sophisticated doping entourages and
networks of suppliers [14015].
The illicit routes to enhanced oxygen transfer capacities in athletes are manifold and the
provision of evidence has been a considerable challenge for doping control laboratories.
Comprehensive reviews on accomplishments as well as unsolved issues were reviewed in
several recent articles. A central aspect of contemporary efforts towards the determination of
autologous blood doping in particular is the Athlete Biological Passport (ABP), which has
been employed as an anti-doping tool since 2009 and enabled various convictions of doped
athletes during the last three years. The ABP's principle relies on the intra-individual stability
of selected blood parameters such as % reticulocytes (%Ret) and hemoglobin concentration
([Hb]), the long-term variation of which was tested over 4 consecutive competition seasons in
elite triathletes. Both parameters were found stable and thus suitable for sports drug testing
purposes, although significant variations among female athletes were detected concerning
%Ret. Since ABP results must allow for comparison of data with other doping control
laboratories, harmonized protocols are important. In that context, the influence of pre-
analytical mixing strategies (manual, mechanical mixing, and automated mixing in the
analyzer autosampler) on full blood counts was assessed, demonstrating that no significant
difference was observed and that 15 min of mechanical shaking as commonly conducted are
more than sufficient [13072].
In the last few years, the increasing number of published studies on variations in
hematological parameters in elite athletes reflects the growing interest in the athlete’s
physiological response to strenuous metabolic effort, especially in the context of the current
debate on blood doping and the ABP. Current knowledge of sports physiology and doping
biomarkers is formalized in the Athlete Biological Passport (ABP) program: an algorithm
tracking the longitudinal record of hematological parameters as a means to define an
individual’s hematological profile and thereby identify potential deviations. The central
concept of the ABP is that a better appreciation of the physiological changes in the
hematological profile related to training, competition, and altitude will allow discrimination of
variations induced by illicit practices from those due to homeostatic response to physical
activity [13072].
During the last four decades, the main instrument at the disposal of anti-doping authorities
has been the detection of prohibited substances in biological samples collected from
athletes. However, the availability of substances identical to those produced by the human
body, such as EPO, testosterone and GH, necessitated a new drug-testing paradigm. From
the early 2000's, the Athlete Biological Passport (ABP) was proposed as an alternative
450
means to drug testing. Doping leaves a characteristic fingerprint on the biology of the athlete
and the ABP is used to prove the act of doping from the detection of that fingerprint. Once a
biomarker of doping is implemented in the ABP, it will continue to remain valid and should be
able to detect the physiological changes brought on by performance-enhancing drugs that
have not yet been invented. However, the sensitivity of the ABP to detect doping is limited if
the physiological result of a low level of doping remains within the individual's own reference
range. Recent advances in proteomics and metabolomics show the huge potential of the
ABP [12040].
Expert evaluation of biological data is a key component of the Athlete Biological Passport
approach in the fight against doping. The evaluation consists of a longitudinal assessment of
biological variables to determine the probability of the data being physiological on the basis
of the athlete's on own previous values (performed by an automated software system using a
Bayesian model) and a subjective evaluation of the results in view of possible causes
(performed by experts). The role of the expert is therefore a key component in the process.
Experts should be qualified to evaluate the data regarding possible explanations related to
the influence of doping products and methods, analytical issues, and the influence of
exercise or pathological conditions. The evaluation provides a scientific basis for the decision
taken by a disciplinary panel. This evaluation should therefore encompass and balance all
possible causes for a given blood profile and provide a likelihood for potential scenarios
(pathology, normal variation, doping) that might have caused the pattern. It should comply
with the standards for the evaluation of scientific evidence in forensics. On the basis of their
evaluation of profiles, experts might provide assistance in planning appropriate target testing
schemes [12041].
The increase of the body's capacity to transport oxygen is a prime target for doping athletes
in all endurance sports. For this purpose, blood transfusions or erythropoiesis stimulating
agents (ESA), such as erythropoietin, NESP, and CERA are used. As direct detection of
such manipulations is difficult, biomarkers that are connected to the haematopoietic system
(haemoglobin concentration, reticulocytes) are monitored over time (Athlete Biological
Passport, ABP) and analyzed using mathematical models to identify patterns suspicious of
doping. With this information, athletes can either be sanctioned directly based on their profile
or targeted with conventional doping tests. Key issues for the appropriate use of the ABP are
correct targeting and use of all available information (e.g. whereabouts, cross sectional
population data) in a forensic manner. Future developments of the passport include the
correction of all concentration-based variables for shifts in plasma volume, which might
considerably increase sensitivity. New passport markers from the genomic, proteomic, and
metabolomic level might add further information, but need to be validated before integration
into the passport procedure. A first assessment of blood data of federations that have
implemented the passport show encouraging signs of a decreased blood-doping prevalence
in their athletes, which adds scientific credibility to this innovative concept in the fight against
ESA- and blood doping [12042].
Steroid profile analyses represent an important resource of information concerning both the
administration of natural (endogenous) steroids as well as those of xenobiotic origin. Steroid
profiling has been utilized in sports drug testing for more than three decades and still much
effort is invested in elaborating and improving this valuable tool, particularly to increase its
screening efficiency and to allow for consideration of more recently clarified (genetically or
pharmacologically induced) variations influencing the steroid profile interpretation. A central
aspect of contemporary efforts towards the determination of autologous blood doping in
particular is the Athlete Biological Passport (ABP), which has been employed as an anti-
doping tool since 2009 and enabled various convictions of doped athletes during the last
three years. The ABP’s principle relies on the intra-individual stability of selected blood
451
parameters such as %reticulocytes (%Ret) and hemoglobin concentration ([Hb]), the long-
term variation of which was tested over 4 consecutive competition seasons in elite triathletes.
Both parameters were found stable and thus suitable for sports drug testing purposes,
although significant variations among female athletes were detected concerning %Ret. Since
ABP results must allow for comparison of data with other doping control laboratories,
harmonized protocols are important. In that context, the influence of pre-analytical mixing
strategies (manual, mechanicalmixing, and automated mixing in the analyzer autosampler)
on full blood counts was assessed, demonstrating that no significant difference was
observed and that 15 min of mechanical shaking as commonly conducted are more than
sufficient [12017].
The Athlete Biological Passport (ABP), is a new testing paradigm with immense potential
value in the current climate of rapid advancement in biomarker discovery. It offers the
enormous advantage of being independent of this endless pharmaceutical race. In addition
to its original aim of providing proof of a doping offense, the ABP can also serve as a
platform for a Rule of Sport, with the presentation before competition of the ABP to
objectively demonstrate that the athlete will participate in a healthy physiological condition
that is unaltered by performance-enhancing drugs. Finally, the decision-support system used
today for the biological monitoring of world top-level athletes can also be advantageously
transferred to other areas of clinical practice to reach the goal of personalized medicine. The
promotion of ethical values and the protection of health in and throughout sports are the
primary objectives of the sport movement. In that context, the abuse of substance doping
represents the most serious threat to the integrity of modern sports. The World Anti-Doping
Code, the reference document that provides the framework for harmonized antidoping rules
within sports organizations, has been written to preserve the core values of natural
performance, protection of health, and the spirit of the sport. Accordingly, a substance or
method is considered for prohibition if it violates at least 2 of these 3 values. Doping triggers
physiological changes that provide physiological enhancements. In the same way that
disease-related biomarkers are invaluable tools that assist physicians in the diagnosis of
pathology, specifically selected biomarkers can be used to detect doping. The primary tool
used by sports authorities to ensure a doping-free sport has been the detection of prohibited
substances in the biological fluids of athletes, specifically urine and blood. This drug-testing
paradigm was introduced in the 1960s and has since been remarkably successful in the
detection of substances that are not naturally produced by the body, such as stimulants,
narcotics, beta2-agonists, and diuretics. This success is largely attributed to the use of
chromatography coupled to mass spectrometry techniques that have revolutionized the
detection of a large number of compounds. As a result of advances in biotechnology, the
pharmaceutical industry continues to market new drugs at a remarkable pace. A substantial
number of these new substances are recombinant proteins or peptides that are strikingly
similar in structure, and in some instances absolutely identical, to those naturally produced by
the human body. The identification of these substances in biological fluids can be difficult or
virtually impossible in some cases. In modern sports, doped athletes are in a constant race
with antidoping researchers, who must employ great ingenuity to develop toxicology tests
capable of distinguishing exogenous substances from their endogenous counterparts. In
addition, detection is further complicated by the medical supervision and increased
sophistication of doping protocols. Contemporary protocols are shifting towards long cycles of
small microdoses taken repeatedly that are difficult to detect by using conventional drug tests.
Worse, designer drugs are currently being produced by black-market laboratories to get
around existing drug tests. Consequently, the drug-testing paradigm established in the 1960s
cannot prevent elite athletes from doping with impunity when using many potent doping
substances such as designer recombinant erythropoietin (rEPO) and designer testosterone.
For these reasons, alternative strategies that are independent of this endless pharmaceutical
race must be developed to maintain fairness in elite sports [11426].
452
Biological fluids, such as blood and urine, contain a treasure trove of potential doping
markers that can be discovered by today’s omics techniques, such as proteomics and
metabolomics. The usefulness of this gold mine for diagnostic purposes has been
recognized, and the same is true for doping biomarkers. By definition, any deviation in a
biomarker from what is expected in a healthy physiological condition according to well-
defined protocols can be attributable only to doping or a medical condition. Interestingly,
these two possible causes are the exact targets of any antidoping program; hence, the
criteria that are used to introduce new biomarkers into the ABP are the same as those that
are used to define a banned substance, more specifically, the criteria of performance and
health. In addition, an eligibility rule becomes a logical consequence of this assumption,
wherein athletes present their passports at the beginning of a competition and individuals are
allowed to participate only if their passport indicates that they are in a healthy and unaltered
physiological condition. Therefore, in addition to proving a doping offense under the World
Anti-Doping Code, the ABP can be a platform for a Rule of Sport enforced by the sport
authorities to prevent athletes from manipulating their physiology to an extent that would
significantly impact their performance and health. It may be foreseen the implementation of a
Rule of Sport in which the athletes who have demonstrated unnatural deviations in
physiology would be temporarily withdrawn from competition to allow a period for return to
normal physiological levels or initiation of appropriate medical controls or treatments. This
short period of debarment could also be used by a panel of experts to determine the cause of
the abnormality and may lead to sanctioning the athlete for a longer period if doping is the
cause [11426].
Although drug tests have been remarkably successful in the detection of synthetic doping
substances, the recent availability of doping substances identical to those naturally produced
by the human body demonstrates the limits of this testing paradigm to ensure fairness and
health protection in elite sports. In that context, the ABP represents the new paradigm in
detection of doping-triggered physiological changes in elite sports. Doping biomarkers
provide a means to deter the athlete from using performance-enhancing drugs that will lead
to deviation from natural baseline values. In contrast to a drug test that returns a result for a
precise moment in time and does not have any memory or perspective, the presentation at
the beginning of competitions of an ABP that demonstrates normal longitudinal profiles will
allow athletes to objectively demonstrate that they will participate in an unaltered
physiological condition clear of any doping suspicion. Scientists are developing methods that
provide an unequalled opportunity to ensure fairness and the protection of health in elite
sports; worldwide ABP implementation is now at the discretion of antidoping organizations.
The same paradigm can be used in the clinics so that personalized medicine will not only be
centered on the deeper molecular makeup of each patient, but also on an interpretation of
existing biomarkers tailored to each individual [11426].
The gatekeepers of fair sport have a whole new way to identify doping: the “athlete biological
passport.” An approach that has evolved over the last several years, it is an electronic record
of test results of the lingering effects of banned substances in the body, rather than the
substances themselves. Like a valid passport required for entry into foreign countries, a valid
(clean) biological passport is nowr equired for many athletes to gain entry into elite
competitions. Hopes are high among sports federations and antidoping agencies that the
biological passport will close some of the biggest loopholes that have let cheaters slip into
the Olympics, the Tour de France, and other major events [10008]
One gaping loophole in such tests is that some drugs, like erythropoietin, can only be
detected in the body for a few days. But the effects of the drugs last for a week or more,
increasing the odds that users will get caught. Another loophole is that banned substances,
453
for a variety of reasons, are sometimes impossible to detect: they can be designed to elude
specific tests, new substances can be made for which there are no tests, and a genetic trait –
missing copies of a gene called UGT2B17, which makes testosterone soluble in urine –
renders testosterone doping invisible to conventional urine tests (he absence of this gene is
strongly related to ethnicity: 81 percent of Asians, 22 percent of Africans, and 10 percent of
Caucasians are missing both copies). Looking for the physiological footprints left by such
drugs, rather than the particular culprits, should reduce these problems [10008].
Few reliable estimate of the prevalence of doping in elite sports has been published. Since
2001, the international governing body for athletics has implemented a blood-testing program
to detect altered hematological profiles in the world's top-level athletes. A total of 7289 blood
samples were collected from 2737 athletes out of and during international athletic
competitions. Data were collected in parallel on each sample, including the age, gender,
nationality, and birth date of the athlete; testing date; sport; venue; and instrument
technology. Period prevalence of blood-doping in samples was estimated by comparing
empirical cumulative distribution functions of the abnormal blood profile score computed for
subpopulations with stratified reference cumulative distribution functions. In addition to an
expected difference between endurance and nonendurance athletes, it was found nationality
to be the major factor of heterogeneity. Estimates of the prevalence of blood doping ranged
from 1 to 48 percent for subpopulations of samples and a mean of 14 percent for the entire
study population. Extreme cases of secondary polycythemia highlighted the health risks
associated with blood manipulations. It was concluded that when applied at a population
level, in this case the population of samples, hematological data can be used to estimate
period prevalence of blood doping in elite sports. It was found that the world's top-level
athletes are not only heterogeneous in physiological and anthropometric factors but also in
their doping behavior, with contrasting attitudes toward doping between countries. When
applied at the individual level, the same biomarkers, as formalized in the Athlete Biological
Passport paradigm, can be used in analysis of the observed different physiological
characteristics and behavioral heterogeneities [11130].
In 1996 several sports federations introduced an upper limit for hemoglobin and hematocrit.
Violation did not lead to a doping conviction, but a temporary suspension for health reasons.
By recording the hematological parameters over time and analyzing the data with a scoring
system based on Bayesian probability theory it is possible to determine whether or not the
changes are physiological. This model is called the Athlete’s Biological Passport (ABP).
Thus, the Athlete Biological Passport (ABP) is principally founded on monitoring an athlete's
biological variables over time, to identify abnormal biases on a longitudinal basis. Several
factors are known to influence the results of these markers. However, the manner in which
the altitude factor is taken into account still needs to be standardized. Causal relationships
between haematological variables should be correctly integrated into ABP software. In
particular, modifications of haematological parameters during and after exposure to different
altitudes/hypoxic protocols need to be properly included within detection models [13072].
Concern for the health of athletes and integrity of sport resulted in the banning of specific
substances although many years passed before analytical testing took place. Soon doping
control programmes became synonymous with urine tests and adverse analytical findings.
This system has its limits due to the detection window of prohibited substances, the timing of
sample collections and the sophistication of some doping regimens. There have been a
number of situations where these limits were demonstrated by athletes who proclaimed
innocence based on passing their analytical tests only to later confess to doping. New
strategies were called for to protect clean athletes. In the current World Anti-Doping Code,
there are eight means to an Anti-Doping Rule Violation (ADRV). Article 2.2 states that the
454
statements, documentary evidence or evaluations of longitudinal profiling. In 2006, the World
Anti-Doping Agency (WADA) with the support of some International Federations (IFs)
gathered a group of experts to develop a harmonised programme on longitudinal profiling, or
serial analysis of indirect biomarkers of doping that was both scientifically and legally robust.
This culminated in the WADA Athlete Biological Passport (ABP) Operating Guidelines and
Technical Documents, published in 2009. The ABP is a paradigm that infers the use of
prohibited substance (or method) by the monitoring of discriminant biomarkers over time.
The haematological module detects blood manipulation by the use of erythropoietic
stimulating agents or via blood transfusions. The steroidal module aims to identify
endogenous anabolic androgenic steroids when administered exogenously and other indirect
steroid doping substances or methods. Other ABP modules (endocrine, “omics”) are being
developed. The term passport, first coined in 2000, is now defined in the ABP Guidelines as
the longitudinal profile and all other relevant information including training, competitions and
information derived from investigations. In the 2015 World Anti-Doping Code, investigations
or enquiries gathered from other sources will play an even more prominent role [14448].
The athlete biological passport (ABP) was recently implemented in anti-doping work and is
based on the individual and longitudinal monitoring of haematological or urine markers.
These may be influenced by illicit procedures performed by some athletes with the intent to
improve exercise performance. Hence the ABP is a valuable tool in the fight against doping.
Actually, the passport has been defined as an individual and longitudinal observation of
markers. These markers need to belong to the biological cascade influenced by the
application of forbidden hormones or more generally, affected by biological manipulations
which can improve the performance of the athlete. So far, the haematological and steroid
profile modules of the ABP have been implemented in major sport organisations, and a
further module is under development. The individual and longitudinal monitoring of some
blood and urine markers are of interest, because the intraindividual variability is lower than
the corresponding interindividual variability. Among the key prerequisites for the
implementation of the ABP is its prospect to resist to the legal and scientific challenges. The
ABP should be implemented in the most transparent way and with the necessary
independence between planning, interpretation and result management of the passport. To
ensure this, the Athlete Passport Management Unit (APMU) was developed and the WADA
implemented different technical documents associated to the passport. This was carried out
to ensure the correct implementation of a profile which can also stand the challenge of any
scientific or legal criticism. This goal can be reached only by following strictly important steps
in the chain of production of the results and in the management of the interpretation of the
passport. Various technical documents have been then associated to the guidelines which
correspond to the requirements for passport operation. The ABP has been completed very
recently by the steroid profile module. As for the haematological module, individual and
longitudinal monitoring have been applied and the interpretation cascade is also managed by
a specific APMU in a similar way as applied in the haematological module. Thus, after
exclusion of any possible pathology, specific variation from the individual norms will be then
considered as a potential misuse of hormones or other modulators to enhance performance
[14449].
As described in the recently published ABP Operating Guidelines and Compilation of

Required Elements, data collection and administration requires specific partners such as
anti-doping organisations (ADOs), Athlete Passport Management Unit (APMU), WADA-
accredited laboratories, expert panel and WADA. Each of these entities has its own
responsibilities to guarantee reliability and credibility of the ABP programme. Briefly, ADOs
are in charge to perform an appropriate and intelligent follow-up of their athletes according to
the International Standard for Testing (IST). In the process they should also consider the
recommendations of the APMUs which are responsible of the passports real-time
455
management through the evaluation of the data of a single sample with respect to the profile
generated by the adaptive model in Anti-Doping Administration & Management System
(ADAMS). In addition, APMUs make connections with the expert panels that are necessary
to bring out any pathology or confounding factors that could impact analytical results
provided by the laboratories which shall adhere to the WADA technical documents
TD2014BAR and TD2014EAAS for haematological and steroidal module, respectively.
Moreover, expert scientists may also request additional testing for a specific athlete to collect
further indications of pathologies or to strengthen an atypical passport finding (ATPF).
Altogether, close cooperation between testing authorities, sample collection authorities and
laboratories is mandatory to ensure a prompt transfer of information and adequate timing of
testing and to allow the ABP programme to be efficient [14450].
Strategies based on the use of upper thresholds of hemoglobin or hematocrit to detect blood
doping in endurance sports have essentially failed to deter this malpractice. With the aim of
establishing a more effective strategy, it was analyzed the biological variations of
hematologic parameters in professional athletes and investigated the possibility of defining
subject-specific reference ranges that could distinguish between physiologic and abnormal
variability. Hemoglobin concentration, hematocrit, reticulocyte count, serum ferritin and
soluble transferrin receptor levels were sequentially evaluated in 923 professional football
players. Using the analysis of variance it was tested the effect of age, ethnicity, exercise
modalities and training phases on hematologic parameters and then estimated components
of variation. The significance of the difference between two measures was obtained from the
distribution of the within-subject variance (the so-called reference change). Subject-specific
reference ranges were centered around the individual mean value with dispersion based on
the 95th percentile of the coefficient of variation distribution. A total of 2,506 hematologic
determinations were made. Exercise modalities were found to have important effects on
hematologic parameters. Hemoglobin and hematocrit values were higher at the beginning of
the competition season, and then declined in well-trained athletes. Aerobic exercise was
clearly associated with lower values, suggesting that marginally low hemoglobin and
hematocrit values should physiologically be found in endurance sports. At least five
determinations were required to define subject-specific reference ranges reliably.
Considering athletes showing normal indices of red cell production (i.e., reticulocyte count
and soluble transferrin receptor), the 95th percentile of the coefficient of variation distribution
was lower than 5 percent for both hemoglobin and hematocrit. Increases exceeding 10
percent in these latter parameters should to be considered abnormal. Score systems capable
of efficiently detecting non-physiologic increases in red cell production were developed.
Thus, using proper sequential determinations of hematologic variables subject-specific
reference ranges can be defined for hemoglobin and hematocrit. Thus, the hematologic
passport is feasible and might be employed to exclude athletes with non-physiologic
increases in hemoglobin and hematocrit from competitions. The hematologic passport should
be used within a global strategy to deter blood doping [03031].
Legal aspects
Professional cycling has suffered from a number of doping scandals. The sport's governing
bodies have responded by implementing an aggressive new antidoping program known as
the biological passport. Cycling's biological passport marks a departure from traditional
antidoping efforts, which have focused on directly detecting prohibited substances in a
cyclist's system. Instead, the biological passport tracks biological variables in a cyclist's blood
and urine over time, monitoring for fluctuations that are thought to indirectly reveal the effects
of doping. Although this method of indirect detection is promising, it also raises serious legal
456
and scientific concerns. Since its introduction, the cycling community has debated the
reliability of indirect biological-passport evidence and the clarity, consistency, and
transparency of its use in proving doping violations. Such uncertainty undermines the
legitimacy of finding cyclists guilty of doping based on this indirect evidence alone.
Antidoping authorities should address these important concerns before continuing to pursue
doping sanctions against cyclists solely on the basis of their biological passports [03032].
Limitations of earlier anti-doping strategies
Concern for the health of athletes and integrity of sport resulted in the banning of specific
substances although many years passed before analytical testing took place. Soon doping
control programmes became synonymous with urine tests and adverse analytical findings.
This system has its limits due to the detection window of prohibited substances, the timing of
sample collections and the sophistication of some doping regimens. There have been a
number of situations where these limits were demonstrated by athletes who proclaimed
innocence based on passing their analytical tests only to later confess to doping. New
strategies were called for to protect clean athletes. In the current World Anti-Doping Code,
there are eight means to an Anti-Doping Rule Violation (ADRV). Article 2.2 states that the
statements, documentary evidence or evaluations of longitudinal profiling. In 2006, the World
Anti-Doping Agency (WADA) with the support of some International Federations (IFs)
gathered a group of experts to develop a harmonised programme on longitudinal profiling, or
serial analysis of indirect biomarkers of doping, that was both scientifically and legally robust.
This culminated in the WADA Athlete Biological Passport (ABP) Operating Guidelines and
Technical Documents, published in 2009. The ABP is a paradigm that infers the use of
prohibited substance (or method) by the monitoring of discriminant biomarkers over time.
The haematological module detects blood manipulation by the use of erythropoietic
stimulating agents or via blood transfusions. The steroidal module aims to identify
endogenous anabolic androgenic steroids when administered exogenously and other indirect
steroid doping substances or methods. Other ABP modules (endocrine, “omics”) are being
developed. The term passport, first coined in 2000, is now defined in the ABP Guidelines as
the longitudinal profile and all other relevant information including training, competitions and
information derived from investigations. In the 2015 World Anti-Doping Code, investigations
or enquiries gathered from other sources will play an even more prominent role [14015].
Individualized statistics
The relevance of indirect biomarkers persisted even after the introduction of a direct test for
rhEPO abuse because the time frame of direct rhEPO detection was short and other forms of
blood doping such as transfusions are believed to have been revived by fraudulent athletes
in that time. Even with the availability of a direct test for homologous transfusions biomarkers
may be used to reveal manipulations induced by blood transfusions regardless of the origin.
It is clear now that the biomarker approach bears another advantage. These markers are
already sensitive to any future compound that elicits a similar physiological response, such
as the increase of the oxygen carrying capacity. Therefore, the concept of a hematologic
passport was developed further and is presented in detail in this section. Early subject-
specific reference ranges were defined for Hb and Hct to encourage the hematologic
passport in a global strategy to deter blood doping. The main theory behind this concept is
that each athlete provides individual reference values that allow a longitudinal analysis by
applying various algorithms [13006].
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Owing to high individuality, the use of absolute reference ranges for hematological
parameters is not really useful for monitoring athletes. However, as the analytical and intra-
individual biological variability of most hematological parameters are both contained, the
definition of a type of ‘‘hematological passport’’ would allow a longitudinal comparison of data
for individual patients, accomplished with major transferability among clinical and antidoping
laboratories So far, this appears suitable for detecting a variety of blood doping practices,
and would also be acceptable in practice, considering that the individuality index of most
hematological parameters (ratio between intra- and inter-individual biological variability) is
always -0.6. The current availability of fully automated hematological systems can easily
provide the traditional parameters of the hematological profile, along with a wide range of
additional parameters for reticulocytes and erythrocytes, increasing the potential use of
laboratory testing in clinical and sports medicine. Repeated evaluation over a period of time
of several of these parameters, including hemoglobin, hematocrit, reticulocyte count and
indexes, would define a highly specific hematological profile, which is supposed to remain
relatively stable over time. At least five sequential determinations should be obtained to
define a reliable subject-specific reference range; substantial variations from the baseline, or
any value exceeding the allowable variation, could highlight either pathologies or unfair
practices, in both cases providing a good reason for an athlete’s withdrawal from
competition. Same major drawbacks of this approach, including collecting samples at
altitude, sample manipulation or the use of plasma expanders, and the type of instrument
used to measure reticulocytes, can be prevented by implementation of standardized
analytical protocols, exclusion of values obtained from samples collected at altitude, specific
instrument calibration, etc. A basic problem is that competitive athletes display significant
differences in hemoglobin, erythrocytes, hematocrit and mean corpuscular volume compared
to the sedentary population, whereas other erythrocyte indexes, reticulocyte counts and
reticulocyte parameters appear to be less influenced by lifestyle [06005].
Personalized monitoring of biomarkers for doping
The Athlete Biological Passport (ABP) is an individual electronic document that collects data
regarding a specific athlete that is useful in differentiating between natural physiologic
variations of selected biomarkers and deviations caused by artificial manipulations. A
subsidiary of the endocrine module of the ABP that which here is called Athlete Steroidal
Passport (ASP), collects data on markers of an altered metabolism of endogenous steroidal
hormones measured in urine samples. The ASP aims to identify not only doping with
anabolic-androgenic steroids, but also most indirect steroid doping strategies such as doping
with estrogen receptor antagonists and aromatase inhibitors. Development of specific
markers of steroid doping, use of the athlete's previous measurements to define individual
limits, with the athlete becoming his or her own reference, the inclusion of heterogeneous
factors such as the UDPglucuronosyltransferase B17 genotype of the athlete, the knowledge
of potentially confounding effects such as heavy alcohol consumption, the development of an
external quality control system to control analytical uncertainty, and finally the use of
Bayesian inferential methods to evaluate the value of indirect evidence have made the ASP
a valuable alternative to deter steroid doping in elite sports. The ASP can be used to target
athletes for gas chromatography/combustion/ isotope ratio mass spectrometry (GC/C/IRMS)
testing, to withdraw temporarily the athlete from competing when an abnormality has been
detected, and ultimately to lead to an antidoping infraction if that abnormality cannot be
explained by a medical condition. Although the ASP has been developed primarily to ensure
fairness in elite sports, its application in endocrinology for clinical purposes is straightforward
in an evidence-based medicine paradigm [10007].
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In elite sports, the growing availability of doping substances identical to those naturally
produced by the human body seriously limits the ability of drug-testing regimes to ensure
fairness and protection of health. The Athlete Biological Passport (ABP), the new paradigm in
testing based on the personalized monitoring of biomarkers of doping, offers the enormous
advantage of being independent of this endless pharmaceutical race. Doping triggers
physiological changes that provide physiological enhancements. In the same way that
disease-related biomarkers are invaluable tools that assist physicians in the diagnosis of
pathology, specifically selected biomarkers can be used to detect doping. The ABP is a new
testing paradigm with immense potential value in the current climate of rapid advancement in
biomarker discovery. In addition to its original aim of providing proof of a doping offense, the
ABP can also serve as a platform for a Rule of Sport, with the presentation before
competition of the ABP to objectively demonstrate that the athlete will participate in a healthy
physiological condition that is unaltered by performance-enhancing drugs. Finally, the
decision-support system used today for the biological monitoring of world top-level athletes
can also be advantageously transferred to other areas of clinical practice to reach the goal of
personalized medicine [11126]
Technical specifications necessary
The prerequisite of valid biomarker data for objective interpretation and legal use is a strict
process for sample collection, transport, and analysis that has been put into place for the
ABP. For instance, sample collection must not occur within 2 h after training or competition
and must be carried out after the athlete has remained in a seated upright position for 10 min
with feet on the floor in order to allow the vascular volumes to equilibrate. In addition,
important minimum information should be included on the ABP control form to review blood
data in an individual context such as and among others blood loss in the 3 months preceding
each sample collection, use of simulated hypoxic conditions (e.g. altitude house, tents), or
exposure to altitude above 1,000 m above sea level. Furthermore, technical documents of
the ABP guideline define the transport and storage conditions, e.g., the type of storage
device, the necessity of a storage temperature data logger, and the rapid transport so that
analysis can ideally be performed within 36 h of sample collection, although there is growing
evidence that this time frame might be extended. Samples should only be analyzed in
WADA-accredited or WADA-approved laboratories or their satellite facilities which must all
be subject to strict regular internal and external quality control procedures and follow the
WADA international standard of laboratories [13006].
Current ABP guidelines describe the particular importance of an athlete passport

management unit (APMU) that is responsible for the administrative management of the ABP.
This includes advising anti-doping organizations about intelligent, targeted testing, liaising
with the expert panel, compiling an ABP documentation package, and reporting adverse
analytical findings to anti-doping organizations and WADA. It seems essential that APMU
personnel has profound knowledge of the ABP concept and indirect doping detection
methods to ensure the intelligent, targeted approach that helps to avoid a waste of
organizational and financial resources. It can be postulated that a main goal is to avoid
parallel passport data collected by different stakeholders such as national anti-doping
organizations (NADO) or governing bodies of each sport. Therefore, harmonization of data
storage with mutual sharing of results that were analyzed with strict adherence to the WADA
protocol is important to use the ABP in its full capacities. The ADAMS (anti-doping
administration and management system) platform could provide a system for all stakeholders
for the planning, collection, and evaluation of all ABP data although the exact responsibility of
each stakeholder remains to be defined. For instance, NADOs could emphasize the testing
of a young and/or nationally active athlete but as the athlete moves on to a regular
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international competition setting, testing could be taken over by the international sporting
federation [13006].
Still an interpretation
Furthermore, it is prudent to consider that blood values may always be subject to biological
variability (e.g. gender, age) – heterogeneous factors – and confounding factors such as
physical activity (e.g. type of sport, competition vs training, or exposure to altitude). Some of
these factors may change over time, such as altitude or the type of instrument used, and
then influence longitudinal monitoring. Yet, time-independent or fixed factors such as ethnic
origin or gender are specific for a given athlete. It has also been considered heterogenous
factors and proposed a model based on a global Bayesian inference approach for the
detection of abnormal blood values over time. In such a Bayesian network, the causal
relationship between a doping activity and the induced alterations of blood markers is
represented as probabilities where every causal relationship is itself a model represented by
a conditional probability density function. WADA and other sports federations observed the
growing scientific knowledge of longitudinal subject-specific monitoring of blood values until,
in 2008, UCI was the first sports organization to introduce the hematological module of the
ABP to detect blood doping. WADA followed in December 2009 and approved the first
version of the WADA ABPoperating guidelines which have since been revised. Nowadays,
the hematological module of the ABP includes the longitudinal monitoring of eight
hematological markers (Hct, Hb, RBC, reticulocyte count (#retics), %retics, MCV, MCH, and
MCHC) to identify abnormal patterns with a subject becoming her or his own reference. From
some of the hematological markers additional models are calculated: OFF-hr and ABPS
(containing 7 markers). According to the WADA ABP guidelines the application of the
“adaptive model” predicts an expected range for an individual within which a series of marker
values falls assuming a normal physiological condition. Outliers correspond to values out of
the 99.9 percent range (0.05-99.95 percentiles) and warrant further attention and review.
Anti-doping organizations may select a value lower than 99.9 percent to identify atypical
samples and/or profiles that warrant further investigation. An expert chosen by the
responsible anti-doping organization shall initially review atypical values or an atypical
longitudinal profile. This expert shall evaluate the anonymous passport data and respond on
the basis of four hypotheses that will trigger the indicted further action:
- the measured values can be considered natural variation and thus normal: normal
testing pattern is continued
- the measured values are suspicious: further data are required and target tests shall
be performed
- the measured values may be the result of the use of a prohibited substance or
prohibited method: two further experts review the passport
- the measured values are indicative of a pathological condition: information of the
athlete or involvement of two further experts.
As the role of scientific experts in doping cases based on indirect evidence will considerably
gain in importance, an emphasis should be made in regular scientific exchange and
continuous education of the experts [13006].
An international tool
The ABP introduces a new form of doping evidence and, as such, paves the way for a more
global and integrated fight against doping. In particular, we foresee a global forensic
460
approach in which multiple pieces of evidence, not restricted to those in the current testing
paradigm, are used to demonstrate the culpability of a suspect. For example, the drug
enforcement agencies and customs departments of many countries seize large quantities of
doping substances with investigations that target illicit drugs, manufacturing companies, and
trafficking networks. Until recently, the lack of collaboration between governmental, public,
and sports authorities has hindered the combination of analytical and nonanalytical evidence
in many countries. In the current fight against doping, a customs agency can learn that a top-
level athlete has received some rEPO by mail before an important competition, but this
information is not shared with sports authorities, so that the athlete is still permitted to
participate in that competition. Interestingly, the methodology developed for the ABP provides
the necessary framework to combine evidence gathered by sports-testing organizations with
nonanalytical evidence gathered by public law enforcement agencies. For example,
knowledge that an athlete received some rEPO by mail can be combined with the information
stored in the ABP to evaluate whether the athlete used that substance before competition. As
such, we do not foresee any scientific limitation for a global fight against doping that is based
on various sources of evidence [11426].
A handful of sports federations have used the biological passport on a trial basis, but it is
becoming more widespread because the World Anti-Doping Agency, which leads the
international effort against banned sports enhancement, just released guidelines on its use.
The U.S. Anti-Doping Agency and similar groups in the United Kingdom and Norway have
adopted the passport for elite athletes in those countries. To prevent false positives,
information is collected about legitimate activities that can affect the readings, such as blood
donation during the previous six months, medications or supplements taken, and training
[10008].
Three complementary items of ABP
Three distinct modules can be distinguished in the ABP: the hematological, steroidal, and
endocrinological modules. The hematological module of the ABP aims to detect any form of
blood doping. As part of a full blood count, 8 hematological variables are considered today in
this module. In 2008, the Union Cycliste Internationale was the first sports organization to
implement the hematological module of the ABP to deter blood doping in elite cycling, and
subsequently, several riders have been prosecuted and sanctioned on the sole basis of their
abnormal hematological profiles. Currently, hematological tracing is performed by several
antidoping organizations for several thousand athletes worldwide. The steroidal module of the
ABP, which aims to detect direct and indirect forms of doping with anabolic agents, is
presently being finalized for implementation in the near future. The endocrinological module
of the ABP aims to detect doping with growth factors, such as growth hormone and insulin
growth factor-1. Despite abundant scientific publications on growth hormone-dependent
markers, the implementation of the endocrinological module in the ABP in the network of
WADA-accredited laboratories requires further validation to fulfill forensic standards [11426].
Aiming for three complementary items, the ABP will consist of a aematological, a steroidal,
and an endocrinological module with the hematological module aiming to detect any form of
blood manipulation. The fact that the ABP relies on indirect evidence for an anti-doping rule
violation, i.e. changes in parameters that are not consistent with natural variations as
determined in extensive validation studies rather than the detection of a prohibited
substance, raised concerns about the validity of the approach and led to several (scientific)
retorts and discussions [12016].
461
The longitudinal tracking and recording of eight hematological parameters should provide a
fingerprint for both the doped and the non-doped athletes, thus representing an approach
that anti-doping organizations have supported in its pioneering endeavour to demonstrate the
use (rather than the presence) of a prohibited substance or method of doping. Limitations
were reported concerning the sensitivity of the ABP analytical approach in case of EPO
microdosing, where a 12-week intravenous EPO injection intervention remained undetected
in ten subjects. The necessity to improve the current approaches was stressed (particularly
in light of the required logistics and costs concerning the ABP) and anecdotal evidence that
athletes might have adapted to the “new situation” was presented. Complementary to the
existing algorithms employing predominantly the blood parameters hemoglobin concentration
([Hb], g/l), hematocrit (Hct), and %reticulocytes (e.g. in the OFF-hr score model) the utility of
haemoglobin mass particularly in combination with %reticulocytes was evaluated in an
original mathematical model. In addition to the subject-based interpretation of blood
parameters collected in the course of ABP programs between 2001 and 2009, the utility of
the available data concerning the estimation of the blood doping prevalence among elite
track and field athletes has been described. Evaluating the empirical cumulative distribution
functions of the abnormal blood profile scores in comparison to stratified reference
cumulative distribution functions differences in blood doping prevalence were observed to be
connected to the type of sports (endurance vs. non-endurance) and, to the athletes'
nationality. That study revealed that the average prevalence of blood doping in the
investigated population is to be estimated at 14 percent [12016].
Regarding blood doping, the traditional analyses based on the detection of a substance in
biologic fluids have major limitations. Presently, only the misuse of allogeneic blood can be
directly detected, whereas retransfused autologous blood is not detectable. There is a
plethora of novel ESAs that are difficult to uncover. To overcome the detection problems, the
“Athletes Biologic Passport” has been developed, which is based on the monitoring of
selected RBC parameters. Blood doping may be suspected, when these parameters change
in a nonphysiologic way. There are methodologic problems because of the lack of clear
standardization and harmonization in antidoping testing. The longitudinal evaluation of
several hematologic variables needs high comparability among various analytical
technologies used by the different accredited laboratories. Although some parameters (i.e.
concentration of hemoglobine and hematocrit) are comparable when measured on different
instrumentations, others (i.e. percentage of macrocytes or reticulocytes parameters) are
peculiar. This bears the risk of false-positive results in athletes. On the other hand, when 400
blood samples obtained from 24 subjects receiving rhEpo injections were screened by the
passport parameters, 42 percent of the subjects were not identified as rhEpo doped. The
statistical approach for evaluating the passport data are focused on the biologic variation of
hematologic values. Critical experts in the analysis of laboratory data have argued that
antidoping tests are based on fraud statistics. Traditional antidoping analyses are based on
the detection of a substance in biologic fluids (“Adverse analytical finding”). This approach
has major limitations in regard to blood doping. As outlined in the previous section,
autologous blood cannot be detected, there is a plethora of ESAs, the detection window is
limited, and there is urine manipulation. Some sports federations earlier introduced upper
concentration of hemoglobin and Hct limits to escape from this dilemma. Athletes tested
above the limits were declared unfit for competition (“No-start rule”). However, concentration
of hemoglobineand hematocrite are influenced by external factors, such as body posture,
exercise, or residence at altitude. In addition, “clean” athletes can have naturally high
hemoglobine and hematocrite values. A large retrospective study on male blood donors in
Denmark revealed that 3.9 percent of nonathletes and 10.4 percent of elite rowers had
462
hematocrit values more than 0.51 (i.e. above the recommended limits for athletic
competition). Hematologic parameters depend on ethnicity, age, and gender. Some blood
parameters, such as the concentration of Epo and reticulocytes (Ret), increase on
administration of ESAs (ON-score), whereas they decrease after RBC transfusion or after the
cessation of ESA administration (OFF-score). The “Abnormal Blood Profile Score” (not
presently used for the assessment of abnormal blood profiles based on the passport data)
regards additional red cell parameters, including the mean corpuscular hemoglobine
concentration (MCHC), mean corpuscular volume (MCV), mean corpuscular hemoglobine
mass (MCH), Ret counts, serum Epo, and soluble transferrin receptor (sTfR).95 Algorithms
have been used that are sensitive during one of the two phases, with ON-score being
sensitive during ESA treatment and OFF-score during the cessation phase. Having become
effective in December 2009, the “Athlete Biologic Passport Operating Guidelines” equip Anti-
Doping Organizations with a framework in which to pursue antidoping rule violations in
accordance with Article 2.2. of the World Anti-Doping (WAD) Code (“Use or Attempted Use
by an Athlete of a Prohibited Substance or a Prohibited Method”). The guidelines include
mandatory requirements for collection, transportation, analysis of blood samples, and results
management. The following markers are considered in the Athlete Biologic Passport
hematologic module: Hct, Hb, RBC count, reticulocyte percentage, reticulocyte number,
MCV, MCH, MCHC, and OFF-hr score (Index of stimulation derived from a formula. In
addition, parameters of interest can be the mean Ret cell volume (MCVr), Ret Hb
concentration (MCHCr) and Ret Hb content (MCHr), as measured by flow cytometry as in
clinical routine. The results reported to the WADA are processed by an “Adaptive Model” that
identifies abnormal blood parameter changes related to the athlete’s individual profile. In
particular, concentration of hemoglobine or OFF-hr score abnormalities with a 99.9
percentage probability or more will be reviewed by experts [11428].
The development and validation of blood-doping biomarkers have also greatly evolved since
the introduction of hematological variables by some international sports federations in the
mid-1990s. With the advent of automated blood analyzers, blood variables can be
quantitatively measured to yield a complete hemogram, either in an accredited laboratory or
directly at the location of the competition, in less than a minute after blood collection. Several
approaches have contributed in recent years to make the use of biomarkers of altered
erythropoiesis an efficient approach to deterring any form of blood doping in sports:
- the introduction of multiparametric blood-doping markers

- the inclusion of heterogeneous factors, such as sex and age, as recommended by the
WHO in the diagnosis of anemia as well as other factors specific to sports
- the add-on of potentially confounding factors, such as the athlete’s exposure to
altitude
- the record of the athlete’s own previous measurements, with the underlying concept
being the use the athlete as his or her own reference
- the adoption of standardized protocols for sample collection and analysis, in addition
to the extensive use of external QC systems to control analytical uncertainty
- the development and validation of probabilistic inference techniques to evaluate the
value of doping evidence
All of the knowledge that has been acquired in recent decades concerning doping biomarkers
has been formalized in the athlete biological passport (ABP) program. The term passport was
first proposed in the early 2000s when the preservation and tracking of a longitudinal record
of hematological variable measurements were planned to be used as a means to define an
individual’s hematological profile. Large disparities between an athlete’s historical values and
the values obtained in a recent test indicate that either doping has taken place or that the
athlete has a potential medical condition that requires closer examination. The concept of the
463
ABP has been discussed and then further elaborated for antidoping application by the World
Anti Doping Agency (WADA) beginning in 2002. Since the 2006 Torino Winter Olympic
Games, several international sports federations have agreed that the WADA should
harmonize the development and validation of the ABP program. As a result, in 2009, the
WADA published the Athlete Biological Passport Operating Guidelines, which can be used as
a reference for any antidoping organizations that are interested in developing a concordant
biological monitoring program [11426].
The World Anti-Doping Agency has implemented the Blood Passport in attempt to detect
blood doping in athletes. The Blood Passport looks for uncommon changes overtime in
reticulocytes percentage (Ret %), as a variable of the OFF-hr score, and hemoglobin
concentration reflecting potential doping violations. Few studies, however, have actually
investigated the concurrent stability of Ret % and hemoglobin concentration in athletes over
extended periods of time, none of which were measured in athletes who undergo strenuous
and prolonged physical exercise. Measurements of Ret % and hemoglobine were assessed
over the course of four competitive seasons in elite triathletes (10 males and seven female).
Blood was obtained at the start of the season, precompetitive period, competitive period and
at the end of the competitive period. Significant differences were observed in both
hemoglobin concentration and Ret % between genders and there was a high variability
between subjects. Neither males nor females exhibited differences in hemoglobine across all
periods within one season. Within gender, analysis revealed that Ret % varied significantly
between periods only in female athletes. It was thus concluded that Ret % and hemoglobin
concentration remain stable over four consecutive seasons in elite triathletes, confirming that
both parameters are valid for antidoping purposes based on the Blood Passport. In addition,
Ret % fluctuations within one season require further investigation in females [11127].
The Athlete's Biological Passport (ABP) is an evaluation of hematological parameters,

hemoglobin (Hb), reticulocytes (Ret), and their combination in the OFF-score. Recently, the
Court of Arbitration for Sport accepted it as a suitable indirect method for detecting blood
doping. There are various topics which are not defined and scientifically completely
explained in ABP, limiting its effectiveness as evidence and as suspect of blood
manipulation. The data source the ABP used for designing a profile is unclear. The variance
used for cyclists is not correct. The covariables which should be calculated together with the
measures of Hb and Ret are not always considered in the statistical program. The pre-
analytical warnings for correct and valid collection, transport, and storage of the specimens
are not assured. Quality control of the instruments is not completely assured. Analytical
variability is not appropriately considered in the program. The seasonal changes of the
hematological parameters, due to training and competitions, are not calculated. Statistical
analysis, based on a Bayesian-like program, not available to the scientific community, does
not follow the classical decision-making approach of medicine and science. Therefore, the
ABP needs of additional evidences and of scientific debate [11128].
The Athlete Blood Passport is the most recent tool adopted by anti-doping authorities to
detect athletes using performance-enhancing drugs such as recombinant human
erythropoietin (rhEPO). This strategy relies on detecting abnormal variations in
haematological variables caused by doping, against a background of biological and analytical
variability. Ten subjects were given twice weekly intravenous injections of rhEPO for up to
12 weeks. Full blood counts were measured using a Sysmex XE-2100 automated
hematology analyser, and total hemoglobin mass via a carbon monoxide rebreathing test.
The sensitivity of the passport to flag abnormal deviations in blood values was evaluated
using dedicated Athlete Blood Passport software. Our treatment regimen elicited a 10%
increase in total haemoglobin mass equivalent to approximately two bags of reinfused blood.
The passport software did not flag any subjects as being suspicious of doping whilst they
464
were receiving rhEPO. It was conclude that it is possible for athletes to use rhEPO without
eliciting abnormal changes in the blood variables currently monitored by the Athlete Blood
Passport [11131].
Manipulation of the blood's oxygen carrying capacity (CaO2) through reinfusion of red blood
cells, injections of recombinant erythropoietin or by other means results in an increased
maximal oxygen uptake and concomitantly enhanced endurance performance. Therefore,
there is a need to establish a system – "A Blood Pass" – through which such illegal and
unethical methods can be detected. Venous blood samples were taken under standardized
conditions from 47 male and female Swedish national and international elite endurance
athletes four times during the athletic year of the individual sport (beginning and end of the
preparation period and at the beginning and during peak performance in the competition
period). In these samples, different hematological values were determined. ON(hes) and
OFF(hre) values were calculated according to the formula of Gore et al. A questionnaire
regarding training at altitude, alcohol use and other important factors for hematological status
was answered by the athletes. There were some individual variations comparing
hematological values obtained at different times of the athletic year or at the same time in the
athletic year but in different years. However, the median values of all individual
hematological, ON(hes) and OFF(hre), values taken at the beginning and the end of the
preparation or at the beginning and the end of the competition period, respectively, as well as
median values for the preparation and competition periods in the respective sport, were all
within the 95% confidence limit (CI) of each comparison. It must be mentioned that there was
no gender difference in this respect. This study shows that even if there are some individual
variations in different hematological values between different sampling times in the athletic
year, median values of important hematological factors are stable over time. It must be
emphasized that for each blood sample, the 95% CI in each athlete will be increasingly
narrower. The conclusion is that there is a physiological basis for establishing an individual-
based "Blood Pass" system, mainly for athletes competing at the international level. On
indications of manipulations of hemoglobin concentration and red cell mass by deviations
from established "Blood Pass" data, more specific methods can be applied [07023].
Indirect markers of altered erythropoiesis can provide enough evidence to differentiate

between natural variations and blood doping. Forensic techniques for the evaluation of the
evidence, and more particularly Bayesian networks, allow antidoping authorities to take into
account firstly the natural variations of indirect markers – through a mathematical formalism
based on probabilities – and secondly the complexity due to the multiplicity of causes and
confounding effects, through a distributed and flexible graphical representation. The
information stored in an athlete's biological passport may be then sufficient to launch a
disciplinary procedure against the athlete. The strength of the passport is that it relies on a
statistical approach based on sound empirical testing on large populations and justifiable
protocols. Interestingly, its introduction coincides with the paradigm shift that is materializing
today in forensic identification science, from archaic assumptions of absolute certainty and
perfection to a more defensible empirical and probabilistic foundation [10010].
Following the doping scandals at the World Championships in cross-country skiing in 2001,
the International Ski Federation decided to generate individual blood profiles. From 2001 to
2007, 7081 blood samples from 1074 male and female elite cross-country skiers were
collected and analyzed for hemoglobin concentration and % reticulocytes. Data were applied
to blood algorithms wherefrom blood model scores were calculated. From 1997-1999 to
2001-2002, the mean hemoglobin concentration was reduced by 0.9 g/dL to 15.3 g/dL in
male skiers and by 0.4 g/dL to 13.8 in female skiers. From 2002-2003 to 2006-2007, the
combination of increases in hemoglobin concentration and decreases in % reticulocytes led
to pronounced increases in mean OFF-model scores. Hemoglobin concentration was 0.2
465
g/dL higher at Olympic Games/World Championships (WOCs) than at World Cups
competitions <4 weeks before and after WOCs. Hemoglobin concentration and %
reticulocytes increased with altitude in both genders. Since the introduction of an enlarged
blood testing program, the mean hemoglobin concentration values were lowered to close to
normal levels, but over the last 2-3 years there has been a small elevation and an increase in
OFF-model scores, which may indicate a change in the manipulations used to elevate the
hemoglobin concentration [08211].
The aim of one study was to investigate an indirect method based on a determination of
absolute norms of variation in biological markers that could be used to identify autologous
blood transfusion within the framework of the fight against doping. The selection of markers
was made from experimental variations obtained during different phases including an
increase in training volume at sea level, high altitude training, blood withdrawal and
autologous blood reinfusion. The global statistical method was then developed in order to fix
absolute norms of variation for each selected marker. The markers selected were
haematocrit (Hct), haemoglobin concentration (Hb), stimulation index (Off-hr) and the
absolute norms of variation (normDelta) established for a maximal 15 days period were
normDeltaHct(0-15) >6%, normDelta[Hb](0-15) >4% and normDeltaOff-hr(0-15) >20 percent.
From analyses between two blood samples spaced at an interval of maximum 15 days, this
method allows to show "abnormal" variation when a variation for one of the selected markers
is strictly superior to the absolute norms of variation established. The legal framework for an
immediate application of this method could be that of the internal regulations implemented by
each international federation in accordance with the health policy in vigour [08212].
An efficient antidoping test designed to obtain direct proof of allogeneic blood transfusion
was developed and validated. This test, based on flow cytometry analysis of red blood cell
(RBCs) phenotypes, was used to determine the absence or the presence of numerous RBCs
populations in a blood sample. A such, it may constitute a direct proof of an abnormal blood
population resulting from homologous transfusion. Single-blind and single-site studies were
carried out to validate this method as a forensic quality standard analysis and to allow
objective interpretation of real cases. The analysis of 140 blood samples containing different
percentages (0-5 %) of a minor RBCs population were carried on by four independent
analysts. Robustness, sensitivity, specificity, precision and stability were assessed. ISO-
accredited controls samples were used to demonstrate that the method was robust, stable
and precise. No false positive results were observed, resulting in a 100 percent specificity of
the method. Most samples containing a 1.5 percent minor RBCs population were
unambiguously detected, yielding a 78 percent sensitivity. These samples mimicked blood
collected from an athlete 3 months after a homologous blood transfusion event where 10
percent of the total RBCs present in the recipient originated in the donor. The observed false
negative results could be explained by differences in antigen expression between the donor
and the recipient. False negatives were more numerous with smaller minor RBCs
populations. The method described here fulfils the ISO-17025 accreditation and validation
requirements. The controls and the methodology are solid enough to determine with certainty
whether a sample contains one or more RBCs populations. This variable is currently the best
indicator for homologous blood transfusion doping [08213].
The aim of one study was to evaluate physical performance loss and underlying mechanisms
following voluntary blood donation. Eleven voluntary subjects (four female) completed a
symptom-limiting cardio-pulmonary exercise test before and after blood donation (500 mL
blood). The haemoglobin value significantly decreased by 1.2 mg/dL (9 %), maximal oxygen
uptake by 9 percent, maximal work rate by 13 percent and duration of exercise fell from 663
down to 607 seconds. Anaerobic transition occurred at 81 percent and 72 percent of maximal
oxygen uptake before and after blood donation, respectively, which was a signifikant
466
difference. Subjects who practise recreational endurance sports appear to be more effected
by endurance loss. The haemoglobin value was the only significant predictor of maximal
oxygen uptake in regression analysis. It was concluded that maximal physical performance is
impaired after blood donation. Haemoglobin decline accounts for the decreased oxygen
uptake. As a consequence thereof the anaerobic transition occurs earlier. Subjects not
engaged in regular sports activity did not experience a decline in their capacity [08214].
Allogeneic transfusions are normally mismatched at one or more minor blood group antigens.
The most sensitive and accurate method known to detect this form of blood doping is flow
cytometry. Low percentages of antigen-positive and antigen-negative red blood cells (RBCs)
can be quantitated using suitable specific alloantibodies and careful analysis. By testing
blood samples taken at various times, a reduction in the percentage of a minor population of
RBCs will indicate transfusion has occurred [08215].
Hemoglobin (Hb) and hematocrit (Hct) are measured as indirect markers of doping in
athletes. We studied the effect of posture on these parameters in a typical antidoping setting.
Venous blood samples were obtained from nine endurance athletes (six males, three
females) and nine control subjects (six males, three females) immediately and after 5, 10, 15,
20 and 30 min after having adopted a seated position from normal daily activity. Hb (CV 0.72
%) and Hct (CV 0.87%) were determined using an automated cell counter, plasma volume
changes were calculated. Differences between the time points, gender and groups were
calculated using a mixed-model procedure. Significant changes were observed in the first 10
min after sitting down but no further changes were noted between 10 and 30 min. Mean
directional change for Hb and Hct between 0 min and the average of the period from 10 to 30
min was -2.4% (-0.35 g/dL) for Hb and -2.7% (-1.2%) for Hct. Plasma volume increased
accordingly. Neither group nor gender had significant effects. Under typical conditions
encountered during blood testing in doping control, a period of 10 min in a seated position is
sufficient for the vascular volumes to re-equilibrate and to adapt to the new posture [10465].
It is interesting to note that biomarkers of doping can be a useful tool to help estimate the
prevalence of doping by comparing haematological results obtained on a population of
athletes of interest with reference data obtained in clinical trials involving clean and doped
volunteer athletes. This was nicely demonstrated by Sottas et al in a study on elite track and
field athletes. Deterrent effects are hard to accurately measure without knowing the
prevalence of doping in different sport populations. However, there have been interesting
changes in blood variables as was demonstrated in a retrospective study from 2000 to 2011.
The percentage of reticulocytes outside the normal range changed significantly over the
years. In 2000–2001, about 10% of cyclists had high reticulocyte percentages (Retic %) >2
percent, then that number dropped and close to the same percentage came in with very low
Retic % as EPO testing became more widespread. Thus, athletes either stopped using EPO
earlier in relation to the competition (and testing) or switched back to blood transfusions.
Then after 2008, there was a return to normal population values as the Union Cycliste
Internationale (UCI) began running the pilot ABP and subsequently the full ABP programme.
This return to normal Retic % in cyclists could be a sign of decreased blood manipulation,
although one cannot exclude highly sophisticated doping including the use of other
substances. Further suggestions that the ABP may be having a deterrent effect come from
performance evaluations (and from anecdotal stories and interviews of athletes). The
normalisation of haematological data and significant reduction of extreme variations could
also be perceived as a health benefit for the sport population [14015].
Direct detection of blood transfusions and ESAs (erythropoietin, NESP, and CERA) is often
difficult. In 1994, it was shown that between-subject variation can be removed in doping tests
by using a series of measurements obtained from the same individual Therefore, there is a
467
growing trend towards monitoring biomarkers of erythropoiesis (hemoglobin, hematocrit, and
reticulocytes) over time (for an individual athlete) and analyzing these data using analytical
models to identify patterns suggestive of doping. This type of monitoring is referred to as the
Athlete Biological Passport. With this information, athletes can either be sanctioned directly
based on their profile, or targeted with conventional doping tests. Both the International
Cycling Union and other federations that have implemented the passport to target athletes
for the presence of erythropoiesis stimulating agents have reported a reduction of blood-
doping among their athletes. Studies are also exploring the excretion of plasticizers as
indicators of autologous blood transfusion [14017].
The haematology module of the Athlete Biological Passport (ABP) will allow authorities to
identify sudden changes in haematological parameters subsequent to a blood transfusion
process. The key is an intraindividual longitudinal evaluation of haematological parameters to
detect manipulations which affect erythropoietic parameters. A subject-specific reference
range is created and a Bayesian statistical approach is chosen to predict which values fall
outside the expected intraindividual range. Collection, transport and analysis of samples
must follow strict protocols. When intraindividual variations are higher than expected, it may
raise a case against an athlete, and then experts such as haematologists will contribute to a
detailed further evaluation. Although the ABP is a big step towards detecting illegal blood
transfusion, it does not, in its present form, provide a final solution. In cases where only a
small amount of blood is transfused, or in participants attempting to hide the effects on
haematological parameters by other means (“masking”), the ability to sentence cheating
athletes will benefit from additional evidence [14419].
The establishment of the haematological athlete's biological passport has offered the
possibility to flag those athletes with abnormal variations that deserve close monitoring. The
ABP also provides a valid drug test for sanctions as indirect markers are now recognised as
a powerful tool to combat doping manipulations that affect erythropoietic parameters.
However, there will be occasions on which an adverse analytical finding will need additional
data to reveal whether an athlete has performed transfusions or not. One example could be
an athlete with abnormal blood values that were suspicious for blood doping, who had
recently lived at altitude. This is a likely scenario as some athletes may spend time at altitude
to mask their blood doping practice. If, however, urine DEHP was also high, this would
strengthen the case for blood doping to have occurred. In summary, given the trend towards
increasing concurrent data allowing a more intelligent interpretation and planning of doping
controls, simultaneous sampling of blood and urine in the framework of the ABP appears to
be a feasible procedure which could be shortly implemented [14419].
The phlebotomy (blood withdrawal) procedure itself causes markers of erythropoiesis to

change. When stored blood is then later reinfused, it also leaves a characteristic “footprint” of
blood parameters. This approach provides the basis to detect this type of doping in the ABP.
The haematological module of the ABP uses blood markers to identify any changes in
erythropoiesis. In the future, the main objectives of ABP must be to refine the use of this
relevant tool and the introduction of new analytical markers in different fields [14419].
The haematological module of the ABP has been implemented in 2008 by certain
international sport federations and since then this indirect methodology has resulted in
sanctioning of numerous athletes for anti-doping rule violation. This achievement stimulated
the expansion of the ABP to establish the intraindividual reference ranges to monitor the
steroid profile of an athlete. In the fight against doping, steroid profiling is the next powerful
tool to detect drug misuse with endogenous anabolic androgenic steroids. Since the advent
of the fight against doping in sports in the 1970s, detection of the prohibited substances has
seen many improvements. Targeted analyses using technologies such as gas
468
chromatography coupled to mass spectrometry (GC-MS) have been the golden standard for
many years. Liquid chromatography coupled to MS (LC-MS) allowed for much easier and
straightforward sample preparation and shortened the turnaround time to complete the
analyses. Constant development in the MS instrumentation has enabled a continuous
increase of performance in terms of sensitivity as well as specificity. To establish sensitive
and reliable models, the factors influencing profiling should be recognised. It was performed
an extensive literature review of the multiple factors that could influence the quantitative
levels and ratios of endogenous steroids in urine matrix. For a comprehensive and scientific
evaluation of the urinary steroid profile, it is necessary to define the target analytes as well as
testosterone metabolism. The two main confounding factors, that is, endogenous and
exogenous factors, are detailed to show the complex process of quantifying the steroid
profile within WADA-accredited laboratories. Technical aspects are also discussed as they
could have a significant impact on the steroid profile, and thus the steroid module of the
athlete biological passport (ABP). The different factors impacting the major components of
the steroid profile must be understood to ensure scientifically sound interpretation through
the Bayesian model of the ABP. Not only should the statistical data be considered but also
the experts in the field must be consulted for successful implementation of the steroidal
module [14450].
Hemoglobin mass
Coinciding with the agreement on the ABP, it was evaluated three passport approaches for
their sensitivity and specificity for the detection of autologous blood transfusion. The best
possible marker for lower dosages of transfused blood (e.g. one bag) was OFF-hr.
Interestingly, a new score (Hbmr) was introduced and showed the best performance for
larger amounts (e.g. three bags), but requires the determination of total hemoglobin mass
(Hb mass) as a further measure. Hb mass determined by the optimized CO rebreathing
method was first suggested as a potential biomarker in the context of blood doping in 2007
mainly because it is independent of plasma volume fluctuations. The usefulness and
applicability in several circumstances were evaluated in several studies thereafter. Hb mass
was also evaluated as a marker in the adaptive model of the ABP in a longitudinal blinded
study, in which a new score (OFFmass including %retics) was likewise published and yielded
a sensitivity of 73 percent without false-positives at the 99.9 percent specificity level. Hb
mass was also evaluated for the potential to detect rhEPO misuse. Various efforts have been
made to improve the problems associated with the CO rebreathing method such as the
administration of a potentially toxic substance and lack of a quality control system.
Nevertheless, it seems that these problems limit the applicability of the method in anti-
doping. Therefore, the search is on for an alternative to Hb mass determination which is
compatible with today’s standards of testing because of its potential to improve the detection
rate of autologous blood transfusion. On such approach could be the indirect modeling of Hb
mass from indirect markers [13006].
Hematocrit
Since the introduction of Hct cut-off values, several studies have evaluated the current limits.
The Hct value is subject to a number of influences and can thereby change significantly
within a short period of time. Diurnal variance of Hct ranges around 2 percent, with higher
values in the morning. Change of body posture from supine to upright position can increase
Hct up to 6 percent within 20 minutes. Dehydration results in a higher Hct due to
hemoconcentration. Physical training has a long-term decreasing and a short-term increasing
effect on Hct. Even a sampling technique is known to have a major impact on Hct. Hct can
increase significantly within minutes when a tourniquet for sampling purposes is applied
469
[01011].
Variability of serum markers of erythropoiesis during intense exercise
The athlete biological passport for the fight against doping is currently based on longitudinal
monitoring for abnormal changes in cellular blood parameters. Serum parameters related to
altered erythropoiesis could be considered for inclusion in the passport. The aim of one study
was to quantify the changes in such parameters in athletes during a period of intense
exercise.12 highly trained cyclists tapered for 3 days before 6 days of simulated intense
stage racing. Morning and afternoon blood samples were taken on most days and analysed
for total protein, albumin, soluble transferrin receptor and ferritin concentrations. Plasma
volume was determined via total haemoglobin mass measured by carbon-monoxide
rebreathing. Percent changes in means from baseline and percent standard errors of
measurement (analytical error plus intra-athlete variation) on each measurement occasion
were estimated with mixed linear modelling of log-transformed measures. Means of all
variables changed substantially in the days following the onset of racing, ranging from -13
percent (haemoglobin concentration) to +27 percent (ferritin). After the second day, errors of
measurement were generally twice those at baseline. Plasma variables were affected by
heavy exercise, either because of changes in plasma volume (total protein, albumin,
haemoglobin), acute phase/inflammatory reactions (ferritin) or both (soluble transferrin
receptor). These effects need to be taken into consideration when integrating a plasma
parameter into the biological passport model for athletes [14008].
One investigation quantified the effect of changes in plasma osmolality on the measurement
of hematocrit (Hct) and the implications for the subsequent use of these data to calculate
changes in plasma volume and application to the World Anti-Doping Agency Athlete
Biological Passport. Two groups of eight male volunteers visited the laboratory after an
overnight fast. In study 1, a 20-mL blood sample was collected and aliquoted into collection
tubes containing saline of varying concentrations to alter the sample osmolality. In study 2,
plasma osmolality was manipulated in vivo through prolonged exercise. Samples were
analyzed for hemoglobin concentration and Hct using manual methods and using an
automated hematology analyzer (AHA). Changes in blood, plasma, and red cell volumes
were calculated. Although AHA Hct values did not change, spun packed cell volume fell
progressively as the osmolality of the sample increased. Consequently, there was a
significant increase in apparent plasma volume as osmolality increased: regression analysis
revealed that a 10 mOsm·kg change in plasma osmolality produced a difference of 0.8 Hct
units and a 1.6% change in plasma volume. In study 2, exercise produced a 12 ± 3 mOsm·kg
increase in plasma osmolality. No difference in Hct was apparent at rest, but spun packed
cell volume was 1.0 ± 0.9 Hct units lower during exercise compared with AHA data. There
was a difference in the degree of plasma volume change calculated, with a reduction of 8.7 ±
3.4 percent and 11.3 ± 3.5 percent reported with the manual and AHA methods, respectively.
It was concluded that conditions or interventions that result in a marked change in plasma
osmolality produce a discrepancy in Hct measured using an AHA, consequently introducing
errors into any calculation of changes in plasma volume using these data. These findings
may also have implications for the measurement of Hct by World Anti-Doping Agency-
accredited laboratories [01420].
Short-term effects of prolonged strenuous endurance exercise

Knowledge is sparse about the extent of potential dehydration due to prolonged strenuous
cycling and its haematological acute effects on the haematocrit (Hct) in study populations
credibly not taking any kind of doping. With increasing training load levels of Hct and
haemoglobin (Hb) decrease in both amateurs and professionals as a long-term consequence
470
due to expanded plasma volume (PV). On a short-term basis, however, counteracting
dehydration potentially brought about by endurance exercise may cause a rise in Hct
bringing competitive cyclists into conflict with the current condition regulations and Hct cut-off
of 50 percent set by the International Cycling Union (UCI) in its fight against erythropoietin
(rhEPO) doping. On the other hand adequate and sufficient fluid substitution being
substantial for a successful endurance performance should prevent any pronounced Hct
rises. To study the haematological acute effects of prolonged strenuous cycling we
measured Hct, Hb, red blood cell (RBC) count and plasma protein in a reliably 'clean'
population of 38 well-trained male amateur cyclists before, immediately after and one day
after an extraordinary ultramarathon. The pre-race levels of Hct, Hb and RBC count were
placed in the lower range of normal distribution and well below the Hct cut-off limit of the UCI.
Immediately post-exercise the mean levels of Hct, Hb, RBC count and protein remained
unchanged. One day after race, however, all four parameters significantly dropped by 3, 7, 7,
10 percent respectively, indicating marked post-exercise PV expansion. The calculated
percentage increase in PV was 12 percent. No evidence for coexisting exercise-induced
haemolysis was found. Our study shows that in "clean, rhEPO-free" amateur cyclists who
involve in strenuous marathon cycling the haematological short-term effects of extraordinary
marathon cycling consist in considerable PV expansion making Hct values fall on the
following day. The findings – gained from amateurs though – suggest that despite all its
disadvantages the UCI Hct cut-off represents an appropriate means to discourage from
excessive rhEPO doping at least as long as the available direct methods for detecting this
kind of misuse are not yet applied by the international sports federations [02020].
Plasma osmolality
Plasma osmolality is known to change during exercise, and depending on the chosen
analytical approach (e.g. manual vs automated measurements) significant differences in
plasma volume-depending parameters such as hematocrit can occur. Since ABP
measurements are harmonized internationally, these observations should not apply to
routine doping controls. Additionally, suggestions concerning the improved accounting for
real and/or simulated altitude in the ABP algorithm were made, especially in the light of the
different scenarios athletes might exploit altitude exposure or the assumed effects thereo
[14715].
Animal studies
The extent to which hematocrit is regulated and the impact of altered hematocrit on blood
oxygen transport in avian embryos are largely unknown. Consequently, it was investigated
how acute blood removal or Ringer solution injection modified hematocrit in day 15 embryos,
and how “blood doping” with erythrocyte-enriched whole blood influenced O2 consumption in
day 15-17 chicken embryos. Mean hematocrit (+ s.e.m.) at day 15, 16 and 17 was 27 + 1
percent, 28 + 0 percent, and 31 + 1 percent, respectively. Blood withdrawal (19 increments of
125 mul each, separated by 30 min) caused a progressive fall in hematocrit to approximately
12 percent at day 15. Hematocrit decline was strictly proportional to the extent of blood
withdrawal. Incremental Ringer solution injection over an 8 h period, transiently increasing
blood volume up to 85 percent over initial values, did not decrease hematocrit, indicating that
injected Ringer solution rapidly left the circulating blood compartment. Blood doping with
erythrocyte-enriched whole blood artificially elevated hematocrit from 27 percent to 38
percent, but caused no significant change in routine O2 consumption (0.35-0.39 ml O2 per
min per egg) at any point over the subsequent 6 h period in day 15-17 embryos. It was
conclude that hematocrit is not protected acutely in day 15 chicken embryos, with no
evidence of erythrocyte sequestration or release. Additionally, at day 15-17, hematocrit
471
increases of approximately 10 percent do not enhance embryonic oxygen consumption,
suggesting that blood oxygen carrying capacity per se is not limiting to oxygen consumption
[08216].
Biking
During the Tour de France 2007, 7 riders were randomly tested on 3 different occasions; the
day before the prologue, and 12 and 19 days after the prologue. Blood was drawn into 3 mL
EDTA covered tubes and kept at 4 degrees Celsius. They were analyzed within 24 hours.
The concentration of hemoglobin and hematocrit were significantly lower on day 12 and day
19 compared to baseline. All 7 riders had lower hemoglobin and hematocrit on day 19
compared to baseline, while this was the case in 6 out of 7 riders already on day 12. The
concentration of hemoglobin and hematocrit were 11.5 percent and 12.1 percent lower on
day 19 compared to baseline. Whether or not this low value is due to decrease in
hemoglobin mass or hemodilution, or the latter solely, increases in hemoglobin and
hematocrit during prolonged stage racing seem unphysiological and should therefore lead to
further examination of the rider [08217].
Possible variables
Blood passport has been suggested as an indirect tool to detect various kinds of blood
manipulations. Autologous blood transfusions are currently undetectable, and the objective of
this study was to examine the sensitivities of different blood markers and blood passport
approaches in order to determine the best approach to detect autologous blood transfusions.
Twenty-nine subjects were transfused with either one (n=8) or three (n=21) bags of
autologous blood. Hemoglobin concentration ([Hb]), percentage of reticulocytes (%ret) and
hemoglobin mass (Hbmass) were measured 1 day before reinfusion and six times after
reinfusion. The sensitivity and specificity of a novel marker, Hbmr (based on Hbmass and %ret),
was evaluated together with [Hb], Hbmass and OFF-hr by different passport methods. The
novel Hbmr marker showed superior sensitivity in detecting the highest dosage of transfused
blood, with OFF-hr showing equal or superior sensitivities at lower dosages. Hbmr and OFF-hr
showed superior but equal sensitivities from 1 to 4 weeks after transfusion compared with
[Hb] and Hbmass, with Hbmass being the only tenable prospect to detect acute transfusions.
Because autologous blood transfusions can be an acute practice with blood withdrawal and
reinfusion within a few days, Hbmass seems to be the only option for revealing this practice
[11132].
Example of the haematological modules
The haematological passport of an athlete is in fact a statistical representation of the

longitudinal follow-up of some of his blood markers. The individual and longitudinal follow-up
is of interest when the intraindividual variability of a biological marker is lower than the
corresponding interindividual variability. This fact is obvious for most of the blood markers in
consideration. Then, from a statistical point of view, the athlete has his own reference values
for a biological biomarker. The indirect detection of blood doping, as described previously,
was one of the major tasks of the antidoping community before the 2000 Sydney Summer
Olympic Games. Many developments in this field were set up by the Australian Institute of
Sport, whose work was mainly dedicated to the detection of rh-EPO for the 2000 Olympics.
The basis of these developments was to get a score using several indirect haematological
and biochemical markers used simultaneously to identify efficiently current or recent users of
rh-EPO [14433].
472
The first models presented by the group integrated a combination of RETHCT, serum EPO,
serum soluble transferrin receptor (sTFR), HCT and percentage of macrocytes. They then
distinguished the so-called ON-model, giving a score composed of these markers describing
the situation they had during the final 2 weeks of rh-EPO administration phase. The OFF-
model integrated RETHCT, EPO and HCT and was applied during the washout phase and,
during the period up to 12–21 days after the last rh-EPO injection. Magnani et al in 2001
demonstrated that HCT, RET count, serum sTFR and concentration of beta-globin mRNA put
together into a new multiparametric formula, could detect rh-EPO abuse in about 60 percent
of the samples examined after low dosage of rh-EPO doping. Altitude and exercise could of
course change the paradigm for multiparametric calculations. The evolution of doping habits
among endurance athletes and their spectacular mode of adaptation to respond to the
detection policy pushed the scientists to propose new models. Gore et al and Sharpe et al
presented the second generation model, which defines better the situation, when lower
dosages of rh-EPO are used [14433].
In 2006, Sottas et al proposed to use and combine all data contained in a single blood profile
of an athlete. The Abnormal Blood Profile Score (ABPS) corresponds to a multiparametric
marker which can include up to 12 different blood markers which are affected by the
administration of rh-EPO or the use of blood transfusion. More recently, two other statistical
tools were suggested to be used for targeting purposes only. Indirect markers used in the
haematological passport can be affected by heterogenous and confounding factors. These
factors will create variability in the multiparametric score which needs to be correctly
managed for the proper use of the system. The role played by factors like age, gender,
altitude, type of sport and instrumentation has been extensively studied. Training at altitude
is one of the factors which can produce the highest variability of the model, affecting then the
specificity and the sensitivity of any longitudinal tool describing the evolution of
haematological markers. It is nowadays generally accepted that an exposure to high altitude
during the 2 weeks preceding the test must be taken into account in establishing individual
limits even if the debate is never ending in regard to the factors influencing the stability of
blood markers and then, the specificity of the system. It has been also argued that seasonal
changes of haematological markers, due to training and competitions, should be taken into
account. A greater intravariability factor, if included in the model, will of course be detrimental
to the sensitivity of the passport, which seems to be already critical. Recently, this sensitivity
to flag abnormal deviations in blood values was evaluated after microdosage of rh-EPO
treatment applied to athletes. The authors demonstrated that it is possible for athletes to use
rh-EPO without eliciting abnormal changes in blood markers currently used by the passport,
questioning its sensitivity [14433].
How does the haematological module work?
The different markers of blood doping on which experience has been acquired in recent
decades have been implemented into the athlete biological passport (ABP). The concept of
the ABP has been discussed and then further elaborated by the WADA. Since the 2006
Torino Winter Olympics, several international federations (IFs) agreed that WADA should
harmonise the development and validation of the ABP programme. As a result, in 2009,
WADA published the ABP operating guidelines which are regularly updated. This can
nowadays be used by anti-doping organisations (ADOs) who want to implement the
passport. In these guidelines, two main objectives are defined in the introduction:
- To identify and target athletes for specific analytical testing (e.g. EPO urine test,
homologous blood transfusion test) by intelligent and timely interpretation of
haematological module data
473
- To pursue possible antidoping rule violations in accordance with the World Anti-
Doping Code Article 2.2 (use or attempted use by an athlete of a prohibited
substance or a prohibited method)
As described in the WADA document, the aim of the ABP, when implemented in accordance
with the appropriate technical documents, is a reliable method for indirectly detecting doping
that can resist to legal and scientific challenges. Furthermore, it can also be used to evaluate
the prevalence of doping within a population of interest [14433].
Haematological markers and athlete's information for the ABP
The haematological module gathers information on the potential markers for blood doping
(e.g. rh-EPO doping blood transfusion and gene manipulation). In the haematological
module, the following markers are considered:
- HCT
- HGB
- RBC: RBC count
- RET%: the percentage of RETs
- RET#: RETs count
- MCV: mean corpuscular volume
- MCH: mean corpuscular haemoglobin¨
- MCHC: mean corpuscular haemoglobin concentration
Some of these markers can be combined to be incorporated into the module such as the
OFFs, as described by the Australian group in their presentation of the second generation
blood test to detect EPO abuse by athletes. The OFFs is calculated by the following formula:
OFFs = HGB−60√(RET%)
- HGB: HGB concentration in g/L

- RET%: RETs in percentage
Another combination, ABPS, introduced by Sottas et al in 2006, is a combination of up to 12

different blood markers such as HCT, HGB, RBC, RET%, MCV, MCHC, serum EPO, serum
sTFR, RBCo (optical RBC), RDW (red cell distribution width), RET# (absolute RET count),
IRF (immature RET fraction). The passport will also take into account the individual athlete
profile information to provide as precisely as possible the context for a better interpretation of
the markers. These additional informations include, but are not restricted to gender, type of
sport, whereabouts information for the month before, competition schedule use of hypoxic
devices or altitude training [14433].
Technical documents associated to the haematological module
To allow a correct implementation of a profile which can resist to any scientific or legal
criticism it is mandatory to follow rigorously some steps in the chain of production of the
results and in the management of the interpretation of the passport. Four technical
documents have been then associated to the guidelines for the haematological module which
correspond to the requirements for the passport operation. These documents are linked to
the International Standards for Testing and International Standards for Laboratories but have
been put in place exclusively for the implementation of the passport. The preanalytical
procedures, which are associated to the two first documents, are certainly among the most
sensitive and critical paths in the overall process. With blood testing and the introduction of
the haematological module, living material for the first time has been introduced in the fight
474
against doping. This is a major change in comparison to the collection and transport of urine,
which is still the most common biological sample used in that field. Many authors studied all
these technical and biological aspects and all proposed necessary improvements in the
procedures [14433].
Regarding the blood collection, beside the technical aspects of the blood draw (which must
be performed after allowing a timeout period in a sitting position), the timing of the sample
collection, the exercise and the exposure of the athlete to altitude (real or simulated) are
regarded as very important for the stability of haematological markers. Then, the main points
to be confirmed are the following:
- No training or competition before the last 2 h of the blood test.

- Did the athlete train, compete or reside at an altitude greater than 1000 m within the
previous 2 weeks?
- Did the athlete use any form of altitude simulation (hypoxic tent, mask, etc) during the
previous 2 weeks?
- Did the athlete receive or donate for any reason blood transfusion the previous 3
months?
For transportation of the sample, the rules are relatively obvious. It must be safe in order to
assure the integrity of the sample and assure the chain of custody. It must be rapid, because
the blood in the laboratory must be analysed within 36–48 h of sample collection. It must be
cooled (2-12°C) in order to preserve the blood cells in the sample and keep them intact
[14433].
Microdoses
The Athlete Biological Passport (ABP) was recently introduced as a new tool to indirectly
detect erythropoiesis-stimulating agents such as rHumanEPO, which can lead to a doping
sanction and at the same time as intelligently targeting athletes for additional testing. The
ABP approach relies on identifying intraindividual abnormal variability over time in selected
haematological parameters. The blood doping behaviour of athletes has changed since the
introduction of the ABP. Extreme reticulocyte values (i.e. <0.4 % or >2 %) have dramatically
decreased and as such, the implementation of the ABP has undeniably been a step forward
in the antidoping field. Although the ABP approach can identify abnormal enhanced
erythropoiesis regardless of the method used, such as autologous blood transfusion, as
shown by Pottgiesser et al in a field-like longitudinal blinded setting, the sensitivity of the ABP
has been questioned. In order to minimise the risk of being caught via the ABP, it is well
known that some athletes are now using “microdoses” of rHumanEPO which allegedly range
from 10 to 40 IU/kg body mass. Microdoses of rHumanEPO aim (1) to increase haemoglobin
mass (Hbmass) while avoiding large fluctuations in the ABP blood markers as well as
minimising the detection window for conventional direct methods and/or (2) to “normalise” the
ABP blood markers after blood manipulations such as autologous blood transfusion. In
addition, variation in ABP haematological parameters due to factors such as training or
hypoxia exposure can influence the interpretation of the ABP results. Adjustments by the
athletes to antidoping methods impose a constant need for scientists and antidoping
authorities to develop and implement new detection methods based on innovative concepts
and cutting-edge technologies [14430].
Erythropoietin
The so-called third-generation tests or the z-score were introduced to distinguish the effect of
475
rhEPO abuse from natural biological fluctuations with longitudinal observations of Hb or OFF-
hr. Assuming a universal within-subject variation only two values from one athlete could be
used for the calculation of the z-score. In a new approach, it was concluded that longitudinal
data might not only be used to ban participation in competition but also to establish target
testing of suspicious athletes. This was important as only positive direct test results could be
used for sanctioning of manipulating athletes. Progress of the passport concept prevailed as
Sottas et al. combined all data contained in a single blood profile of an athlete in a universal
multiparametric score (abnormal blood profile score, ABPS) that was initially presented in
various versions using between three and 12 different biomarkers responding to either
rhEPO administration or blood transfusion. In the current version of the WADA ABP
operating guidelines, ABPS calculated from Hct, Hb, RBC count, reticulocyte percentage
(RET%), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean
corpuscular hemoglobin concentration (MCHC) is recommended as a marker in addition to
OFF-hr. In a universal marker such as ABPS various doped states might equally lead to a
high score, e.g., the donation and reinfusion of autologous blood both will elevate the score,
whereas this will lead to a one-directional change only from a low to a high value for the non-
universal marker OFF-hr [13006].
Steroid profile for athlete biological passport
The athlete's steroid profile analysis represents a powerful tool to identify the administration
of AAS (including those of endogenous as well as xenobiotic nature). This is achievable by
monitoring urinary concentrations of selected steroids and comparing their levels and specific
steroid ratios to inter-individual and intra-individual reference ranges. Traditionally,
population-based reference ranges are applied as defined in respective technical documents
issued by WADA. The five-year data review from a single laboratory demonstrated a rather
limited gain in sensitivity for identifying testosterone abuse along with a de facto considerably
increased workload concerning the confirmatory analyses required in cases of T/EpiT
ratios > 4. Alternatively, subject-based reference ranges were favoured and the consideration
of additional hormone concentrations (e.g. of luteinizing hormone, LH) was recommended. In
another study, the threshold values for testosterone (200 ng/mL), epitestosterone
(200 ng/mL), androsterone (10,000 ng/mL) etiocholanolone (10,000 ng/mL), dehydroep-
iandrosterone (DHEA, 100 ng/mL), 5alpha-androstane-3alpha,17beta-diol (200 ng/mL) and
5beta-androstane-3alpha,17beta-diol (200 ng/mL) were evaluated. The consideration of
subject-based (intra-individual) reference ranges in particular has gained much attention and
several initiatives pursuing this route for complementing modern anti-doping efforts have
been initiated. In a recent pilot study, new potential biomarkers to support the identification of
testosterone misuse in sports were described. Besides the established
testosterone/epitestosterone ratio (T/EpiT), other additional steroid ratios have been found to
possess the potential to support the detection of testosterone abuse, namely 6alpha-OH-
androstenedione/16alpha-OH-dehydroepiandrosterone, 4-OH-androstenedione/16alpha-OH-
androstenedione, 7alpha-OH-testosterone/7beta-OH-dehydroepiandrosterone, and dihydro-
testosterone/5beta-androstane-3alpha,17beta-diol. Since these findings represent results of
a pilot study, the variability and susceptibility of the identified marker ratios will require follow-
up studies under different conditions to demonstrate their long-term stability and influences
caused for example by acute ethanol consumption or bacterial activity. The same approach
was also used to screen for additional biomarker ratios supporting the identification of
dihydrotestosterone (DHT) or DHEA misuse in sports. Besides the detection of steroid
abuse, steroid profiles have also been shown to provide valuable information concerning
possible degradation processes occurring in urine specimens due to the non-sterile collection
conditions. The measurement of free testosterone and/or epitestosterone (the percentage of
476
which should be below 5 % of the glucuronic acid-bound counterpart) and free 5alpha- and
5beta-androstane-3,17-dione (elevated concentrations of which are considered as indicative
for urine degradation) is used to test for the validity of doping control samples regarding the
steroid profile interpretation [12016].
The Athlete Biological Passport (ABP) is an individual electronic document that collects data
regarding a specific athlete that is useful in differentiating between natural physiologic
variations of selected biomarkers and deviations caused by artificial manipulations. A
subsidiary of the endocrine module of the ABP that which here is called Athlete Steroidal
Passport (ASP), collects data on markers of an altered metabolism of endogenous steroidal
hormones measured in urine samples. The ASP aims to identify not only doping with
anabolic-androgenic steroids, but also most indirect steroid doping strategies such as doping
with estrogen receptor antagonists and aromatase inhibitors. Development of specific
markers of steroid doping, use of the athlete's previous measurements to define individual
limits, with the athlete becoming his or her own reference, the inclusion of heterogeneous
factors such as the UDPglucuronosyltransferase B17 genotype of the athlete, the knowledge
of potentially confounding effects such as heavy alcohol consumption, the development of an
external quality control system to control analytical uncertainty, and finally the use of
Bayesian inferential methods to evaluate the value of indirect evidence have made the ASP
a valuable alternative to deter steroid doping in elite sports. The ASP can be used to target
athletes for gas chromatography/combustion/ isotope ratio mass spectrometry (GC/C/IRMS)
testing, to withdraw temporarily the athlete from competing when an abnormality has been
detected, and ultimately to lead to an antidoping infraction if that abnormality cannot be
explained by a medical condition. Although the ASP has been developed primarily to ensure
fairness in elite sports, its application in endocrinology for clinical purposes is straightforward
in an evidence-based medicine paradigm [10009].
The steroidal module took effect on 1 January 2014; it uses the same principles and
processes as the haematological module and can be used for targeting or can directly result
in an ADRV. A further feature of the current steroidal module is an enhanced individualised
approach to testosterone:estrogen (T:E) ratios for IRMS confirmation. Since the 1980s, a T:E
ratio of initially >6:1 and then >4:1 was considered suspicious of doping. IRMS confirmation
was then recommended on the sample in order to confirm the presence of an exogenous
steroid. It is known that there are certain genetic polymorphisms, such as the deletion of the
UGTB217 gene, that result in very low T:E ratios far below the typical 1:1.11 Applying a
longitudinal approach with personalised upper and lower limits would take into account those
with low but fluctuating T:E ratios who may have otherwise avoided this confirmation test and
also decrease excessive IRMS testing in those with naturally T:E high ratios. Therefore the
steroidal module will result in a more effective and efficient use of IRMS [14015].
A factor to be considered in steroid profiling, the construction of regionally relevant reference
ranges was discussed. In one study the urinary steroid profiles measured from 2454 and
1181 specimens obtained from male and female Latin American athletes, respectively, were
presented. The specimens originated from Cuba, Venezuela, Mexico, Dominican Republic,
Guatemala, and Chile and a total of 17 analytes were evaluated (including 10 androgens, 3
estrogens, 2 pregnanes and 2 corticosteroids) using established GC-MS methodologies. The
fact of significant differences in steroid profiles of males and females was confirmed also
within the studied population, which included (under consideration of the overall population)
Amerindians, Creoles, Afro-Americans, Africans, and Asians. Moreover, the upper limit of the
calculated 98 percent confidence range was in agreement with the recommendations on
steroid profile analyte concentrations and ratios as defined in the prevailing WADA technical
document TD2004EAAS for further confirmatory actions [14009].
477
In the light of the continuously growing knowledge on the relevance of steroid conjugation
(i.e. phase-II metabolism reactions), various new studies were initiated and reported
concerning steroid glucuronides and sulfates, factors arguably confining their urinary
concentrations, and the accurate and sensitive determination of these steroid conjugates for
potential evaluation as a complement to the current steroid profiling approaches. It was
investigated the utility of combining analytical results of 7 steroid glucuronides and 5 steroid
sulfates for the detection of transdermal and oral administrations of testosterone and
testosterone undecanoate, respectively. A total of 19 volunteers were subjected to
genotyping concerning the insertion/deletion of the UGT2B17 gene yielding 7 ins/ins, 7
ins/del, and 5 del/del genotypes. All participants underwent a transdermal testosterone
application (via patches providing 2.4 mg of testosterone/24 h) and, after washout, oral
testosterone undecanoate administration (2 x 40 mg), and urine samples were collected over
a period of 96 h. By means of LC-MS/MS, relevant steroid conjugates were quantified,
corroborating the issue of common GC-MS-based steroid profile approaches that population-
based reference ranges barely allow the identification of topical and oral testosterone
administration. Employing an intra-individual profiling strategy, the administration of
testosterone via patches was identified, particularly by means of the ratios of testosterone
glucuronide (TG)/epitestosterone glucuronide (epiTG) as well as androsterone glucuronide
(AG)/etiocholanolone glucuronide (EG). The ingestion of testosterone undecanoate was
detectable predominantly by means of etiocholanolone sulfate (ES), especially in UGT2B17
del/del genotypes [14009].
Steroid profile analyses represent an important resource of information concerning both the
administration of natural (endogenous) steroids as well as those of xenobiotic origin. Steroid
profiling has been utilized in sports drug testing for more than three decades and still much
effort is invested in elaborating and improving this valuable tool, particularly to increase its
screening efficiency and to allow for consideration of more recently clarified (genetically or
pharmacologically induced) variations influencing the steroid profile interpretation [13009].
GC-MS(/MS)-based methods with electron ionization (EI) are still preferred over alternative
options to produce steroid profile data; nevertheless, the utility of chemical ionization (CI) in
combination with comprehensive 2-dimensional GC (GCxGC) and a fast-scanning
quadrupole-MS was evaluated and found to be competitive with commonly used GC-MS
benchtop systems concerning steroid quantification. The advantage of this approach was
mentioned to be the superior GCxGC separation of analytes with full-scan EI-MS data
recording, which supports the detection of presumably unknown anabolic agents. Here, the
employed model steroids were measured mainly underivatized or acetylated, which is
common to IRMS analyses but (yet) seldom to generic steroid screening assays [13012].
Steroid profile consists of the quantification of several glucuroconjugated and free urinary
compounds linked to T and its metabolism, and is well known as a potent tool to uncover
doping with endogenous anabolic steroids. However, due to a wide interindividual variability
in absolute endogenous steroid concentrations originating from various factors, it has been
proven that population-based reference values, which were considered for years by every
protagonist in the fight against doping, are not always sensitive enough to reveal the
potential misuse of anabolic androgenic steroids at an individual level. For these reasons,
there is an obvious need of individual monitoring of the steroid profile to allow a fair
evaluation. Before the steroidal module, ABP has been developed using a Bayesian
approach to deter blood doping based on haematological data obtained in whole blood
sample. Historically, anti-doping laboratories and sport authorities detect misuse of
endogenous steroids based on the ratio between T and its 17alpha-epimer, epitestosterone
(E; T/E ratio). A threshold based on previous anti-doping data and population studies was
first set at 6 by the International Olympic Committee in 1983 and later lowered to 4 to
478
discriminate between normal and abnormal values. Urine samples showing a T/E ratio above
the threshold were then submitted to further analyses such as gas chromatography-
combustion-isotope ratio mass spectrometry to evaluate the steroid profile and the
endogenous or exogenous origin of the target compounds. The sensitivity of the T/E ratio
approach based on population-based reference ranges has been questioned since 1994. At
that time, individual reference ranges, instead of population-based references, have already
been proposed and used in steroid profiling. However, there are now a new and very
effective mathematical tool came into this field which allowed an optimised evaluation of the
longitudinal data. This mathematical model is, currently, one of the basic tools of the ABP
[14450].
Knowing that every athlete has his/her own metabolism and responds differently after any
drug misuse, this profiling approach is relevant for the results management in the fight
against doping. Even if the follow-up of secondary markers indicating a drug intake or a
manipulation to increase the performance skills is becoming essential, direct detection of
prohibited substances is still necessary to prevent athlete from cheating, and the biological
passport profile may assist in targeting the doping control analysis to particular additional
tests, such as GC-C-IRMS. As the urinary steroid profile, especially the T/E ratio, is well
known as being a stable marker within an individual, the integration of the adaptive steroidal
module was a natural evolution of the ABP. This module aims to identify endogenous
anabolic androgenic steroids when administered exogenously and other anabolic agents,
such as selective androgen receptor modulators categorised under section S1 of the
Prohibited List. Six markers are considered within the steroidal module which are T, E,
androsterone, etiocholanolone, 5alpha-androstane-3alpha,17beta-diol (5alpha-diol) and
5beta-androstane-3alpha,17beta-diol (5beta-diol) [14450].
In the fight against doping, steroid profiling is a powerful tool to detect drug misuse with
endogenous anabolic androgenic steroids. To establish sensitive and reliable models, the
factors influencing profiling should be recognised. It was performed an extensive literature
review of the multiple factors that could influence the quantitative levels and ratios of
endogenous steroids in urine matrix. For a comprehensive and scientific evaluation of the
urinary steroid profile, it is necessary to define the target analytes as well as testosterone
metabolism. The two main confounding factors, that is, endogenous and exogenous factors,
are detailed to show the complex process of quantifying the steroid profile within WADA-
accredited laboratories. Technical aspects are also discussed as they could have a
significant impact on the steroid profile, and thus the steroid module of the athlete biological
passport (ABP). The different factors impacting the major components of the steroid profile
must be understood to ensure scientifically sound interpretation through the Bayesian model
of the ABP. Not only should the statistical data be considered but also the experts in the field
must be consulted for successful implementation of the steroidal module [14450].
Anabolic steroids are mainly excreted through the urinary route, requiring modifications of
their hydrophobic chemical structures. Phase I and phase II metabolic reactions are
responsible for, respectively, functionalisation and addition of conjugates (i.e. glucuronides or
sulfates) to steroids, thereby increasing their hydrophilicity and allowing their dissolution and
elimination in urine mixture. Since steroid conjugates analysis is not compatible with GC-MS,
the only analytical technique recognised by WADA for endogenous steroids quantification in
urine, deconjugation of the conjugated moiety by enzymatic hydrolysis (beta-glucuronidase)
is a crucial step during sample preparation and prior to GC-MS measurement [14450].
Steroid profile consists of the quantification of several glucuroconjugated and free urinary
compounds linked to T and its metabolism, and is well known as a potent tool to uncover
doping with endogenous anabolic steroids. However, due to a wide interindividual variability
479
in absolute endogenous steroid concentrations originating from various factors, it has been
proven that population-based reference values, which were considered for years by every
protagonist in the fight against doping, are not always sensitive enough to reveal the
potential misuse of anabolic androgenic steroids at an individual level. For these reasons,
there is an obvious need of individual monitoring of the steroid profile to allow a fair
evaluation [14450].
The in-competition and out-of-competition testing programmes are the best strategies to
screen and confirm adverse analytical findings of exogenous and endogenous steroids. From
the basis of these routine analyses, the WADA-accredited laboratories provide harmonised
and robust analytical data for steroid profile. Recently, a new technical document
TD2014EAAS has been edited to ensure this harmonisation and is in force from January
2014. A detailed description of selected aspects of TD2014EAAS is given later in this review.
The application of these rules should enable a suitable application of steroid module of the
athlete biological passport (ABP) and the assessment of steroid profile using the adaptive
model [14450].
Before the steroidal module, ABP has been developed using a Bayesian approach to deter
blood doping based on haematological data obtained in whole blood sample. The
haematological module of the ABP has been implemented in 2008 by certain international
sport federations and since then this indirect methodology has resulted in sanctioning of
numerous athletes for anti-doping rule violation. This achievement stimulated the expansion
of the ABP to establish the intraindividual reference ranges to monitor the steroid profile of an
athlete. Historically, anti-doping laboratories and sport authorities detect misuse of
endogenous steroids based on the ratio between T and its 17alpha-epimer, epitestosterone
(E; T/E ratio). A threshold based on previous anti-doping data and population studies was
first set at 6 by the IOC in 1983 and later lowered to 4 to discriminate between normal and
abnormal values. Urine samples showing a T/E ratio above the threshold were then
submitted to further analyses such as gas chromatography-combustion-isotope ratio mass
spectrometry to evaluate the steroid profile and the endogenous or exogenous origin of the
target compounds. The sensitivity of the T/E ratio approach based on population-based
reference ranges has been questioned since 1994. At that time, individual reference ranges,
instead of population-based references, have already been proposed and used in steroid
profiling. By the work of Sottas et al, a new and very effective mathematical tool came into
this field which allowed an optimised evaluation of the longitudinal data. This mathematical
model is, currently, one of the basic tools of the ABP [14450].
In 2008, the ABP has been implemented for haematological parameters based on a
Bayesian statistical model that allows monitoring of intraindividual fluctuations of blood
doping markers. Knowing that every athlete has his/her own metabolism and responds
differently after any drug misuse, this profiling approach is relevant for the results
management in the fight against doping. Even if the follow-up of secondary markers
indicating a drug intake or a manipulation to increase the performance skills is becoming
essential, direct detection of prohibited substances is still necessary to prevent athlete from
cheating, and the biological passport profile may assist in targeting the doping control
analysis to particular additional tests, such as GC-C-IRMS [14450].
As the urinary steroid profile, especially the T/E ratio, is well known as being a stable marker
within an individual, the integration of the adaptive steroidal module was a natural evolution
of the ABP. This module aims to identify endogenous anabolic androgenic steroids when
administered exogenously and other anabolic agents, such as selective androgen receptor
modulators categorised under section S1 of the Prohibited List. Six markers are considered
within the steroidal module which are T, E, androsterone, etiocholanolone, 5alpha-
480
androstane-3alpha,17beta-diol (5alpha-diol) and 5beta-androstane-3alpha,17beta-diol
(5beta-diol), although Van Renterghem et al proposed additional compounds to be integrated
in the ABP [14450].
As described in the recently published ABP Operating Guidelines and Compilation of

Required Elements, data collection and administration requires specific partners such as
anti-doping organisations (ADOs), Athlete Passport Management Unit (APMU), WADA-
accredited laboratories, expert panel and WADA. Each of these entities has its own
responsibilities to guarantee reliability and credibility of the ABP programme. Briefly, ADOs
are in charge to perform an appropriate and intelligent follow-up of their athletes according to
the International Standard for Testing (IST). In the process they should also consider the
recommendations of the APMUs which are responsible of the passports real-time
management through the evaluation of the data of a single sample with respect to the profile
generated by the adaptive model in Anti-Doping Administration and Management System
(ADAMS). In addition, APMUs make connections with the expert panels that are necessary
to bring out any pathology or confounding factors that could impact analytical results
provided by the laboratories which shall adhere to the WADA technical documents
TD2014BAR and TD2014EAAS for haematological and steroidal module, respectively.
Moreover, expert scientists may also request additional testing for a specific athlete to collect
further indications of pathologies or to strengthen an atypical passport finding (ATPF).
Altogether, close cooperation between testing authorities, sample collection authorities and
laboratories is mandatory to ensure a prompt transfer of information and adequate timing of
testing and to allow the ABP programme to be efficient [14450].
Despite the fact that much is known about the absorption, distribution, metabolism, and
elimination (ADME) of approved therapeutics, the human metabolism particularly concerning
androgens has been shown to be substantially influenced by various different
pharmacological interventions as well as other confounding factors. In consideration of the
significance of steroid profiling in the anti-doping field and incorporation of “steroidal module”
to the athlete biological passport (ABP), continuous research is conducted to expand the
knowledge on aspects potentially or evidently affecting urinary steroid concentrations and
result interpretation [14715]
Endogenous factors, general considerations
The ABP aims at monitoring of an individual athlete with respect to his/her own, long-term
steroid profile. Interesting parameters with this respect are the general endogenous factors
which, on one hand, set the baseline of an individual, and, on the other hand, may lead to
“natural” variation of the profile within a long period of time. Among these factors are, for
example, age and gender of the athlete. A major role is played also by ethnicity, but as these
interindividual differences are linked essentially to genetic polymorphism, these properties
are discussed in connection to androgen metabolism. As a general remark for the
interpretation of the results originating before year 2005, it should be notified that the critical
value of T/E for doping control purposes was >6, instead of >4 [14450].
It was carried out a study of 141 normal male participants (aged 8-26 years), categorised the
population into five groups based on the development stages according to Tanner's scale
and compared the excretion profiles of T and E between different age groups. According to
their report, excretion of both markers increased significantly during development and
correlated highly with age. However, a significant difference was observed between the
481
increase of T and E relative to age, T excretion increasing much faster than E and indicating
the potential instability of T/E during puberty. In another study, originating from approximately
same time, Dehennin et al studied a population of 140 male participants (aged 13-20 years)
with respect to urinary excretion of several endogenous steroids and luteinising hormone
(LH). Although they concluded that the increase in excretion rates of glucuronide-conjugated
T and E correlated with pubertal development, the result was somewhat contrary to earlier
one with respect to T/E, where the observed differences were not significant. In this study,
ratio of T-glucuronide to LH, which has been proposed as additional information on T misuse,
increased throughout puberty. An independent study from Schweizer et al with 100 male
participants (aged 10-17) supports the results of Dehennin et al, as their study showed
insignificant change in T/E between different stages, although higher instability of the ratio
was associated to prepubertal stages. In a group of adolescent girls (aged 6-17, n=256), the
same research group observed a decreasing T/E ratio during development, most obviously
due to larger relative increase in E excretion. The results were similar between exercising
and control group of participants [14450].
Interindividual variation in genetics, in enzyme distribution and, consequently, in drug

metabolism are discussed later in this review in detail. Briefly, two main families of enzymes
contributing the drug metabolism in humans are cytochrome P450 (CYP450), which is
responsible for phase I reactions, and uridine diphosphate glucuronosyltransferase (UGT)
enzymes, which catalyse the phase II conjugation reaction with glucuronic acid. Gender-
dependent differences in enzyme activity have been demonstrated for several CYP
isoenzymes and for UGTs, supporting the possibility of quantitative differences between
female and male athletes. However, the genes for CYP and UGT proteins are not linked to
X-chromosome, and, thus, the prevalence of poor metabolisers should not be expected to be
different between genders. In fact, reference concentration ranges of urinary T and excreted
metabolites have been published previously with lower levels in female participants than in
male participants [14450].
Periodical variations in hormones concentrations are well established in different species and
matrices. In humans, T is also subjected to these fluctuations, as is previously shown in
serum, saliva and urine. This daily, monthly and even yearly based variability of steroid
hormones concentrations should not significantly impact the longitudinal follow-up of
participants, and is included within the normal intraindividual variation of the steroid profile
components [14450].
Regarding the urinary steroid profile and physical exercise there are studies concluding
differences between sedentary and exercising individuals, and that the physical activity may
influence the elimination of androgens due to changes in sex hormone binding globulin
(SHBG). A group of trained female athletes was investigated by Bricout et al with respect to
urinary steroid profiles during menstrual cycle and compared with non-athlete (sedentary)
group. T and E were measured from glucuronide-conjugated fraction by radioimmunoassay
(RIA), and based on this study, the T/E remained stable between the follicular phase and
luteal phase of menstrual cycle within athlete (0.66 ± 0.05 vs 0.69 ± 0.33) and non-athlete
(0.72 ± 0.26 vs 0.67 ± 0.31) groups. As a conclusion, it was stated that although physical
training may have an effect on androgen metabolism, active sportswomen can be considered
as members of normal population as long as there are no signs of secondary amenorrhoea
induced by physical activity. Regarding male participants, similar results were previously
published by Donike et al, who showed that high workload during the Tour de France does
not influence the T/E ratio in top-level athletes [14450].
482
During pregnancy, however, female athletes encounter much more dramatic changes.
Controlled longitudinal studies of steroid profile during pregnancy are scarce, but according
to available data, significant alterations occur not only in the production of progesterone and
oestriol, but also in androgen concentrations. For the status of the steroid profile and its
interpretation, the most significant factors are pregnandiol (PD) and T itself. According to
Mareck-Engleke et al the PD concentration may increase up to 10–100 fold (to 10 000
ng/mL) from the baseline levels during the early pregnancy, and despite being quite
theoretical in performance-sport context, the levels of 20 000 ng/mL concentration can be
reached just before delivery. In their recent work, Fabregat et al conducted a longitudinal
study in three pregnant women, and focused on cysteine-conjugated androgens and
glucuronide-conjugated androgens and oestrogens during different trimesters of pregnancy.
From a steroid profile perspective, there was a significant increase in urinary oestrogen
levels and moderate decrease in urinary androgen concentration, and thus alteration in
general profiles due to pregnancy. Interesting results were obtained for E glucuronide
concentrations, which were elevated during the first trimester, and thus a feature to take into
account in interpreting of T/E in steroid profiles of female athletes. The results of this study
were also well in accordance with the earlier ones describing the formation of
norandrosterone, a nandrolone metabolite, during pregnancy [14450].
Metabolism, genetics and interindividual variation
Androgens are an essential part of endocrinological homeostasis in human body and their
dual effects are associated mainly to masculinisation (androgenic effects) and protein
synthesis (anabolic effects). There are several mechanisms and functions which mediate the
androgen action, control the transport and binding of T and other androgens or activate the
expression of androgen-responsive genes. In human genome, two or more variants can be
encountered for a particular DNA sequence. In its simplest form, this natural variation,
polymorphism, involves not only a single nucleotide (SNP), but also longer DNA stretches
can be involved. The outcome of the complex network of these bioprocesses and
interindividual as well as interethnic variations within them leads to a steroid profile with an
individual baseline of endogenous steroids. Massive amounts of research results are
available on the clinical and pathological relevance of androgens and the factors contributing
to the phenotype of an individual. For example, low serum T concentration is associated to
several pathological conditions, for example, cardiovascular morbidity, type 2 diabetes and
increased risk of mortality. The studies indicate strong heritability of serum T levels and
clinical studies have focused on T as a biomarker of male health status and on the effects of
genetic variants on serum T concentrations. Although sports and doping control involve only
minor fraction of population, the atypical patterns, anomalies and pathological conditions are
factors to keep in mind when evaluating individual athlete profiles [14450].
The steroidal markers
In the 1980s, based on the work of Donike and colleagues, an upper limit of 6.0 for the
testosterone/epistestosterone (T/E) ratio was introduced to deter testosterone administration.
After exogenous testosterone administration, the clear effect of the latter is an increase in the
T/E ratio. The T/E has been the first widely used indirect marker of doping with anabolic
steroids, with a discrimination principle not based on the distinction between the exogenous
substance and its endogenous counterpart, but rather on the effect induced by the intake of
the exogenous substance on some selected biological markers. Since then, the T/E ratio has
been used as a screening test, with any positive result requiring a subsequent confirmation
analysis by GC/C/IRMS. GC/C/IRMS allows measurement of slight differences in 13C/12C
ratio of testosterone metabolites. Discrimination between pharmaceutical and natural
483
testosterone is possible because synthetic testosterone is known to display different 13C
content than its human counterpart produced by means of cholesterol metabolism [14433].
UGT2B17 gene deletion influence on Athletes Biological Passport
The newly implemented Steroid Module of the Athlete Biological Passport has improved
doping tests for steroids. A biomarker included in this passport is the urinary testosterone
glucuronide to epitestosterone glucuronide (T/E) ratio, a ratio greatly affected by a deletion
polymorphism in UGT2B17. Suspect urine doping tests are further analyzed with gas
chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) to determine the
origin of the androgen. In this study, we investigated the sensitivity of the steroidal module
and the IRMS analysis, in subjects administered with three doses of testosterone enanthate
(500, 250, and 125 mg), in relation to the UGT2B17 polymorphism. All subjects carrying the
UGT2B17 enzyme reached the traditionally used threshold, a T/E ratio of 4, after all three
administered doses, whereas none of the subjects devoid of this enzyme reached a T/E of 4.
On the other hand, using the athlete biological passport and IRMS analysis, all three doses
could be detected to a high degree of sensitivity. The concentrations of all steroids included
in the steroidal module were dose dependently increased, except for epitestosterone which
decreased independent of dose. The decrease in epitestosterone was significantly
associated with circulatory levels of testosterone post dose. In conclusion, these results
demonstrate that administration of a single dose of 125-500 mg testosterone enanthate could
be detected using the athlete biological passport, together with IRMS. Since IRMS is
sensitive to testosterone doping independent of UGT2B17 genotype, also very small
changes in the steroidal passport should be investigated with IRMS [150104].
Testosterone is mainly excreted as a glucuronide conjugate after its metabolism by uridine

diphosphate (UDP) and glucuronosyl transferase (UGT). UGT2B7, UGT2B15, and UGT2B17
are known to be the main glucuronidation catalysts of androgens and their metabolites in
humans. Testosterone is mainly conjugated by UGT2B17 and to a lesser extent by
UGT2B15. The main androgen substrate of UGT2B15 is androstane‐3alpha 17beta‐diol. The
actions of UGT2B17 are 96 percent in common with those of UGT2B15. The enzyme
UGT2B7 also has the capacity to conjugate epitestosterone, while testosterone is a poor
substrate for this enzyme. It has been established that a deletion polymorphism in the gene
that codes for UGT2B17 correlates highly with testosterone levels in urine. Thus, subjects
lacking this gene have been found to show a T/E ratio lower than 0.4. This polymorphism is
much more common in Asian subjects than Caucasian subjects and its prevalence has been
estimated at 67 percent in Asians versus 9 percent in Caucasians. The measurement of the
testosterone to epitestosterone ratio (T/E ratio) in urine is often used as a marker for
testosterone administration in the doping control field. This study examines the frequencies
of the different expression forms of the UGT2B17 gene, and assesses their effects on this
marker in volunteer subjects. The sample for this descriptive study was composed of male
and female athletes aged between 16 and 55 years old who practiced different sports
disciplines. All participants underwent a sports-medical physical examination, and
subsequently provided 10 urine samples consecutively over a period of 48 h. The dependent
variable examined was T/E and the main independent variable was the UGT2B17 gene
polymorphism. During 1 year, 1410 urine samples were obtained from 141 athletes. The
frequencies of the three genotypes were as follows: wt homozygotes (ins/ins) 48 percent
(n=68), mutant homozygotes (del/del) 12 percent (n=17), and heterozygotes (ins/del) 40
percent (n=56). Genotype distributions varied significantly according to ethnicity, 80 percent
of Asian subjects being homozygous for the gene deletion (del/del) compared to 7 percent of
Caucasian subjects. A multivariate analysis adjusted for genotype, age, sex, and sports
discipline revealed that athletes with the del/del polymorphism showed a significantly lower
mean T/E than heterozygotes (ins/del). In contrast, homozygous athletes for the gene
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insertion (ins/ins) showed higher mean T/E ratios than heterozygotes (ins/del). UGT2B17
gene deletion has a strong influence on the T/E ratio in urine, which is the most efficient
indicator of testosterone prohormone misuse. Others factors studied seem not to have such
an impact. The genotyping of UGT2B17 is an important source of information for
understanding steroid profiling in the doping control field; therefore it is suggested that it be
included in the Athletes Biological Passport [150148].
Dose-dependent testosterone sensitivity related to the UGT2B17 polymorphism

The newly implemented Steroid Module of the Athlete Biological Passport has improved
doping tests for steroids. A biomarker included in this passport is the urinary testosterone
glucuronide to epitestosterone glucuronide (T/E) ratio, a ratio greatly affected by a deletion
polymorphism in UGT2B17. Suspect urine doping tests are further analyzed with gas
chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) to determine the
origin of the androgen. In this study, we investigated the sensitivity of the steroidal module
and the IRMS analysis, in subjects administered with three doses of testosterone enanthate
(500, 250, and 125 mg), in relation to the UGT2B17 polymorphism. All subjects carrying the
UGT2B17 enzyme reached the traditionally used threshold, a T/E ratio of 4, after all three
administered doses, whereas none of the subjects devoid of this enzyme reached a T/E of 4.
On the other hand, using the athlete biological passport and IRMS analysis, all three doses
could be detected to a high degree of sensitivity. The concentrations of all steroids included
in the steroidal module were dose dependently increased, except for epitestosterone which
decreased independent of dose. The decrease in epitestosterone was significantly
associated with circulatory levels of testosterone post dose. In conclusion, these results
demonstrate that administration of a single dose of 125-500 mg testosterone enanthate could
be detected using the athlete biological passport, together with IRMS. Since IRMS is
sensitive to testosterone doping independent of UGT2B17 genotype, also very small
changes in the steroidal passport should be investigated with IRMS [150106].
Effect of Ramadan
One study investigated the effect of Ramadan on the haematological and steroid module of
the Athletes Biological Passport (ABP) of the World Anti-Doping Agency (WADA). Nine
healthy physically active subjects were tested in the morning and afternoon for two days
before and three days during Ramadan. Sample collection and all analyses were performed
according to WADA technical documents. Although there were significant changes in the
haemoglobin concentration during Ramadan, especially during the first fasting week, none of
the subjects in this study exceeded the individually calculated thresholds of the ABP. No
significant effects on testosterone/epitestosterone (T/E) ratio were observed but only the
afternoon specific gravity (SG) of the urine was elevated. Thus, when urinary steroid
concentrations are required, SG corrections need to be performed. The haematological and
the steroid module of the ABP can be reliably applied during Ramadan as the observed
changes are only marginal [150107].
Impact of genetics and oral contraceptives on the steroid profile in female athletes
The steroid module of the Athlete Biological Passport, the newest innovation in doping
testing, is currently being finalized for implementation. Several factors, other than doping,
can affect the longitudinal steroid profile. In one study, it was investigated the effect of
hormonal contraceptives (HC) as well as the effect of three polymorphisms on female steroid
profiles in relation to doping controls. The study population consisted of 79 female elite
athletes between the ages of 18 and 45. HC were used by 32 percent of the subjects. A full
urinary steroid profile was obtained using World Anti-Doping Agency accredited methods. In
485
addition all subjects were genotyped for copy number variation of UGT2B17 and SNPs in
UGT2B7 and CYP17. Subjects using HC excreted 40 percent less epitestosterone as
compared to non-users but showed no difference in testosterone excretion. When removing
individuals homozygous for the deletion in UGT2B17, the testosterone to epitestosterone
(T/E) ratio was 29 percent higher in the HC group. In agreement with previous findings in
men, copy number variation of UGT2B17 had significant effect on female urinary
testosterone excretion and therefore also the T/E ratio. Subjects homozygous for the T allele
of CYP17 showed a lower urinary epitestosterone concentration than the other CYP17
genotypes. It is of great importance that the athlete's steroidal passport can compensate for
all possible normal variability in steroid profiles from women. Therefore, considering the large
impact of HC on female steroid profiles, it was suggest that the use of HC should be a
mandatory question on the doping control form [150108].
It was known in the 1990s that participant-based reference ranges are more reliable than
population-based reference ranges for androgens and that individual T/E values do not
deviate from the mean value by more than 30 percent. It has only been recently that a
method was proposed to take into account formally these characteristics. Based on empirical
Bayesian inferential techniques for longitudinal profiling completely similar to what is used for
the haematological markers, the test progressively switches the focus from comparison with
a population to the determination of individual values. Interestingly, this test is neither a
purely population-based nor a purely subject-based approach, but an intermediate approach
that makes the best decision in function of the between-subject and within-subject variance
components of the marker and actual individual test results. Using the athlete as his own
reference is particularly interesting when the marker presents a low ratio of within-subject to
between-subject variations. In a population composed of male Caucasian athletes, this ratio
has been estimated to be as low as 0.04 for the T/E. Such a low ratio already questions the
pertinence of a population-based threshold for the T/E ratio (fixed at 4.0 for a long time by
WADA). While the terminology “steroid profiling” is used in the literature to denote a follow-up
over time, a steroid profile includes concentration levels of endogenous steroids in urine and
their respective ratios. Steroid profiles are employed widely in endocrinology to detect
enzyme deficiencies or adrenal problems. In antidoping laboratories, the urinary steroid
profile usually includes the concentration levels of testosterone, the testosterone's inactive
epimer, epistestosterone and four testosterone metabolites, androsterone (A),
etiocholanolone (Etio), 5alpha-androstane-3alpha,17beta-diol (alpha-diol) and 5beta-
androstane-3alpha,17beta-diol (beta-diol). Ratios such as T/E, A/Etio, A/T, alpha-diol/E and
alpha-diol/beta-diol are robust and do not change due to circadian rhythm or physiological
conditions such as exercise workload for athletes. On the other hand, these markers may be
altered significantly according to the administered steroid and its application mode [14433].
The detection of testosterone abuse in sports is routinely achieved through the “steroidal
module” of the Athlete Biological Passport by GC-MS(/MS) quantification of selected
endogenous anabolic androgenic steroids (EAAS) from athletes' urines. To overcome some
limitations of the "urinary steroid profile" such as the presence of confounding factors
(ethnicity, enzyme polymorphism, bacterial contamination, and ethanol), ultrahigh
performance liquid chromatography (UHPLC) measurements of blood concentrations of
testosterone, its major metabolites, and precursors could represent an interesting and
complementary strategy. In this work, two UHPLC-MS/MS methods were developed for the
quantification of testosterone and related compounds in human serum, including major
progestogens, corticoids, and estrogens. The validated methods were then used for the
analyses of serum samples collected from 19 healthy male volunteers after oral and
transdermal testosterone administration. Results from unsupervised multiway analysis
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allowed variations of target analytes to be assessed simultaneously over a 96-h time period.
Except for alteration of concentration values due to the circadian rhythm, which concerns
mainly corticosteroids, DHEA, and progesterone, significant variations linked to the oral and
transdermal testosterone administration were observed for testosterone, DHT, and
androstenedione. As a second step of analysis, the longitudinal monitoring of these
biomarkers using intra-individual thresholds showed, in comparison to urine, significant
improvements in the detection of testosterone administration, especially for volunteers with
del/del genotype for phase II UGT2B17 enzyme, not sensitive to the main urinary marker,
T/E ratio. A substantial extension of the detection window after transdermal testosterone
administration was also observed in serum matrix. The longitudinal follow-up proposed in this
study represents a first example of ”blood steroid profile” in doping control analysis, which
can be proposed in the future as a complement to the “urinary module” for improving steroid
abuse detection capabilities [150105].
In summary there are some conclusion that can be of interest regarding the steroid module in
the ABS [14450]:
- New steroidal module of the athlete biological passport in place since January 2014
- Two distinct classes of factors can influence the quantification of endogenous steroid
compounds linked to testosterone and its metabolism
- Endogenous factors include ethnicity, gender, age and genetic polymorphisms
whereas exogenous factors comprise medications, diet, matrix composition and
analytical tools used for the quantification
- Implementation of the steroidal module depends on the evaluation of steroid profiles
through a dedicated statistical model but also on the expertise given by specialised
scientists.
Korean values of urinary levels of testosterone and epitestosterone
Cannabis, or marijuana, the most commonly used illicit drug in the world, has been shown to
be responsible for suppressing the production and secretion of androgens, particularly
testosterone. However, despite such findings in animals, the chronic effects of marijuana use
on human endocrine systems have proved to be inconsistent. Here, it was investigated the
reference ranges of urinary levels of testosterone (T) and epitestosterone (E) as well as their
metabolic ratio of T/E in a Korean male population (n=337), which would enable an
evaluation of abnormal changes in steroid metabolism induced by habitually administered
cannabis. The T/E ratio was significantly decreased in the marijuana group (n=18), while the
urinary testosterone concentrations were also tended to decrease. This study is the first to
provide data for the reference values of two urinary androgens and T/E values among control
Korean males, and, furthermore, suggests that the T/E ratio, though not testosterone levels,
might be used to understand the suppression of human male gonadal function affected by
smoking marijuana [14058].
The growth hormone module
The hematological module has been in place in some federation since 2008; the steroid
module is in place since the beginning of 2014 with the experience accumulated for many
years in the analysis of steroid profile in urine in antidoping laboratories. The third part
dedicated to the hGH line and called “endocrine module” is certainly the one which still needs
to be structured and validated with some emphasis. There is evidence that growth hormone
is used by athletes for its anabolic and lipolytic properties. It is often abused in combination
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with anabolic steroids and insulin, even with insulin-like growth factor-I (IGF-I). The GH-2000
project developed a methodology to detect its abuse using the concentrations of two GH-
dependent markers, IGF-I and type 3 procollagen (P-III-nP). Even if this approach is claimed
to be used for a single serum sample collected by the ADO, there is no doubt that a
longitudinal sequence of those markers will be more selective to prove a manipulation with
human growth hormone [14433].
Nowadays, the only official method which has been officially introduced in the hGH doping
detection with some success is the one based on the so-called “isoform aproach”. It is known
that the pituitary gland secretes a variable spectrum of hGH isoforms, whereas recombinant
growth hormone (rh-hGH) is the monomeric 22 000 Da isoform only. This isoform becomes
predominant after injection of rh-hGH. The isoform test is built on specific immunoassays
with preference for one or the other isoform allowing the analysis of the relative abundance
of the 22 000 Da isoform. Application of rh-hGH can be proven when the ratio of this isoform
relative to the others is increased above a certain threshold. The different detection windows
of the so called ‘marker method’ and the ‘isoform method’ makes them complementary and
could increase the overall detection window of hGH abuse. It is known that both approaches
must overcome the interindividual and intraindividual variability, but, as for other hormones,
the longitudinal study of markers influenced by the application of rh-hGH can be the right
answer to the question of the biological variability. This will then complete the ABP with its
endocrine module [14433].
Potential confounding factors
Physical exertion, training periodization, time of year, dehydration and altitude may all affect
different hematological parameters and lead to false positives. Even though the ABP protocol
is designed to account for these factors, the addition of an expert panel minimizes this risk
[13006].
Although the ABP had the initial exclusive intent of biological monitoring, today, the ABP
contains more than a simple series of individual biomarker values. Heterogeneous factors,
such as age, gender, and genotype; confounding factors, such as exposure to higher
altitudes for the hematological module; and some information regarding the conditions of
sample collection, transport, and analysis are also stored in the passport for improved
decision making. As such, the ABP becomes a platform for the evaluation of multiple pieces
of scientific evidence, which is similar to a forensic approach. Similarly to forensic
identification science, the strength of the ABP is that it relies on sound empirical testing in
large populations by use of justifiable protocols. The decision support system that is used
routinely to interpret the biomarker data stored in an ABP heavily relies on Bayesian
inference techniques. Every element of information that constitutes doping evidence can be
incorporated into other elements and/or corroborated by additional evidence. For example,
the result of traditional drug tests, such as the detection of rEPO in urine; some
characteristics of athletes, such as a particular genotype; and the longitudinal monitoring of
individual performance are evidentiary values that can be incorporated into the ABP decision
support system for improved detection of doping [11426].
Various factors are capable of influencing either the quantification of the urinary steroid
profile or its interpretation. The endogenous or exogenous origin of those issues allows for
their classification into two main categories [14450].
The ABP aims at monitoring of an individual athlete with respect to his/her own, long-term
steroid profile. Interesting parameters with this respect are the general endogenous factors
488
which, on one hand, set the baseline of an individual, and, on the other hand, may lead to
“natural” variation of the profile within a long period of time. Among these factors are, for
example, age and gender of the athlete. A major role is played also by ethnicity, but as these
interindividual differences are linked essentially to genetic polymorphism [14450].
The establishment of urinary steroid profile through analytical quantification of T and its
related compounds has been proven to be a reliable and efficient tool for endogenous
anabolic androgenic steroids misuse detection. An additional and significant step in the
steroid profile application in the fight against doping is the integration of the steroidal module
within the ABP. Although the steroid profile components are quite stable against physical
exercise, menstrual cycle or biological rhythms (circadian or annual), many exogenous and
endogenous influencing parameters exist. These confounding factors could not be monitored
only by the ABP steroidal module but need scientific expertise to be evaluated and to avoid
any sanction of athlete simply based on the statistical and mechanical approach of steroid
profile monitoring. Essential part of the steroid profile is a representative number of samples,
well-planned testing strategies and significant effort from sample collection authorities, as
well as smooth cooperation between ADOs. Sample collection and transportation conditions
should be organised in an appropriate manner to preserve the sample integrity, and the
laboratories should be harmonised in analytical methodologies to provide reliable and
comparable results. WADA-accredited laboratories should not only focus on endogenous
steroids quantification but also on the detection of exogenous factors such as drugs
interfering with metabolic pathways or adulteration markers. Genetics factors are much more
sensitive considering the ethical issues. As the final stage, the passport management units
as well as the scientific expert panels should be well trained and experienced in the
interpretation of analytical data and profiles in order to distinguish between ATPFs and
pathological or clinical conditions which may alter the individual passport results [14450].
- New steroidal module of the athlete biological passport in place since January 2014
- Two distinct classes of factors can influence the quantification of endogenous steroid
compounds linked to testosterone and its metabolism
- Endogenous factors include ethnicity, gender, age and genetic polymorphisms
whereas exogenous factors comprise medications, diet, matrix composition and
analytical tools used for the quantification
- Implementation of the steroidal module depends on the evaluation of steroid profiles
through a dedicated statistical model but also on the expertise given by specialised
scientists.
Plasma osmolality
One investigation quantified the effect of changes in plasma osmolality on the measurement
of hematocrit (Hct) and the implications for the subsequent use of these data to calculate
changes in plasma volume and application to the WADA Athlete Biological Passport. Two
groups of eight male volunteers visited the laboratory after an overnight fast. In study 1, a 20
mL blood sample was collected and aliquoted into collection tubes containing saline of
varying concentrations to alter the sample osmolality. In study 2, plasma osmolality was
manipulated in vivo through prolonged exercise. Samples were analysed for hemoglobin
concentration and Hct using manual methods and using an automated hematology analyser
(AHA). Changes in blood, plasma and red cell volumes were calculated. While AHA Hct
values did not change spun plasma volume changes (PCV) fell progressively as the
osmolality of the sample increased. Consequently, there was a significant increase in
apparent plasma volume as osmolality increased: regression analysis revealed that a 10
mOsmol/kg change in plasma osmolality produced a difference of 0.8 Hct units and a 1.6
489
percent change in plasma volume. In study 2 exercise produced a 12 ± 3 mOsmol/kg
increase in plasma osmolality. No difference in Hct was apparent at rest, but spun PCV was
1.0 ± 0.9 Hct units lower during exercise compared to AHA data . There was a difference in
the degree of plasma volume change calculated, with a reduction of 8.7 ± 3.4 percent and
11.3 ± 3.5 percent reported with the manual and AHA methods respectively. It was concluded
that conditions or interventions which result in a marked change to plasma osmolality
produce a discrepancy in Hct measured using an AHA, consequently introducing errors into
any calculation of changes in plasma volume using these data. These findings may also
have implications for the measurement of Hct by WADA-accredited laboratories [13074].
Stability of athlete blood passport parameters during air freight
Fluctuations in ambient temperature and pressure, as well as physical jostling, may affect the
stability of whole blood samples transported by air freight. The aim of one study was to
characterize the stability of key blood variables during air freight and to investigate whether
vibration or reduced pressure alone affected results. Over a 72-h interval, it was evaluated
the stability of full blood count indices (plus reticulocytes) in tubes that were air-freighted a
total of 2, 10 and 28 h. It was also examined the impact of 24 h of reduced atmospheric
pressure (750 hpa or approximately 2500 m.a.s.l) and vibration (5 Hz). Samples were
measured on a Sysmex XT-2000i instrument. The two key variables in the context of
antidoping (haemoglobin concentration, reticulocytes) remained stable over a 72-h period
regardless of the duration of air freight. Atmospheric pressure and vibration had no
discernible effect. It was concluded that whole blood samples stored in NanoCool devices
can be relied upon to remain stable for at least 72 h despite interim air freight [13075].
Gastroenteritis
The hematological module of the "Athletes Biological Passport" (ABP) is used to detect blood
doping through the longitudinal variation of blood variables, such as hemoglobin
concentration (Hb). Sporting federations have opened disciplinary procedures against
athletes based on ABP results. Suspicious athletes try to explain the variations in their blood
values with dehydration caused by gastrointestinal (GI) problems. The aim of one report was
to describe hemoglobin concentration, a key variable of the ABP, during acute gastroenteritis
in athletes. 5 athletes with severe gastroenteritis were studied in retrospective. Blood test
results (Hb, white blood cell count (WBC) and differential, CRP) obtained on hospital
admission for GI problems were compared to data obtained from the same athletes in states
of good health on previous occasions. During GI problems, athletes displayed marked
inflammatory constellations with increased CRP and typical WBC shifts. Hb was not affected
and remained mostly unchanged. This is in line with basic physiologic fluid regulation, where
plasma volume is kept constant, even under conditions of severe dehydration. It is therefore
unlikely that fluid loss associated with gastroenteritis will cause athletes blood data to reach
levels of abnormality that will be suspicious of blood doping [11133].
Oral contraceptives
The steroid module of the Athlete Biological Passport, the newest innovation in doping
testing, is currently being finalized for implementation. Several factors, other than doping,
can affect the longitudinal steroid profile. In one study, it was investigated the effect of
hormonal contraceptives (HC) as well as the effect of three polymorphisms on female steroid
profiles in relation to doping controls. The study population consisted of 79 female elite
athletes between the ages of 18 and 45. HC were used by 32 percent of the subjects. A full
urinary steroid profile was obtained using World Anti-Doping Agency accredited methods. In
490
addition all subjects were genotyped for copy number variation of UGT2B17 and SNPs in
UGT2B7 and CYP17. Subjects using HC excreted 40 percent less epitestosterone as
compared to non-users but showed no difference in testosterone excretion. When removing
individuals homozygous for the deletion in UGT2B17, the testosterone to epitestosterone
(T/E) ratio was 29 percent higher in the HC group. In agreement with previous findings in
men, copy number variation of UGT2B17 had significant effect on female urinary
testosterone excretion and therefore also the T/E ratio. Subjects homozygous for the T allele
of CYP17 showed a lower urinary epitestosterone concentration than the other CYP17
genotypes. It is of great importance that the athlete's steroidal passport can compensate for
all possible normal variability in steroid profiles from women. Therefore, considering the large
impact of HC on female steroid profiles, it was suggest that the use of HC should be a
mandatory question on the doping control form [14076].
Influence of transport and time on blood variables
Some recent studies have characterized the stability of blood variables commonly measured
for the Athlete Biological Passport. The aim of this study was to characterize the impact of
different shipments conditions and the quality of the results returned by the haematological
analyzer. Twenty-two healthy male subjects provided five EDTA tubes each. Four shipment
conditions (24, 36, 48, 72 h) under refrigerated conditions were tested and compared to a set
of samples left in the laboratory also under refrigerated conditions (group control). All
measurements were conducted using two Sysmex XT-2000i analyzers. Haemoglobin
concentration, reticulocytes percentage, and OFF-score numerical data were the same for
samples analyzed just after collection and after a shipment under refrigerated conditions up
to 72 h. Detailed information reported especially by the differential (DIFF) channel scatterplot
of the Sysmex XT-2000i indicated that there were signs of blood deterioration, but were not
of relevance for the variables used in the Athlete Biological Passport. As long as the cold
chain is guaranteed, the time delay between the collection and the analyses of blood
variables can be extended [150101].
High altitude training
The Athlete Biological Passport (ABP) detects blood doping in athletes through longitudinal
monitoring of erythropoietic markers. Mathematical algorithms are used to define individual
reference ranges for these markers for each athlete. It is unclear if altitude and exercise can
affect the variables included in these calculations in a way that the changes might be
mistaken for blood manipulation. The aim of this study was to investigate the influence of the
simultaneous strenuous exercise and low to high altitude exposure on the calculation
algorithms of the ABP. Forteen sea level (SL) and 11 altitude native (ALT) highly trained
athletes participated in a 14-day cycling stage race taking place at an average altitude of
2496 m above sea level (min. 1014 m, max. 4120 m), race distances ranged between 96 and
227 km per day. ABP blood measures were taken on days -1, 3, 6, 10, 14 (SL) and -1, 9, 15
(ALT) of the race. Four results from three samples of two different SL athletes exceeded the
individual limits at the 99 percent specificity threshold and one value at 99.9 percent. In ALT,
three results from three samples of three different athletes were beyond the individual limits
at 99 percent, one at 99.9 percent. The variations could be explained by the expected
physiological reaction to exercise and altitude. In summary, the abnormalities observed in the
haematological ABP’s of well-trained athletes during extensive exercise at altitude are limited
and in line with expected physiological changes [150102].
Impact of altitude training on hematological parameters

The impact of altitude training on hematological parameters and the Athlete Biological
491
Passport (ABP) was evaluated in international-level elite athletes. One group of swimmers
lived high and trained high (LHTH, n=10) for three to four weeks at 2130 m or higher whereas
a control group (n=10) completed a three-week training camp at sea-level. Haematological
parameters were determined weekly three times before and four times after the training
camps. ABP thresholds for haemoglobin concentration ([Hb]), reticulocyte percentage
(RET%), OFF score and the abnormal blood profile score (ABPS) were calculated using the
Bayesian model. After altitude training, six swimmers exceeded the 99% ABP thresholds:
two swimmers exceeded the OFF score thresholds at day +7; one swimmer exceeded the
OFF score threshold at day +28; one swimmer exceeded the threshold for RET% at day +14;
and one swimmer surpassed the ABPS threshold at day +14. In the control group, no values
exceeded the individual ABP reference range. In conclusion, LHTH induces haematological
changes in Olympic-level elite athletes which can exceed the individually generated
references in the ABP. Training at altitude should be considered a confounding factor for
ABP interpretation for up to four weeks after altitude exposure but does not consistently
cause abnormal values in the ABP [150102].
Hyperhydration
Anecdotal evidence suggests that athletes hyperhydrate to mask prohibited substances in

urine and potentially counteract suspicious fluctuations in blood parameters in the athlete
biological passport (ABP). It is examined if acute hyperhydration changes parameters
included in the ABP. Twenty subjects received recombinant human erythropoietin (rhEPO)
for 3 weeks. After 10 days of rhEPO washout, 10 subjects ingested normal amount of water
(270 mL), whereas the remaining 10 ingested a 1000 mL bolus of water. Blood variables
were measured 20, 40, 60, and 80 min after ingestion. Three days later, the subjects were
crossed-over with regard to water ingestion and the procedure was repeated. OFF-hr was
reduced by 4, 3, and 2 percent at 40, 60, and 80 min, respectively, after drinking 1000 mL of
water, compared with normal water ingestion. Forty percent of the subjects were identified
with atypical blood profiles (99 % specificity level) before drinking 1000 mL of water, whereas
11 percent (n=18), 10 percent and 11 percent (n=18) were identified 40, 60, and 80 min,
respectively, after ingestion. This was different compared with normal water intake, where 45
percent of the subjects were identified before ingestion, and 54 percent (n=19), 45 percent,
and 47 percent (n = 19) were identified 40, 60, and 80 min, respectively, after ingestion. In
conclusion, acute hyperhydration reduces ABP OFF-hr and reduces ABP sensitivity
[150103].
Heterogeneous factors
Heterogeneous factors refer to the factors specific to an individual that are known to have an
influence on a biomarker. For example, sex and age are well-known heterogeneous factors
used in the evaluation of a steroid profile. It has long been known for a long time that urinary
testosterone glucuronides present a bimodal distribution, this effect being particularly marked
between Caucasian and Asian populations. It only was recently, however, that it was
demonstrated that the significant differences observed in testosterone glucuronide excretion
are associated with a deletion mutation in the UDP-glucuronide transferase 2B17 (UGT2B17)
gene. This discovery has important implications for doping tests. For example, when
participants deficient in the UGT2B17 gene (del/del) receive exogenous testosterone, it has
been shown that their T/E ratio does not rise significantly, remaining well below current
threshold at 4.0. This suggests that the knowledge of genetic differences in metabolism and
excretion is important in the evaluation of urinary steroid profiles. These studies confirm,
again, that unique and non-specific thresholds on markers of steroid doping do not fit for
indicating anabolic-androgenic steroids misuse [14433].
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Recently, Van Renterghem et al as well as Boccard et al made proposals of new markers for
the detection of testosterone in sports using extensive steroid profiling and by using an
adaptive model based on Bayesian inference. Apart from T/E ratio, four other steroid ratios
(6alpha-OH-androstenedione/16alpha-OH-dehydroepiandrostenedione (DHEA), 4-OH-
androstenedione/16alpha-OH-androstenedione, 7alpha-OH-testosterone/7beta-OH-DHEA
and dihydrotestosterone (DHT)/5beta-androstane-3alpha,17beta-diol) were identified as
sensitive urinary markers for testosterone misuse. These selected markers were found
suitable for individual referencing within the concept of the ABP. The markers showed
improved detection time and discriminative power compared to solely T/E ratio. These
markers were supporting the evidence of doping with small oral doses of testosterone.
Another subject-based steroid profiling was shown to determine misuse of precursors of
testosterone like DHEA or DHT. Thus, there is a great potential in using a similar approach
as developed for the haematological module to implement the steroid module of the ABP.
Like in the first module, there is still need for harmonisation for its reliability. This is not so
much in the preanalytical conditions, but rather in the analytical production of results that the
laboratories shall pursue to decrease the interlaboratory variability [14433].
The analytical procedure
Many aspects of blood analyses have been debated since the beginning of blood tests in
sports. The need for standardisation appeared obviously due to different techniques of flow
cytometry applied in cell counting and identification of categories of erythrocytes. As this is a
quantitative measurement, it is clear that harmonised reference material must be available.
The quality control system should be applied in a similar manner in the network of
laboratories in order to decrease the variability in these longitudinal measurements. Still,
some clinical haematologists discuss or are critical to the system in place nowadays. One of
the best solution to standardise the analytical results for all samples included in a passport is
to analyse blood samples in an appropriate dedicated network (WADA accredited
laboratories) using analysers with comparable technical characteristics – if not identical. The
preanalytical procedure including the calibration of the analyser and the chain of custody of
the samples in the laboratory must be standardised and well documented [14433].
Each blood sample shall be analysed twice after proper homogenisation. This is not usual in
clinical haematology laboratory, but was decided in order to ensure legal validity of the result.
This is why the preferable institutions to perform that type of work are the WADA accredited
antidoping laboratories. They are accredited for the analyses of biological samples and also
used to the legal aspects of the procedure. Each pair of result for the sample must be
examined and must correspond to certain criteria. Absolute differences between the results
of the two analyses shall be equal or less than the following for the analyses to be accepted:
- 0.1 g/dL for HGB analysis.

- 0.15 absolute difference for RET% analysis (if both measurements are lower or equal
to 1%).
- 0.25 absolute difference for RET% analysis (if both measurements are higher than 1
%).
The data from the second analysis are used to confirm the first analysis data and only the
results of the first analysis are reported. The laboratory is requested to report immediately
the result into the international ADAMS web database [14433].
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Result management: the role of the APMU
The APMU is a key element to make the system work properly and efficiently. This unit
works on behalf of the ADO. APMU has the responsibility to manage the biological data and
such information shall be stored in ADAMS and/or the ABP software which implement the
blood data into the adaptive model. The APMU will review all profiles in order to provide
targeting recommendations to the ADO, when suitable or to refer to the expert panel if
appropriate. After verifying that the results included in the adaptive model are valid (ie, when
all the technical documents have been properly applied for all data), the APMU will evaluate
the profiles provided by the adaptive model. The latter predicts for an individual an expected
range within which marker values fall assuming a normal physiological condition. A sample
or a longitudinal profile will be considered as atypical if it returns a HGB and/or OFFs value
outside the expected individual range as defined by the adaptive model [14433].
The expert review
In case of an atypical result or profile, the APMU is responsible to contact one expert from its
expert panel to review the passport properly and suggest further action. Several situations
can appear and necessitate actions from the APMU. Once a pathology has been excluded
and if the profile is providing a high suspicion of blood manipulation, the review of the group
will follow the same procedure as for the first step of evaluation. The expert panel can
request any additional information which is needed for their expertise, including the
competition programme and/or training schedule of the particular athlete. The APMU will be
the link between the ADO and the experts, in order to keep the entire procedure anonymous
and confidential. A unanimous opinion among the three experts is necessary. If the three
experts determine whether abnormal or suspicious passport profiles are the result of a
medical condition or doping, then the APMU is responsible to compile all the documentation
supporting the so-called “Adverse Passport Finding” (APF). APMU will thus constitute the
ABP documentation package. This shall contain the following information:
- The age, gender, sport and discipline of the athlete

- The biological data and the results obtained by the adaptive model
- Information on possible exposure to altitude (whereabouts)
- The competition information
- The documentation of the entire chain of custody (including temperature conditions
and transport) of each sample making the passport
- The laboratory documentation for each blood sample, including the scattergrams, the
chain of custody and the internal/external quality controls
- All information collected on the doping control forms from each sample
- Any other additional information provided by the ADO
- The reports of experts’ opinions.
The APF will be then reported by the APMU to the ADO and to WADA in a similar manner as
the antidoping laboratories report an Adverse Analytical Finding after detecting the use of a
forbidden substance or a forbidden method. Then the ADO result management will be in
charge to review the case and decide if the APF constitutes an anti-doping rule violation as it
is defined in the World Anti-Doping Code [14433].
Also for research
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Marion Jones, who were tested for years and were never found to be cheating, admitted in
2007 to using “the clear,” a steroid formulated by the Bay Area Laboratory Cooperative,
known as BALCO, to elude steroid testing. It is probable that the passport approach will
identify athletes like Jones. If the athletes consented to have their test samples used for
research, it would improve scientific understanding of the range of readings that are normal.
But support for a voluntary program has not caught on. But culture change takes time, and
the need to secure the borders of fair sport is urgent [10008].
Law issues
The Athletes Biological Passport (ABP) has received both criticisms and support during this
year. In an issue of The Lancet, Michael Wozny considered that the use of the ABP makes it
more difficult to take banned substances and that it was successfully used against the Italian
elite cyclist Franco Pellizotti. After that, Italy's anti-doping tribunal considered that there was
not enough evidence to prove manipulation of his own blood profile in Pellizotti's case.
However, the UCI appealed to the Court of Arbitration for Sport (CAS) that sanctioned
Pellizotti with a suspension of 2 years. Since its implementation, some problems have
emerged. From 2010 to date, a large number of reports regarding the stability of the blood
variables used to determine the ABP have been published, showing mixed results. One
study considered that there is a risk of misinterpreting the physiological variations of the
hematological parameters determined by the anti-doping authorities in the ABP. The
analytical variability due to exercise training and competitions and/or to different metabolic
energy demands, hypoxia treatments, etc. could lead to an increase in false-positives when
using the ABP with the dramatic consequences that they might cause in major sports events
like the forthcoming London Olympic Games. Moreover, the ABP characteristics, procedures,
thresholds, or individual determination of reference ranges, abnormal out-comes, strikes,
"how the profile differs from what is expected in clean athletes" should be clearly stated and
explained in a new public technical document to avoid misunderstandings and to promote
transparency [11129].
Software
Substances and methods used to increase oxygen blood transport and physical performance
can be detected in the blood, but the screening of the athletes to be tested remains a critical
issue for the International Federations. One project, AR.I.E.T.T.A., aimed to develop a
software capable of analysing athletes' hematological and performance profiles to detect
abnormal patterns. One-hundred eighty athletes belonging to the International Biathlon Union
gave written informed consent to have their hematological data, previously collected
according to anti-doping rules, used to develop the AR.I.E.T.T.A. software. Software was
developed with the included sections: 1) log-in; 2) data-entry: where data are loaded, stored
and grouped; 3) analysis: where data are analysed, validated scores are calculated, and
parameters are simultaneously displayed as statistics, tables and graphs, and individual or
subpopulation profiles; 4) screening: where an immediate evaluation of the risk score of the
present sample and/or the athlete under study is obtained. The sample risk score or
AR.I.E.T.T.A. score is calculated by a simple computational system combining different
parameters (absolute values and intra-individual variations) considered concurrently. The
AR.I.E.T.T.A. score is obtained by the sum of the deviation units derived from each
parameter, considering the shift of the present value from the reference values, based on the
number of standard deviations. Future studies aiming to validate the AR.I.E.T.T.A. score and
improve the diagnostic accuracy will improve the system [11134].
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Passports in practice
The 2011 International Association of Athletics Federation (IAAF) World Championships took
place in Daegu, Korea. For the first time, all athletes were blood tested prior to the
competition in order to give a clear signal to the world athletic community of the wish to enter
into the era of the Athlete Biological Passport and fight against doping in their sport. The
hematological parameters were measured on site. Thus, a mobile-accredited laboratory for
blood testing was created in Daegu. Two serum tubes were collected for clinical chemistry
and hormonal analyses in order to build the bases of the endocrine and the androgen
(steroid) modules of the Athlete Biological Passport in blood. One paper described some of
the main challenges the project faced with regard to the large number of athletes, competing
in different disciplines, and the logistic problems that had to be solved for smart
implementation of one of the most complex operations organized in the last decade in the
The most promising attempt to reveal otherwise undetectable autologous blood doping is the
Athlete Biological Passport enabling a longitudinal monitoring of hematological measures.
Recently, the determination of hemoglobin mass (tHb) was suggested to be incorporated in
the adaptive model of the Athlete Biological Passport. The purpose therefore was to evaluate
the performance of tHb as part of the adaptive model for the detection of autologous blood
transfusions in a longitudinal blinded study. Twenty-one subjects were divided into a doped
group (n=11) and a control group (n=10). During the time course of a simulated cycling
season (42 weeks) including three major competitions (Classics, Grand Tour, World
Championships), multiple autologous transfusions of erythrocyte concentrates were assigned
in the doped group. A blinded investigator ordered up to 10 tHb measurements (carbon
monoxide rebreathing) per subject, mimicking an intelligent doping testing approach in
obtaining hematological data (tHb, OFFmass (novel marker including reticulocytes), and
respective sequences) for the adaptive model. The final analysis included 199 of 206 overall
tHb measurements. The use of tHb, OFFmass, and their sequences as markers of the
adaptive model at the 99 percent specificity level allowed identification of 10 of 11 doped
subjects (91 % sensitivity) including one false positive in the control group. At the 99.9
percent specificity level, 8 of 11 subjects were identified without false positives (73 %
sensitivity). It seems that the problems of tHb determination by carbon monoxide rebreathing
limit the application of this method in antidoping. Because of its potential to detect individual
abnormalities associated with autologous blood transfusions shown in this study, a method
for tHb determination that is compatible with today's standards of testing should be the focus
of future research [12044].
2014 Commonwealth Games in Glasgow

In summer 2014, the world watched as Glasgow hosted the 2014 Commonwealth Games
and athletes pushed the boundaries of human performance. Sport has developed into a
multi-billion pound industry leading to the development of a “win at any cost” mentality in
some individuals. The abuse of performance-enhancing drugs has developed into a
sophisticated arms race between those unfairly enhancing performance and those wishing to
preserve the dignity of sport and the health of the competitors. The challenge for the
Commonwealth games organising committee was to ensure that competition remained fair
and that athletes were kept safe. The athlete biological passport is a system implemented by
the World Anti-Doping Agency directed towards enhancing the identification of those athletes
accountable for the misuse of performance-enhancing substances. One article exemplified
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which drugs are currently being exploited and how the athlete biological passport has
evolved to improve their detection [14630].
Erythropoesis stimulating agents
Blood doping, through the increase of red cells, induces changes of hematological
parameters. The aim of the Biological Passport is first to analyse individual longitudinal
profiles in order to identify, through variations of the specific parameters, doping
manipulations. Additionally, on the basis of abnormal values or profiles, athletes can be
targeted for traditional anti-doping tests in order to detect forbidden substances or methods.
We report the experience of the International Cycling Union in applying the Biological
Passport to target athletes for the presence of erythropoiesis stimulating agents. All positive
results which have been reported between 2008 and 2010 concerning athletes enrolled in
the Biological Passport program are presented. Four cases are discussed more in details. To
conclude, it was propose possible ways of using the Biological Passport in order to better
understand athletes' doping modalities, so that testing programs efficiency can be improved
[12045].
Testing during cycle tournament
Cycling stage races are among the most strenuous of endurance events. The exercise-
induced variations observed in hematological parameters appear to be consistent with the
rider’s physiological response to maintain and improve highly demanding performances day-
after-day. During training and competition, an essential part of evaluating the health and
performance of professional and recreational athletes is periodic assessment of the
hematological profile. Together with evaluation of iron metabolism, serial blood chemistry
analysis can point to whether an out-of-range shift in blood parameters may be attributable to
the response to physical effort or to an index of abnormal response. A starting point for
determining irregular and suspect behavior in athletes is a better appreciation of the
hematological response to vigorous physical activity. This is of particular interest in the
context of the Athlete’s Biological Passport (ABP), which was devised to detect abnormal
variation(s), even at a single time-point, versus a subject-specific physiological range
deduced from the athlete’s own previous data. Because the variations during a competitive
season affect the behavior of hematological parameters over a season, knowing their
variability could help to define the physiological ranges in an athlete. The GiroBio, held in
mid-June every year in northern Italy, is the “under-27 s amateur Giro d’Italia”, a surrogate
for the Giro d’Italia and other international road races (Tour de France, Vuelta a Espana) for
young cyclists. It attracts more than 150 professional cyclists from all over the world annually.
Since 2005 it has been included in the Union Cycliste Internationale (UCI) Europe Tour
circuit, category 2.2. About half the duration of its major counterpart, the GiroBio format is 10
stages over 11 days. The GiroBio race represents the entry step to a fully professional career
for most cyclists. The race aims to promote the values of sport and fair play in healthy
competition, counteracting the doping culture, through the adoption of innovative
organizational aspects, as discussed below. The aims of one study were to determine the
hematological response to middle-term strenuous endurance and to determine whether a
relationship exists between the athlete-specific hematological profile and final placement in a
cycling stage race. The study population was male professional cyclists (n = 253) competing
in the 2010 (n = 144) and 2012 (n = 109) GiroBio 10-day stage races. Blood draws taken
before the start of the race, at mid-race, and at end-race were performed in strict compliance
with academic and anti-doping pre-analytical warnings. Blood chemistry included white blood
cell, red blood cell, hemoglobin concentration, hematocrit, mean corpuscular volume (MCV),
mean hemoglobin content (MCH), mean corpuscular hemoglobin content (MCHC), platelets,
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and reticulocyte relative and absolute counts. Compared to baseline values, erythrocyte,
hemoglobin, hematocrit, MCHC, platelet and reticulocyte counts were all consistently lower
at mid-race, but returned to normal by race-end, while leukocytes were increased in the final
phase. MCV increased during both events. MCH increased in the first part to then return to
baseline in the 2012 race. The calculated OFF-score consistently decreased in the first half
of the race before increasing, but remained lower than the baseline value. The trends of
variation in hematological parameters were substantially similar in both events. There was an
inverse, albeit weak, relationship between placement and erythrocyte, platelet, hemoglobin,
hematocrit and OFF-score values in the 2010, but not in the 2012 race. In conclusion, the
data confirm that, in this large series of elite road cyclists, the strenuous effort a rider
sustains during a stage race induces appreciable changes in the hematological profile
[13296].
Positive cases in biking
Blood doping, through the increase of red cells, induces changes of hematological
parameters. The aim of the Biological Passport is first to analyse individual longitudinal
profiles in order to identify, through variations of the specific parameters, doping
manipulations. Additionally, on the basis of abnormal values or profiles, athletes can be
targeted for traditional anti-doping tests in order to detect forbidden substances or methods.
We report the experience of the International Cycling Union in applying the Biological
Passport to target athletes for the presence of erythropoiesis stimulating agents. All positive
results which have been reported between 2008 and 2010 concerning athletes enrolled in
the Biological Passport program are presented. Four cases were discussed more in details.
To conclude, it was proposed possible ways of using the Biological Passport in order to
better understand athletes' doping modalities, so that testing programs efficiency can be
improved [12046].
Practical experience with athletics, cycling, football, swimming and biathlon
The introduction of the athlete's biological passport (ABP) has been a milestone in the fight
against doping. The ABP is a collection of measurements of different biological parameters
influenced by the administration of doping agents through the time and for each athlete. Two
different modules have been developed and validated so far: the haematological module,
which aims to identify enhancement of oxygen transport, including use of erythropoiesis-
stimulating agents and any form of blood transfusion or manipulation, which became
effective in 2010; and the steroidal module, which intends to detect the use of endogenous
anabolic androgenic steroids when administered exogenously and other anabolic agents,
which was introduced in 2014. Prior to the implementation of the haematological module, it is
important to define an athlete's testing pool on whom to collect blood and/or urine in-
competition and out-of-competition (for the steroidal module, this is irrelevant because all
collected urine samples will be subjected to analysis for the steroidal variables) and to be
compliant with the strict requirements of the World Anti-Doping Agency ABP Operating
Guidelines. The established individual profile can be used either to target traditional
antidoping tests (recombinant erythropoietins, or homologous blood transfusion tests for the
haematological module; isotope ratio mass spectrometry (IRMS) for the steroidal module) or
to support an antidoping rule violation due to the use of a forbidden substance or method. In
this article, we present the experience of four major International Federations which have
implemented an ABP programme, focusing on the haematological module. They constitute
examples which could be followed by other antidoping organisations wishing to introduce this
new, efficient and innovative antidoping tool [14451].
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UCI
In 2008, the Union Cycliste Internationale (UCI) became the first IF to introduce an APB
programme by launching a pilot project (in conjunction with WADA and the Lausanne Anti-
Doping Laboratory) in order to evaluate the feasibility of such approaches. There are strict
technical requirements surrounding the introduction of an ABP programme which can impose
additional logistical and financial constraints on an antidoping authority. Because the
haematological module of an ABP involves the measurement of a variety of blood
parameters, blood has to be collected, handled and transported under special conditions
(particularly with regard to temperature) and analysed within a strict time limit. An additional
difficulty surrounding the initial introduction of the programme was that only a few antidoping
laboratories were accredited to perform the required analyses. Finally, because samples
have to be collected both in-competition and out-of-competition, it is important that all
athletes selected to be part of an ABP programme provide daily whereabouts information to
the ADO in order to permit out-of-competition testing. Despite these challenges, the UCI pilot
programme was a success. It has continued to evolve and expand [14451].
Athlete’s testing pool and whereabouts

In 2013, the registered testing pool of athletes included in the ABP programme consisted of
954 professional male road riders and 78 athletes from all other cycling disciplines. All riders
were required to provide information regarding their daily whereabouts, the vast majority
through ADAMS (Anti-Doping Administration and Management System, an antidoping
administrative database administered by WADA) or using other electronic systems provided
by their National Anti-Doping Agencies (NADOs). It is important to emphasise that following
the introduction of ADAMS in 2008, UCI invested resources to ensure face-to-face meetings
with the teams, at which the use of the ADAMS whereabouts module could be introduced
and explained; written resources were prepared for riders and team managers and have
been updated regularly to further facilitate the provision of whereabouts information. The
outcome of these initiatives has been that cyclists are comfortable and confident in providing
their whereabouts information using the ADAMS resource; notwithstanding the large
numbers of competitors and out-of-competition tests, the number of whereabouts “failures”
has been low [14451].
Sample collection
ABP samples are used to determine the level of a number of haematological parameters. For
this reason, the WADA ABP Operating Guidelines recommend collecting an A and a B
sample (although only the A sample is required). This allows, in case of an abnormal result,
an immediate antidoping analysis for the detection of ESA, synthetic haemoglobin or a
homologous blood transfusion. Since 2008, more than 30 000 blood passport samples have
been collected. In 2013, the Cycling Anti-Doping Foundation (CADF), responsible for all
antidoping operational activities on behalf of UCI, collected 5399 ABP blood samples; 2267
samples were collected in-competition and 3132 obtained out-of-competition. CADF relies on
its own antidoping and blood collection officers (BCO) to administer the in-competition tests;
other service providers are engaged in the collection of out-of-competition samples [14451].
As a UCI rule, a new athlete entering the programme has to be tested at least three times
over a period of minimum 6 weeks before he is allowed to compete in a UCI World Tour
event. In a year, each athlete is tested from a minimum of 2–3 times (rider with a normal
profile established on several samples) to a maximum of 10–15 times (rider who is part of a
special testing programme because of his ranking or his performances or with an abnormal
profile); therefore, their profile can contain up to 70–80 tests [14451].
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APMU and the panel of experts
UCI has appointed the Lausanne APMU to administer the ABP programme. The APMU
evaluates each new result/profile and forwards it to haematological experts whenever the
abnormality is higher than the 99th centile (frequently, profiles exceeding a lower threshold of
abnormality are also sent for review). Typically, this means that the probability that the same
value/profile would be observed in a similar healthy, non-doping athlete is approximately 1 in
a 100. Additionally, the APMU can recommend target testing to CADF whenever appropriate;
request additional documentation from CADF when profiles are considered suspicious; and
coordinate the work of the panel of experts when dealing with a possible adverse passport
finding [14451].
FIFA/UEFA
Testosterone and related compounds are the most recurrent doping substances. The steroid
profile, consisting of the quantification of testosterone and its metabolites, has been
described as the most significant biomarker to detect doping with pseudo-endogenous
anabolic steroids. The steroidal module of the Athlete Biological Passport (ABP) was
launched by the World Anti-Doping Agency (WADA) in 2014. To assess the value of
introducing the module to its anti-doping programme, the Union of European Football
Associations (UEFA) decided to analyze retrospectively the steroid profile data of 4195 urine
samples, collected from 879 male football players and analyzed in 12 WADA-accredited
laboratories between 2008 and mid-2013. This study focused on the evaluation of T/E ratios.
The coefficient of variation (CV) and the adaptive model were the two statistical models used
to study the longitudinal follow-up. A CV of 46 percent was determined to be the maximal
natural intra-individual variation of the T/E when the sequence consisted of single data points
analyzed in different laboratories. The adaptive model showed some profiles with an atypical
T/E sequence and also enabled an estimate of the prevalence of external factors impacting
the T/E sequences. Despite the limitations of this retrospective study, it clearly showed that
the longitudinal and individual follow-up of the T/E biomarker of the players is a good tool for
target testing in football. UEFA has therefore decided to implement the steroidal module of
the ABP from the start of the next European football season in September 2015 [150111].
The International Federation of Football Associations (FIFA) has introduced blood sampling
for the first time during the FIFA World Cup 2002 Korea/Japan. A total of 256 samples have
been collected and analysed for blood parameters (haemoglobin, haematocrit, erythrocytes
and reticulocytes). None of the collected samples presented suspicious values indicating
blood manipulation. The Union of European Football Association (UEFA) introduced the
concept of the biological passport at the final tournament of the UEFA European
Championship in 2008 (EURO 2008) [14451].
The EURO 2008 final tournament antidoping programme ensured the simultaneous
collection of blood (whole blood and serum) and urine from every tested football player,
permitting both the responsible antidoping laboratories in Lausanne and Seibersdorf to
record and store blood profiles, as well as endogenous steroid parameters. All possible
WADA-accredited analytical methods were implemented in testing labs and applied to all
samples, including GH testing as well as testing for homologous blood transfusion and the
evaluation of other blood parameters [14451].
In addition and prior to the start of the final tournament, UEFA conducted out-of-competition
testing of 160 players (10 players from each of the 16 participating teams). Blood and urine
samples were delivered to the laboratories within 12 h of their collection. During the
competition, 124 players (2 from each team) were tested in all 31 matches of the tournament.
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Six teams of two UEFA DCOs were appointed to carry out in-competition testing. Each pair
included a BCO in charge of blood collection and a doping control officer (DCO) in charge of
urine collection [14451].
UEFA secured an agreement with all NADOs concerned in order to ensure coordination
between the AD stakeholders. There were no positive tests, no team whereabouts failures
(filing failures/missed tests) and the acceptance of the programme as well as the cooperation
with the players and the teams was excellent. The UEFA EURO 2012 antidoping
programme reflected UEFA's ongoing commitment to ensuring that the highest possible
standards of doping deterrence and detection are applied. Consistent with the process begun
in 2008, all samples were analysed for the entire range of prohibited substances, using blood
and urine samples processed at the WADA accredited laboratory in Warsaw under the
direction of some of the leading antidoping laboratories in Europe [14451].
In total, 284 players (160 out-of-competitions and 124 in-competitions) were tested. The
menu included partial screening for out-of-competition samples and, in addition, testing for
artificial haemoglobin, blood transfusion (whole blood), blood parameters (whole blood), hGH
(serum), EPO (urine), CERA (serum), SARMS (urine – as part of the partial OOC screen)
and plasticisers (urine), as well as IRMS analysis (which can identify endogenous and
exogenous intake of anabolic steroids, providing a complete individual steroid profile). None
of the samples collected returned positive results, and although one team whereabouts
failure occurred, the collaboration with the players and teams was very good. The collection
of samples in this manner represents the beginning of an ongoing approach in which profiles
will be developed for players as sequential testing results accumulate [14451].
FIFA introduced the ABP in a pilot project during the FIFA Club World Cup 2011 Japan. All
collected samples were analysed for the steroid profile. Prior to and during the FIFA Club
World Cup 2012 Japan and 2013 Morocco, samples of blood and urine from all players have
been collected prior to the competition and routinely during competition. The same
procedures have been performed prior to and during the FIFA Confederation Cup Brazil
2013. The out-of-competition controls (blood and urine samples) will be collected from all
players participating in the FIFA World Cup 2014 Brazil and compared with the blood and
urine samples collected during the competition. Combining the data from FIFA and UEFA will
result in a database of more than 2000 top football players from around the world. The
analysis of these data will have a major impact on the future antidoping strategy in football
and possibly in other team sports [14451].
FINA
FINA – the Federation Internationale de Natation – is the governing body regulating

competition in swimming, open water swimming, diving, water polo and synchronised
swimming. In 2012, competitors in FINA regulated sports constituted the fourth largest group
of competitors in the world to undergo doping controls. Testing of FINA competitors involves
doping controls conducted by FINA itself in addition to testing programmes administered in
association with Continental competitions or by National Federations and National Anti-
Doping Organisations [14451].
In 2012, FINA, committed to the continual improvement of its antidoping programme,

introduced a pilot ABP programme in one of its disciplines – swimming – to ensure that its
doping control programmes were the leading edge. In preparing to introduce the pilot, FINA
sought the advice, and benefited from the counsel, of others who had introduced ABP
programmes in their sports [14451].
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For several years, the majority of doping control tests conducted by FINA have been
administered on an “out-of-competition” basis. On a quarterly basis, competitors in swimming
are ranked according to their performance times in their event; testing programmes focus –
though not exclusively – on the most highly ranked. Thus, the highest ranking competitors in
swimming have always been subject to a much greater chance of selection in out-of-
competition testing programmes; they are also, by virtue of their competitive successes,
more likely to be selected during in-competition testing programmes [14451].
In 2012, in order to initiate its pilot ABP programme, 32 highly ranked elite swimmers, drawn
from across the globe with equal numbers of male and female competitors, were entered into
the ABP pool. These competitors then became the focus of testing using blood and urine.
The opportunity to conduct additional numbers of blood tests on many other swimmers at the
time of the 2012 London Olympics permitted the acquisition of baseline and ongoing
haematological measurements of 83 more competitors; a similar opportunity at the time of
the 2013 Barcelona World Championships permitted further blood sampling to be performed
on an additional 442 swimmers (and competitors in other FINA disciplines). As a
consequence, the acquisition of baseline haematological data on a large number of
swimmers has already begun and will facilitate the further expansion of the ABP programme
[14451].
FINA's antidoping activities are overseen and coordinated by its Doping Control Review
Board (DCRB) – a body composed of internationally acknowledged experts in analytical
science, doping control and sport medicine. The results of the haematological investigations
performed on those in the pilot ABP pool are monitored by the Chair of the DCRB serving as
the APMU for the pilot project; any anomalous profiles are forwarded to haematological
experts for their review. The conclusions of the haematological experts would then be
forwarded to the DCRB for further review. To this point, that has not been necessary as no
adverse profiles have been identified. The ABP programme has run smoothly with no
evidence of concerns or complaints. By September 2013, of the 32 competitors currently the
focus of the pilot ABP testing, 22 have undergone 5 or more sample collections; 11 have
undergone 7 or more sample collections and there are 5 competitors who have been tested
on 10 or more occasions [14451].
As the programme has evolved, we have also sought to develop strategic agreements with
other ADOs in order to ensure that our ABP testing might be coordinated with testing
conducted by other agencies – typically NADOs – in order to achieve efficiencies and
enhanced effectiveness while optimising the timing and coordination of testing. Such
approaches are entirely consistent with the theme of the new World Anti-Doping Code which
emphasises cooperation and coordination and the implementation of “intelligent” testing
strategies. Our experience has demonstrated that an ABP programme can be successfully
introduced on a global basis; it is anticipated that the programme will now evolve to include a
greater number of swimmers as well as being expanded to include competitors within other
FINA disciplines [14451].
Finally, it should be noted that FINA has, in recent years, been assembling “steroid profiles”
on all competitors who have been noted to have elevated T/E ratios. In this respect, the
application of an ABP-like approach to testing is already an important element of FINA's
antidoping programmes [14451].
IAAF
IAAF – the International Association of Athletics Federations – is the governing body for the
sport of track and field Athletics. In 2009, following the UCI experience and the publication by
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WADA of the first formal ABP Operating Guidelines and standards, the IAAF started its own
ABP programme in collaboration with the Lausanne Anti-Doping Laboratory [14451].
Before 2009 (from 2001 to 2008), the IAAF conducted roughly 7300 haematological blood
tests in precompetition settings at major events, with the objective of detecting blood
manipulation, and identifying the misuse of EPO administration by athletes (during this
period, a positive urine EPO test was the only way to pursue an antidoping rule violation
(ADRV), and ultimately to apply a sanction) [14451].
Particularities of athletics
Athletics is a truly universal sport which draws competitors from around the world. Members
of 212 National Federations compete in 47 different disciplines, each of which may
emphasise a specific combination of endurance, power, strength and speed. There is an
ongoing and important turnover of top competitors in many events. Considering the network
of WADA accredited Laboratories involved in the IAAF ABP programme, the required time
delay of 36 h between collection and analysis, and the necessity to transport samples at a
carefully regulated temperature, the administration of un-announced out-of-competition ABP
tests in some countries can be extremely difficult, if not impossible [14451].
Athlete’s testing pool and whereabouts

The IAAF ABP programme is principally focused on top athletes in all endurance events
(race walking, steeplechase, cross-country and long-distance and middle-distance events),
with a dedicated Registered Testing Pool (RTP) for ABP testing purposes of approximately
150 athletes. The majority of these athletes – approximately 95 percent – directly enter their
whereabouts details in ADAMS. For the remaining 5 percent, the work is performed internally
(with information being submitted by email, fax or SMS) [14451].
In addition, a list of 60 athletes, whose previous results have shown a suspicion of blood
doping, are followed throughout the year, without any formal inclusion in the RTP. Alongside
individual testing plans, “mass screening” ABP tests are conducted prior to major IAAF
events (eg, World Championships, IAAF Calendar meetings, World Marathon Majors) for
purposes of deterrence and detection. All athletes competing at the IAAF World
Championships in Daegu (2011) and Moscow (2013) underwent a blood ABP test [14451].
Sample collection
Since 2009, the IAAF has conducted 9524 ABP tests and incorporated 874 test results
provided by data sharing with 18 participating NADOs. These data are managed internally,
both with ADAMS and a dedicated IAAF Database. They represent, in total, approximately
4800 different biological profiles (of an average 2 samples/athlete). To organise blood
collection, the IAAF collaborates with NADOs and three private stakeholders. On average,
about 25 percent of such tests are conducted on an out-of-competition basis, whereas the
remainders are collected in a precompetition setting [14451].
Athlete passport management

An internal IAAF APMU, assisted by an ABP medical expert, manages and follows up the
ABP programme for Athletics (except for the review of the laboratory documentation
packages, which is performed by the APMU of the Lausanne anti-doping laboratory). A panel
of three independent medical experts provides an analytical interpretation of suspicious
patterns of abnormality whose score is greater than 99.9 percent certainty, and when these
profiles have a sufficient number of samples collected under appropriate conditions (in
practice, at least 5) [14451].
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Antidoping rule violations
Since 2009, the IAAF has concluded 92 ADRV involving blood manipulation: 27 ABP cases
with several resulting in the imposition of 4-year sanctions (other cases are ongoing); and 65
ESA cases related to positive urine tests. In conclusion, the IAAF started its ABP programme
with two approaches: one focused on the top athletes in endurance events, the other focused
on large-scale screening – for a deterrence effect – on numerous athletes [14451].
It has also been mentioned that IAAF will develop the closest collaboration possible with
NADOs who have developed a national ABP programme, in order to delegate the ABP
monitoring of their national level athletes, thus focusing its efforts on top level international
athletes [14451].
Biathlon
In recent years, some international sports federations have introduced blood testing
procedures that can lead to suspension from competition for athletes whose haematologic
values exceed certain established limits. In 1994 the International Biathlon Union initiated a
three-phase blood testing program to safeguard athletes' health and ensure fair competition.
The first phase, lasting three years, was aimed at measuring the haematocrit values of
biathletes in order to determine statistically acceptable limits for participation in competition.
The second phase, lasting four years, consisted of pre-race testing for an increasing number
of athletes and suspension from competition for those whose haematocrit values exceeded
52 percent for males and 48 percent for females. The results of this second phase (third
phase now in progress) are reported. Progressive increases have been made in the numbers
of countries examined, athletes tested, and tests performed. This retrospective study reveals
a reassuring trend in average values for haematocrit and haemoglobin in the entire study
population, a minimal number of athletes with excessive values and a consequent low risk of
false positive results, an acceptable incidence of relatively high values (50 % for males and
45 % for females), and constant non-elevated haematological profiles for elite athletes. The
variability in individual haematocrit levels among all biathletes with a minimum of four
observations during the four-year period is also evaluated and discussed [03033].
Future modules and integration
There are new modules in the process of development to complement the haematological
and steroidal modules of the ABP. Research has been directed to develop an endocrine
module to look at substances that control various hormone axes, such as growth hormone.
Longitudinal profiles of high level athletes are being collected to establish population-based
comparisons; individual endocrine profiles for various hormones are being tested to see
whether they have the potential to reveal doping. Furthermore, WADA has directed and
supported research into “omics” strategies from genomic to proteomic evaluations. Some -
omic variables could be integrated in the ABP following due validation and assessment of
confounding factors. Eventually the ABP may have quite a different look and these modules
which are presently operating and being developed separately will become evaluated
together as part of the athletes’ passport [14015].
It is known that the use of anabolic steroids may enhance erythropoiesis, therefore it could
be interesting for some athletes who are at high risk of steroid doping to have a
haematological profile as well. Competition, performance results and other information such
as that obtained from investigations will all be layered on top of the longitudinal profiles. It
504
should be noted that the definition of a “passport” in the Guidelines is the longitudinal profile
and all other information [14015].
If the theoretical aspects of the biological passport have been established and are applied
already with the haematological and steroid modules by some IFs and national anti-doping
agencies, there are still many factors to be solved in order to implement it in an efficient way
for the benefits of the fight against doping. The first implementation into several international
sports federations or NADO shows clearly that “one size fit all” cannot be applied here.
Depending on the type of sport (individual or team sport) and how it is organised throughout
the year, the approach may change. Some sports have many competitions spread over a
very long season; some others concentrate on major events for which the athlete prepares
the rest of the season. Some sports are practiced at the international level over the entire
planet like soccer or track and field. Some others like cycling are concentrated in a relatively
limited geographical area and small population of interest. For these reasons, the strategy in
the organisation of this biological profiling will change drastically from a federation to another,
mainly due to logistic and financial constraints. The main objective of the implementation of
the passport has been created by WADA as a tool to define an antidoping rule violation, but
the experience of the last seasons shows that the ABP is also a very good tool for prevention
and deterrence. As the number of out of competition tests has generally increased, the
athlete will react automatically by being more cautious in his behaviour [14433].
This preventive and deterrent approach could be even improved if an abnormal biological
profile prior to an event, can lead to non-participation to a race event. This would certainly
bring a lot to the fairness of the competition. ABP is still in its infancy and thanks to recent
development and advances in circulating microRNA analyses, proteomics and especially
metabolomics; the specific fingerprints left by doping could also be included in an individual
follow-up and be part of the biological passport. Current status of the athlete biological
passport [14433]:
- The steroidal module to fight anabolic steroid abuse is the second module, after the
blood module to fight erythropoiesis-stimulating agents and blood transfusions, to be
implemented in the athlete biological passport (ABP)
- Urine and blood data can be implemented in the ABP if the collection, transport,
analyses follow rigorous protocols
- The evaluation of data included in the ABP by the panel of experts must take into
account additional information such as heterogeneous factors which could influence
the behaviour of biological variables
- The indirect approach of the biological passport has been fully accepted by the Court
of Arbitration for Sport (CAS) during previous hearings.
General scheme of the athlete biological passport (ABP)
The anti-doping organisation (ADO) testing authority has a pool of athletes to be tested.
Mainly, international sports federations (IFs) and national anti-doping organisations (NADOs)
will order for sample collection on athletes from their registered testing pool (RTP). Official
Doping and/or Blood Control Officers (DCOs/BCOs) are designated to proceed with the
appropriate sample collection. They are in charge then to assure the transport of the samples
in the appropriate conditions and time (in less than 36 h) to the accredited laboratory. The
analysis results are then immediately introduced in the passport of the athlete, which is
constituted of at least 4-6 values per season. The Athlete Passport Management Unit
(APMU), linked to a WADA accredited laboratory, is in charge of processing the passport,
control its validity and the quality of the data and if presenting abnormality in the sequence of
biological data (biological values being significantly outside the individual norms), the full
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ABP documentation package will be examined by three independent experts who shall
provide an unanimous decision to the APMU to release a certificate of “Adverse Passport
Finding” (APF) for the ADO result management unit [14433].
Key points for the implementation of the ABP
To resist to legal and scientific challenges, the ABP should be a transparent process with the
necessary independence between planning, interpretation and result management of the
passport. A new major actor has been introduced in the system to create a framework of
independence: the Athlete Passport Management Unit (APMU). This unit should be the
central hub connecting laboratory-generated biological data with active test planning
intelligence. Ideally, this central hub should be associated with a WADA accredited
laboratory, because the employees are independent, trained and used to all steps of the
legal procedure engaged in case of doping offences and they must report to the recognised
ADO as well as WADA. For the time being, APMUs can either be integrated to WADA
accredited laboratories, national anti-doping organisations (NADOs) or Ifs [14433].
Moreover, a mathematical model has been designed to identify non-subjectively, unusual

longitudinal results of the athlete. This is the adaptive model which calculates the probability
of a longitudinal profile of marker values assuming that the athlete has a normal physiological
condition. The adaptive model has been introduced into the ABP software, which has been
produced by the Lausanne Laboratory scientists. The APMU will liaise with a panel of
experts (as agreed with the ADO) in order to interpret in an independent manner the results
of the adaptive model in cases of significant abnormality of the profile. These experts should
be knowledgeable in one or more of the fields of clinical haematology, sports medicine or
exercise physiology. The ideal (and recommended) administrative sequence of the ABP is
the following (adapted from WADA guidelines) [14433]:
- Identification of the athlete by the ADO:

o Identification of the suitable time for collection based on recommendation of
the APMU.
o Sample collection request delivered by the ADO to a sample collection agency
or to appropriate doping control personnel.
o The sample collection authority accesses the whereabouts information of the
athlete (localisation).
- The blood collection officer locates the athlete and collects the biological sample:
o The sample collection personnel are responsible for the transport of the
biological sample to the WADA accredited laboratory.
o The sample collection personnel are responsible to transcribe the doping
control form immediately after the collection into web-based database ADAMS
(Anti-Doping Administration and Management System) to provide direct
access to the relevant data for the laboratory, APMU and the ADO.
- The WADA accredited laboratory analyses the sample and reports to ADAMS:
o Notification is carried out to APMU which updates the ABP and applies the
adaptive model using the ABP software.
o The APMU reviews the updated passport including the results in the adaptive
model, and advises the ADO on intelligent testing strategies.
o When the markers (HGB, and/or OFF-hr Score (OFFs) are beyond the 99th
centile of the expected ranges returned by the adaptive model, the APMU
shall proceed with the steps of evaluation liaising with the expert panel.
o The APMU will also regularly provide profiles that do not exceed the 99th
centile, in order to provide experts with a more balanced view of the
considered athlete population.
506
Database and sharing of information
Athletes may be tested by different testing authorities which may be National Anti-Doping
Organisations, IFs or Major Events Organisers. Data should be entered into a secure central
database system to ensure that there are no gaps in the longitudinal profiles which would
interfere with the interpretation of the individual data. The goal is to have one passport for
one athlete with all the data points included. All WADA accredited and approved laboratories
enter the results into a central database called Anti-Doping Administration and Management
System (ADAMS) where each athlete gets assigned an anonymous BP ID number. The
Adaptive Model, a Bayesian type of statistical analysis that uses prior information, is
automatically applied to these longitudinal profiles and notifications are sent to the
appropriate agencies when the profile is considered atypical. However, currently not all
stakeholders are using ADAMS which means that some results entered by laboratories are
orphaned and not associated with the appropriate BP identity number. This diminishes the
global efficacy of the ABP and prevents WADA from fulfilling its duty as a monitoring agency.
WADA encourages Anti-Doping Organisations to share information and testing to increase
the efficiency of the ABP, avoid duplication and to decrease costs. However, athletes’ rights
must be fully respected and protected through this process which is why WADA consults
ethicists, data-protection and other legal experts to ensure that basic principles such as
privacy and proportionality remain entrenched [14015].
Continuous evaluation of the ABP
Further studies evaluated the strength and possible limitations of the ABP after introduction
of the hematological module by UCI and WADA which will be presented in the subsequent
paragraph. As the ABP depends on analytically flawless data, the pre-analytical conditions
and the laboratory testing were put into question. Thus, it seemed of great importance
whether the adherence to the ABP guidelines will allow high quality results respecting
forensic standards and thus assure their applicability in a juridical context. It was confirmed
that the rigorous adherence to WADA’s guidelines yields excellent results required in the
anti-doping context. According to previous studies on the topic, the limit of 36 h from blood
collection to analysis is reasonable to guarantee analytical quality, when the samples are
transported at 4 °C which seemed to improve the stability of hematological parameters
independently from the analytical methodology. Further studies on the stability of blood
parameters used within an anti-doping setting were available before the ABP was officially
introduced. In addition, pursuing the goal to provide a sound foundation for the interpretation
of blood profiles in athletes, more data were published on various confounding factors and
analytical aspects which may help to refine existing guidelines. For instance, from a practical
point of view, it was reported that long-haul air travel leads to normal diurnal variations of Hb
without any indication that travel will affect the hematological measures in a way that might
be interpreted as blood doping. Despite the general appreciation of the concept, it also
became clear that the ABP has some limitations. It has also been discussed that “the
sensitivity of the ABP to detect doping is limited if the physiological result of a low level of
doping remains within the individual’s own reference range”. It fits in this context that
microdosing of rhEPO does not lead to abnormal changes in the current ABP markers during
rhEPO administration. Nevertheless, for example, blood counts were measured only during
the titration and following maintenance phase, but not longitudinally after rhEPO was
discontinued for which OFF-hr was originally described [13006].
507
In order to evaluate the best possible realistic performance of the ABP adaptive model for a
blood doping technique (autologous blood doping), a longitudinal blinded study design
included 21 subjects that were divided into two groups of which one was transfused with 1-2
bags of blood at 2-3 time points during a simulated time course of a professional cycling
season. A blinded investigator who was well trained in the use of the ABP was requested to
apply an intelligent testing approach which allowed a sensitivity of 82 percent at the
99 percent threshold of the adaptive model as 9 of 11 subjects in the doped group had at
least one value outside of the individual limits for Hb and OFF-hr or a sequence (Hb and
OFF-hr) above the 99.9 percent probability threshold. The specificity was 90 percent for Hb
(one false-positive value in 1 of 11 athletes) and 100 % for OFF-hr, sequence Hb as well as
sequence OFF-hr [13006].
Several aspects allude to the effectiveness of the ABP hematological module. First,
individual non-physiological data from the ABP have been used to sanction athletes and
were recognized by the Court of Arbitration in Sport (CAS) in a final hearing. Second, these
precedents might have had a deterrent effect on future behavior of athletes as the value of
%retics has significantly changed and normalized since the introduction of the ABP in 2008,
which may reflect a decreased prevalence of blood manipulations in the professional cycling
peloton. From a practical and more measurable perspective, the introduction of the ABP
hematological module and dedicated target testing based on the ABP data led to a
250 percent increase in the number of positive rhEPO cases in 2008 and 2009. In 2011 the
number of cases was still about 300 percent higher than it was before the introduction of the
ABP (data available from www.wada-ama.org) [13006].
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SOCIO-MEDICAL, SOCIAL AND PSYCHOLOGICAL ASPECTS ON
DOPING
Doping in sport has been a focus of medical, physiology and social science research in
recent years. Whereas medical and physiology researchers focus on improving methods
(e.g. blood, urine and gene tests) for detecting the use of prohibited substances and to deter
athletes from their use, social science researchers strive to better understand the
psychosocial factors (e.g. attitudes, environment and beliefs) that may offer targets for
educational programms aimed at preventing this behaviour [13017].
Costs of antidoping programs are high, and programs are increasingly complex and difficult
to manage. As the bureaucratic burden imposed on athletes, physicians and sport
organizations grows, concerns about sustainability abound. But the resolve of athletes and
sport organizations to maintain integrity and eradicate cheating is strong [014]. However,
taken together it is obvious that the fight against doping not only is played on a morality
ground but also have economical implications.
Although difficult to document, it has been postulated that more steroid use takes place
outside organized sport, within body-building and other subcultures, where no attempt is
made to address drug-taking behaviour. Adolescent athletes, often eager to gain weight and
muscle mass, are ripe targets for those who offer easy approaches that promise startling
results in just a few weeks. To some, performance and size are paramount; they are seen to
be the keys to success and stardom. And therein lies a special challenge for physicians
interested and involved in sport: one must ensure that those athletes and, perhaps more
importantly those who coach them, develop approaches to sporting success that emphasize
excellence in every domain. Pharmacologic approaches to enhanced performance represent
a witches' brew of distorted values and maligned perspectives with potentially devastating
health risks [08014]. This means that doping is not a problem only for the athletic
movements.
There is a growing body of empirical evidence on demographic and psychosocial predictors

of doping intentions and behaviors utilizing a variety of variables and conceptual models.
However, to date there has been no attempt to quantitatively synthesize the available
evidence and identify the strongest predictors of doping. Using meta-analysis, it was aimed
to (i) determine effect sizes of psychological (e.g. attitudes) and social-contextual factors
(e.g. social norms), and demographic (e.g. gender and age) variables on doping intentions
and use; (ii) examine variables that moderate such effect sizes; and (iii) test a path analysis
model, using the meta-analyzed effect sizes, based on variables from the theory of planned
behavior (TPB). Articles were identified from online databases, by contacting experts in the
field, and searching the World Anti-Doping Agency website. Studies that measured doping
behaviors and/or doping intentions, and at least one other demographic, psychological, or
social-contextual variable were included. It was identified 63 independent datasets. Study
information was extracted by using predefined data fields and taking into account study
quality indicators. A random effects meta-analysis was carried out, correcting for sampling
and measurement error, and identifying moderator variables. Path analysis was conducted
on a subset of studies that utilized the TPB. Use of legal supplements, perceived social
norms, and positive attitudes towards doping were the strongest positive correlates of doping
intentions and behaviors. In contrast, morality and self-efficacy to refrain from doping had the
strongest negative association with doping intentions and behaviors. Furthermore, path
analysis suggested that attitudes, perceived norms, and self-efficacy to refrain from doping
509
predicted intentions to dope and, indirectly, doping behaviors. Various meta-analyzed effect
sizes were based on a small number of studies, which were correlational in nature. This is a
limitation of the extant literature. The review identified a number of important correlates of
doping intention and behavior, many of which were measured via self-reports and were
drawn from an extended TPB framework. Future research might benefit from embracing
other conceptual models of doping behavior and adopting experimental methodologies that
will test some of the identified correlates in an effort to develop targeted anti-doping policies
and programs [14655].
Semantics in doping
Several studies have looked at the unsupervised drug habits of AAS users, but these are
clearly subject to different types of bias. Nevertheless, these field studies should not be
overlooked. According to the studies, drug regimens follow typical patterns. Different oral and
injectable compounds are generally combined (“stacked”), creating large dose regimens
usually self-administered during periods (“cycles”) lasting 4-12 weeks. “Stacking” is based on
the idea that smaller dosages of multiple drugs might reduce the chance of complications
than larger dosages of a single drug. This may also facilitate the administration of multiple
AASs (necessary to achieve supraphysiological doses) for longer periods, and so minimizing
the plateauing effect. The aim of “stacking” is to rationally combine different characteristics,
avoiding overlap of benefits or side effects. Combinations of testosterone and nandrolone (or
similar drugs) are the basis of the “massbuilding stacks” used to maximize muscular and
strength gains. Combinations containing potent androgens are preferred for dieting and body
definition, because of their lack of estrogenic activity (less water, salt and fat retention).
These are the “cutting stacks”. Heavy users may combine a “mass-building cycle” with a
subsequent “cutting cycle”, finishing with a ‘post-cycle therapy’, with anti-estrogens or hCG,
in an attempt to restart androgen production. Frequent users may also combine AASs with
other “performance drugs”, such as painkillers (including opioids), diuretics, insulin, growth
hormone, stimulants, aromatase inhibitors and thyroxine [11028].
Political and economical aspects of anti-doping
One article examines the processes by which the Anabolic Steroid Control Act of 2004, an
act that added steroid precursors such as androstenedione to the list of Schedule III
Controlled Substances in the United States, came to pass in both the House of
Representatives and the Senate. Grounded theoretically in political economy, the article
addressed how the interplay of political pressures and economic influences stands to affect
the actions of public officials, and how "tougher" drug policies-those touted to be more
substantive and efficacious than existing regulations-often fail to effect change. The article
concludes with implications for those involved in the regulation of anabolic steroids and
steroid precursors [06011].
Social and socio-medical issues
An integrative model of doping use with adolescent athletes
One study assessed adolescent athletes' intentions toward doping by using an integrative
theoretical model. Overall, 650 adolescent athletes from team and individual sports
completed an anonymous structured questionnaire including demographic information, social
510
desirability, achievement goals, motivational regulations, sportspersonship orientations,
social cognitive variables, and anticipated regret. Hierarchical regression analysis showed
that the integrative model predicted 57 percent of the variance in doping intentions. Social
cognitive variables and anticipated regret directly predicted doping intentions. Anticipated
regret added 3 percent incremental variance on top of other predictors. Multiple mediation
analyses showed that the effects of achievement goals on intentions were mediated by self-
efficacy beliefs, whereas the effects of sportspersonship were mediated by attitudes and
anticipated regret. The study confirmed the dual structure of an integrative model of doping
intentions and further highlighted the role of anticipated regret in the study of adolescent
doping use [150012].
Adolescent use of anabolic steroids: social, personality and health aspects

Since the 1950s and 1960s use of anabolic androgenic steroids (AAS) – endogenous and
synthetic testosterone, as well as synthetic derivatives of testosterone – has broadened
beyond athletes and body-builders to include adolescent males seeking an idealised
appearance and adolescents associated with multiple drug use. It has been shown that
adolescent AAS use is connected to strength training, injected drug use, and die use of
multiple drugs respectively, even after controlling for sports participation and poor academic
performance. However, knowledge about tiie importance of personality and healdi factors
with regard to patterns of AAS use is still inadequate, in particular among potential AAS
users. Aspects of personality and health are of great concern for several reasons. Because
of the suggested associations between AAS use and die desire to improve appearance and
gain body mass, the impact of personality aspects such as self-esteem have to be examined
further. Bodybuilders have been reported to being at risk for body image disturbances as well
as for eating disorders such as anorexia nervosa and “reverse anorexia”, illnesses that have
been connected with low self-esteem among adolescent females. It has also been assumed
drat patterns of AAS use include some psychological aspects also seen with the use of
psychotropic substances (i.e. cannabis, opiates, cocaine, amphetamine, methamphetamine,
LSD, MDMA (ecstasy) and psilocybin). Some studies have indicated that AAS might act on
biological mechanisms similar to those affected by psychotropic substances, and other
studies have pointed to a tendency to use both AAS and psychotropics. Reports have
documented various adverse effects associated with use of AAS on mental health. As is the
case with psychotropic substances, biological research has shown that AAS influence the
reward regions of the brain as well as leading to the development of dependence, aggression
and violent behaviour. Several multifactor theoretical models have been constructed in order
to explain the phenomena of alcohol and psychotropic substance use and these may be
important to keep in mind in order to gain a better understanding of AAS use. Among the
factors shown to be relevant for psychotropic substance use are general attributes of the
interpersonal environment, including sociodemographics such as family circumstances and
socio economic status. Other variables of importance in substance use are aspects of peer
influence. In the stress-coping model, life stress is posited as a general risk factor
predisposing to various kinds of problems. Coping processes are proposed to operate by
either retarding or accelerating the development of problems in combination with buffering
protective factors such as certain types of competence variables or personality related
variables. In accordance with the stress-coping model, it has been shown that low social,
behavioural and academic competencies are directly related to substance use [01010].
Over the last decade adolescent males have been shown to use anabolic-androgenic
steroids (AAS) in order to improve their sports performance and appearance, as well as in
combination with alcohol and psychotropic drugs. However, the risk profile of AAS use is still
not well understood. This study analysed the importance of social, personality and health
factors for the use of AAS. More than 2,700 senior high school students in Uppsala, Sweden,
filled out an anonymous closed-response questionnaire. The findings from multiple logistic
511
regression analyses of adolescent males (n=1,353) showed that immigrant status,
average/low self-esteem, average/low perceived school achievement and use of prescription
tranquillisers/sedatives had independent significant associations with the use of AAS after
controlling for age and previously known factors such as strength training, truancy and heavy
alcohol consumption. It was concluded that the characteristics of AAS users extend beyond
activities such as strength training and multiple drug use to include social, personality and
health aspects [01010].
Doping in sport: a test of the strength-energy model
It was applied the strength-energy model of self-control to understand the relationship

between self-control and young athletes' behavioral responses to taking illegal performance-
enhancing substances, or "doping." Measures of trait self-control, attitude and intention
toward doping, intention toward, and adherence to, doping-avoidant behaviors, and the
prevention of unintended doping behaviors were administered to 410 young Australian
athletes. Participants also completed a "lollipop" decision-making protocol that simulated
avoidance of unintended doping. Hierarchical linear multiple regression analyses revealed
that self-control was negatively associated with doping attitude and intention, and positively
associated with the intention and adherence to doping-avoidant behaviors, and refusal to
take or eat the unfamiliar candy offered in the "lollipop" protocol. Consistent with the
strength-energy model, athletes with low self-control were more likely to have heightened
attitude and intention toward doping, and reduced intention, behavioral adherence, and
awareness of doping avoidance [150024].
Social and psychology characteristics of users of anabolic steroids
It was compared and contrasted the characteristics of 2 groups of men ≥40 years old:
reported anabolic-androgenic steroid (AAS) users and nonusers. A total of 67 male AAS
users and 76 male nonusers ≥40 years old were recruited. Demographics, utilization of AAS
and other performance-enhancing agents (PEAs), exercise patterns, history of illicit drugs
and alcohol use, and psychiatric traits/diagnoses were noted. The majority of AAS users ≥40
years old were caucasian (93 %), heterosexual (97 %), and classified themselves as
recreational exercisers (79 %). AAS users took more PEAs (12 ± 6 vs 5 ± 3), were more
likely to binge drink (48 % vs 29 %), report heavy alcohol use (21 % vs 8 %), meet criteria for
substance dependence disorder (27 % vs 4 %), and reported an anxiety disorder diagnosis
(12 % vs 3 %) than nonusers. It was concluded that AAS misuse is prevalent among older
men and is associated with polypharmacy, more aggressive alcohol use, and a higher
incidence of substance dependence and anxiety disorders compared to nonusers. This
information may help clinicians and researchers identify and develop appropriate intervention
strategies for AAS abuse among older men [14021].
The aim of one study was to identify the social psychological determinants of the use of
performance-enhancing drugs by gym users who practice bodybuilding, fitness, powerlifting
or combat sports. In this questionnaire-based study, 144 respondents answered questions
on their actual use and intention to use such drugs and also on their background
characteristics and beliefs, such as their attitudes, social influences and self-efficacy. While
all social psychological determinants correlated with intention to use these drugs, the most
important predictors were personal norms, beliefs about performance outcomes and the
perceived behavior of others. Non-users held more restrictive norms about using
performance-enhancing drugs, were less optimistic about the performance-enhancing
outcomes and believed that fewer significant others used performance-enhancing drugs than
users and ex-users. The results of this study indicate that users attribute advantages to
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performance-enhancing drugs and are inclined to overlook the risks of using them.
Preventive interventions should focus on influencing personal norms and social processes
[07019].
Risk-taking individuals, such as those who abuse alcohol or drugs, engage in criminality or
have eating disorders, have been shown to have an increased predisposition for doping use.
Despite this evidence, questions remain about the relationship between risk behaviours such
as abuse of alcohol and narcotics and doping. For example, in a study on 15,000 sports
active high school students in the US, there was no correlation between doping use and risk
behaviour [Dodge & Jaccard, 2006]. Coakley and Pike [2009] argue that such results reflect
young people’s use of alcohol and drugs to escape from reality being a completely different
phenomenon to athletes doping to improve athletic performance. Coakley and Pike argue
doping is not about violating social and legal norms, typically seen when abusing alcohol and
drugs, but rather a manifestation of an “over conformity” in relation to basic norms in sport –
the “sport ethic” (which not should be confused with norms of fair play). The “sport ethic” is
characterised as how the athlete should prioritise sport and their team before everything else
in life, continually striving to improve sporting performances, struggle for victories. Doping
becomes a tool in this endeavour. One article investigated doping use in relation to
substance abuse, health risks, risks of being caught in a doping control, and other risk
behaviours, in a qualitative interview study of 11 Swedish elite athletes sanctioned for using
anabolic androgenic steroids (AAS) during their sports careers. Most respondents grew up in
secure, intact families with siblings. Virtually all respondents were from working class homes
where the father typically worked in industry and the mother a housewife who worked part
time in retail when the children become older. The majority of respondents were also
workers, including business owners, clerks and managers. Most attended a vocational
program at high school, although some have no secondary education. No respondent had a
university degree. Respondents reported a positive school experience, with good friends and
mediocre or good school performances. Most respondents had excellent results in junior
and/or senior national championships. Several respondents had a good international record,
participating in national teams, with some winning national and international championships.
Ten of the respondents were convicted 1990–1996 and one in the mid-2000s. AAS was the
only doping agent used by the athletes in this study. None of the respondents reported using
other doping agents; some reported amphetamine and cocaine use for social rather than
sporting reasons. Respondents in this study were not using AAS in cycles, and were instead
using preparations on a daily basis. However, respondents did interrupt use before
competitions to avoid being caught in a doping control. Some respondents mentioned that
they stopped using AAS when they were injured and unable to carry out their training
sessions. Taking AAS without training was typically described as a complete waste of time,
effort and drugs. The “gateway effect” seen in other substance use behaviour (where
abusing one substance is a risk factor for developing other forms of more harmful drug
behaviours) has also been established in doping. Patterns of other substance abuse were
investigated in this study. Overall the respondents were very restricted in their use of alcohol
and drugs. Substance abuse was unusual among the respondents, and in that sense they
could not be regarded as typical risk takers. Their AAS use could characterise them as risk-
takers with regards to doping control and health. Overall, the respondents perceived the
health risks as very limited. Even though respondents did not perceive any serious health
risks due to their use of AAS, they would arguably have been better off if they refrained
entirely. However, this depends on what benefits the AAS provide. If athletes have a lot to
gain in terms of medals, money, less pain or making exercise easier, and the risks are
moderate or small – then doping could be considered a rational choice of action instead of a
irrational risk behavior. Only two respondents were uncertain about the positive effects of the
steroids. Comparing the positive and negative effects of AAS use, most respondents
perceived distinct positive effects from using. It was easier to work-out and they achieved
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better results. The negative effects of doping were, except for one respondent, very small.
Another characteristic of risk-taking behaviour, in addition to the lack rationality, is the failure
to analyse the consequences of different opportunities before action. This was also
investigated among the respondents by probing how they reasoned in relation to the risk of
being caught in doping control, and in connection with possible health risks. Almost all
respondents said that they were cautious and used moderate doses. Respondents were
asked to self-assess their propensity to take different risks in life using a broad
understanding of the concept of risk-taking. To do this, respondents were asked how they
see themselves in relation to risk-taking using examples like drug use, crime, gambling,
speeding, driving without seat belt, skydiving and bungee jumping. Not one of the
respondents describes themselves as risk-takers. It is clear that results focusing on risk
behaviour to explain doping use are not relevant to explain AAS use among most of the
respondents in this study. The respondent who was the heaviest user went on regular
medical surveillance to insure against health risks. Finally, none of the respondents in the
study assessed themselves as risk-takers; they described themselves as cautious,
calculating, planning and cowardly. The findings around risk and doping may also be a result
of the methodological approach taken by the study. Most studies on this topic have a
quantitative design. Evaluating different methodological approaches is an important but
complex question for doping related research [13034].
The use of anabolic-androgenic steroids (AASs) by professional and recreational athletes is

increasing worldwide. The underlying motivations are mainly performance enhancement and
body image improvement. AAS abuse and dependence, which are specifically classified and
coded by the DSM-5, are not uncommon. AAS-using athletes are frequently present with
psychiatric symptoms and disorders, mainly somatoform and eating, but also mood, and
schizophrenia-related disorders. Some psychiatric disorders are typical of athletes, like
muscle dysmorphia. This raises the issue of whether AAS use causes these disorders in
athletes, by determining neuroadaptive changes in the reward neural circuit or by
exacerbating stress vulnerability, or rather these are athletes with premorbid abnormal
personalities or a history of psychiatric disorders who are attracted to AAS use, prompted by
the desire to improve their appearance and control their weights. This may predispose to
eating disorders, but AASs also show mood destabilizing effects, with longterm use inducing
depression and short-term hypomania; withdrawal/discontinuation may be accompanied by
depression. The effects of AASs on anxiety behavior are unclear and studies are
inconsistent. AASs are also linked to psychotic behavior. The psychological characteristics
that could prompt athletes to use AASs have not been elucidated [150031].
Vocabularies of motive for illicit steroid use among bodybuilders

Illicit steroid use, for purposes of performance and physique enhancement, is widely deemed
unnecessary, wrong and dangerous. Such activity would appear especially foolhardy when
engaged in by non-professional athletes who otherwise adhere to “healthy” exercise
regimens. Here a gap exists between many illicit steroid users' actions and societal
expectations. Using qualitative data generated in South Wales, this paper explores
bodybuilders' vocabularies of motive for illicit steroid use. These accounts which justified,
rather than excused, steroid use were predominant during question situations between the
participant observer and the researched. In supporting the fundamental tenets of their drug
subculture, and as part of the underlying negotiation of self-identity, respondents espoused
three main justifications for their own and/or other bodybuilders' illicit steroid use; namely:
self-fulfilment accounts, condemnation of condemners and a denial of injury. Here steroid
use was rationalised as a legitimate means to an end, observers passing negative
judgements were rejected and it was claimed steroids do not (seriously) harm the user's
health or threaten society more generally. These vocabularies of motive, acquired and
honoured within bodybuilding settings, comprise a complex of subjective meanings which
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seem to the actor to be an adequate ground for the conduct in question. Similar to other
sociological studies, this paper states that it is imperative to explore the social meanings
which illicit drug users attach to their “risk” practices. Without these understandings,
researchers and health promoters may struggle to appreciate fully why illicit drug users
behave as they do [02011].
Bodybuilders as gurus on anabolic steroids

The number of athletes self-administering ergogenic pharmacological agents to increase
their competitive edge continues to be a problem. Most athletes using anabolic steroids (AS)
have acquired a crude pharmacological database regarding these drugs. Their opinions
regarding steroids have been derived from their subjective experiences and anecdotal
information. For this reason, traditional warnings regarding the lack of efficacy and potential
dangers of steroid misuse are disregarded. A common widely held opinion among
bodybuilders is that the anabolic steroid experts are the athletic gurus who for years have
utilised themselves as the experimental participants and then dispensed their empirical
findings. One review will address the common anabolic steroid misconceptions held by many
of today's athletes by providing an evaluation of the scientific literature related to AS in
athletic performance [02012].
Moral disengagement and associated processes
One study investigated psychosocial processes associated with avoidance of health- and
morality-based deterrents to performance-enhancing drug (PED) use. In-depth semi-
structured interviews were conducted with 64 English male bodybuilders with experience of
doping. Resultant data were content analysed deductively using definitions for the eight
mechanisms of moral disengagement [Bandura A, 1991. Social cognitive theory of moral
thought and action], and three further themes from Boardley and Grix [2013. Doping in
bodybuilders: A qualitative investigation of facilitative psychosocial processes]. These
analyses evidenced six MD mechanisms, and all three of the themes from Boardley and Grix
[2013. Doping in bodybuilders: A qualitative investigation of facilitative psychosocial
processes]. Subsequent frequency analyses revealed six of the eight MD mechanisms, and
two of the three additional themes, were common across the sample. Overall, the findings
suggest MD may help athletes circumvent health- and morality-based deterrents to doping,
describe a process linking supplement and PED use and detail how some athletes may
actively avoid social censure for doping by only discussing PED use with other PED users
from within their training environment [14025].
Psychological background
Drugs and methods to improve physical performance among athletes have been used since
the beginning of sport history, but the use of performance enhancing drugs has not always
been regarded as cheating. In short, the motives for doping are improving and maintaining
physical functioning, coping with the social/psychological pressures and striving for social
and psychological goals, including economic benefits. Factors such as, "doping dilemma",
"win at all costs", cost versus benefit, and the specificity of some specific doping agents, also
play major roles. It seems that action on the athletes' attitude about the achievement of
physical improvement and creating effective methods to reveal the drug abuse, are two main
ways in winning the struggle against doping [09005].
One of the major justifications for the ban on the use of performance-enhancing drugs in
sport has been that relating to the protection of the health of athletes. One paper subjected
this argument to critical analysis by putting it in the context of the broader relationship
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between sport and health. More particularly, the paper seeked to unravel some of the
complexities of this relationship by an examination of some aspects of sports sponsorship,
particularly with alcohol and tobacco companies; (the health risks associated with elite level
sport; and the widespread and legal use within the sporting context of drugs that can have
dangerous side effects. The paper concluded with an examination of some aspects of anti-
doping policies within sport and it is suggested that a more imaginative approach to athlete
education is needed to prevent the misuse of drugs [09006].
A total of 40 talented male and female athletes (mean average age 20 years) from 13
different sports attended 12 focus groups held over the UK intended to investigate athletes'
attitudes toward doping. Athletes in general did not report a significant national doping
problem in their sport, but exhibited sporting xenophobia with regard to both doping practices
and the stringency of testing procedures outside of the UK. Athletes often viewed doping as
“unnatural” and considered the shame associated with doping to be a significant deterrent.
Athletes perceived no external pressure to use performance enhancing drugs. In response to
hypothetical questions, however, various factors were acknowledged as potential pressure
points: most notably injury recovery and the economic pressures of elite sport. Finally, a
significant minority of athletes entertained the possibility of taking a banned hypothetical
performance enhancing drug under conditions of guaranteed success and undetectability. It
was concluded that the athletes in this study generally embraced those values promoted in
anti-doping educational programmes, although there were some notable exceptions. That the
social emotion of shame was considered a significant deterrent suggests anti-doping efforts
that cultivate a shared sense of responsibility to remain “clean” and emphasise the social
sanctions associated with being deemed a 'drugs cheat', resonate with this atypical social
group [10013].
Athletes' interpersonal antisocial behaviour

The link between fear of failure and students' antisocial behaviour has received scant
research attention despite associations between fear of failure, hostility, and aggression.
Also, the effect of sport experience on antisocial behaviour has not been considered outside
of the sport context in adult populations. Further, to date, gender differences have not been
considered in fear of failure research. To examine whether fear of failure and sport
experience predict antisocial behaviour in the university and sport contexts in student
athletes, and whether this prediction is the same in males and females; and gender
differences exist in antisocial behaviour and fear of failure British university student athletes
(n=176 male; n=155 female; median age 20 year) completed questionnaires assessing fear
of failure, sport experience, and antisocial behaviour in both contexts. Fear of failure and
sport experience positively predicted antisocial behaviour in university and sport and the
strength of these predictions did not differ between males and females; females reported
higher levels of fear of devaluing one's self-estimate than males whereas males reported
higher levels of fear of important others losing interest than females. Males engaged more
frequently than females in antisocial behaviour in both contexts. It was concluded that fear of
failure and sport experience may be important considerations when trying to understand
antisocial behaviour in student athletes in education and sport; moreover, the potential effect
of overall fear of failure and of sport experience on this frequency does not differ by sex. The
findings make an important contribution to the fear of failure and morality literatures [11421].
An analysis from the athletes' perspective

Doping has developed into a widespread problem in competitive and high-performance
sports due to increasing professionalism in, and commercialization of sports. In contrast,
governments and sports organizations have limited financial resources to support all
competitive sports. Therefore, further improvement of anti-doping measures can only be
achieved through the inclusion and active participation of the athletes themselves. In one
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study, 101 German athletes who are subject to national and international anti-doping tests
were asked if doping in sports should be combatted, and which anti-doping measures
appeared effective from an athlete's perspective. Ninety-eight point zero two per cent of
those questioned felt that measures should be taken against doping in sports. Improved
methods of detection and more information on the health risks were favored, as opposed to
more severe punishments. In addition, more than two thirds of the athletes supported the
introduction of an anti-doping law. The desire for more frequent drug testing was also
expressed, despite the distinct invasion of the athletes' privacy. It was concluded that an anti-
doping law, as requested by the athletes, should include measures for educating the public
about the health risks involved with doping. In addition, such a law would also make it
possible to develop suitable methods of detection [02013].
The performance enhancement attitude scale
The aim of one study was to cross-culturally adapt and validate the Spanish version of the
Performance Enhancement Attitude Scale (PEAS). A cross-sectional multi-sample survey
with 17 independent datasets was carried out. Cross-cultural adaptation of the PEAS into
Spanish was conducted through forward/backward translations, consensus panels and
comparative analyses of known-groups to establish evidence for its reliability and validity.
Weighted Kappa coefficients with quadratic weighting were used to assess the reliability of
each item, with Cronbach's internal consistency coefficients for overall scale's reliability and
Spearman's correlation coefficient for test-retest reliability over a one-week period.
Confirmatory factor analysis (CFA) was performed to assess the scale's structure.
Differences between self-admitted doping users and non-users were analysed to verify the
PEAS' construct validity in 8 datasets. Spearman's correlation coefficient was also used to
assess the relationships between the PEAS and self-esteem, self-efficacy and perceived
descriptive norm to establish convergent validity. The scale showed satisfactory levels of
internal consistency, reliability of each item and temporal stability. CFA showed acceptable fit
for all but one samples. As expected, self-admitted doping users showed more positive
attitude toward doping than non-users. Significant and strong negative relationship was
found between PEAS and self-efficacy; weak negative correlation with self-esteem and and
positive correlation with perceived descriptive norm. The Spanish version of PEAS showed
satisfactory psychometric properties. Considerations for application and improvement are
outlined. Key pointsFirst study that crosses culturally adapted the PEAS to the Spanish
language.The Spanish version of PEAS has satisfactory psychometric properties.Users
scored higher than non-users indicating a satisfactory construct validity. Significant positive
correlation was found between PEAS and projected use.Significant negative correlation
between PEAS and self-esteem and self-efficacy [14443].
Cerebral correlates of automatic associations of performance enhancing substances
The direct assessment of explicit attitudes toward performance enhancing substances, for
example Neuroenhancement or doping in sports, can be affected by social desirability biases
and cheating attempts. According to Dual Process Theories of cognition, indirect measures
like the Implicit Association Test (IAT) measure automatic associations toward a topic (as
opposed to explicit attitudes measured by self-report measures). Such automatic
associations are thought to occur rapidly and to evade voluntary control. However, whether
or not such indirect tests actually reflect automatic associations is difficult to validate.
Electroencephalography (EEG) has a superior time resolution which can differentiate
between highly automatic compared to more elaborate processing stages. We therefore
used EEG to examine on which processing stages cortical differences between negative or
positive attitudes to doping occur, and whether or not these differences can be related to
517
BIAT scores. It was tested 42 university students (31 females, 24 years old), who were
requested to complete a brief doping IAT (BIAT) on attitudes toward doping. Cerebral activity
during doping BIAT completion was assessed using high-density EEG. Behaviorally,
participants D-scores exhibited negative attitudes toward doping, represented by faster
reaction times in the doping + dislike pairing task. Event-related potentials (ERPs) revealed
earliest effects between 200 and 300 ms. Here, a relatively larger occipital positivity was
found for the doping + dislike pairing task. Further, in the LPP time range between 400 and
600 ms a larger late positive potential was found for the doping + dislike pairing task over
central regions. These LPP amplitude differences were successfully predicting participants'
BIAT D-scores. Results indicate that event-related potentials differentiate between positive
and negative doping attitudes at stages of mid-latency. However, it seems that IAT scores
can be predicted only by the later occurring LPP. The study is the first to investigate the
cerebral correlates that contribute to test scores obtained in the indirect testing of automatic
associations toward doping. The implications of our results for the broader NE concept are
discussed in light of the conceptual similarity of doping and NE [150051].
Athletes confessions on doping
Commercialization of emotions is not a new phenomenon but in Denmark there is a new

general trend to tell and sell personal stories in the media. Personal deprivation and crises
are also major topics in sports media. One paper focused on sports biographies as a book
genre that is reviving in popularity. The paper approaches the topic through the biographies
of one Danish athlete: the former professional cyclist, Jesper Skibby, who writes about his
doping disclosure and shares his personal dilemmas as a former elite sportsman. The
thematic text analysis orientates around social interactions, emotions, and personality
constructions. Inspired by microsociology with a Durkheimian flavor of Goffman and
Hochschild, themes including "face work," "interaction rituals," and "emotions management"
are discussed. The analysis claims that sharing personal information in the media is not only
a means of confession and reclaiming status but is also business and management - on an
intimate level. Telling the story of the corrosion of a sporting character has become a hot
issue, an entertainment, and not least a commercial commitment [14004].
Reporting doping: national level athletes' perceptions

One paper qualitatively explored national level athletes' willingness to report doping in sport.
Following ethical approval, semi-structured interviews were conducted with nine national
level athletes from rugby league (n=5) and track and field athletics (n=4). Thematic analysis
established the main themes within the data. Contextual differences existed around the role
that athletes perceived they would play if they became aware of doping. Specifically, track
and field athletes would adopt the role of a whistle-blower and report individuals who were
doping in their sport. In comparison, the rugby league players highlighted a moral dilemma.
Despite disagreeing with their teammates' actions, the players would adhere to a code of
silence and refrain from reporting doping. Taking these findings into account, prevention
programs might focus on changing broader group and community norms around doping. In
doing so, community members' receptivity to prevention messages may increase. Moreover,
developing skills to intervene (e.g. speaking out against social norms that support doping
behavior) or increasing awareness of reporting lines could enhance community responsibility
for doping prevention. In sum, the findings highlight the need to consider the context of sport
and emphasize that a one-size-fits-all approach to anti-doping is problematic [14931].
Word-of-mouth testimony
Anecdotal evidence suggests the widespread usage of anabolic steroids among athletes (20-
90 %), particularly at the professional and elite amateur levels. In contrast, scientific studies
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indicate that usage is rare and no higher than 6 percent. Conclusions from scientific studies
suggest that anabolic steroid usage declines progressively from high school to college and
beyond; however, anecdotal evidence claims the opposite trend. In this clash between "hard"
scientific data versus "soft" anecdotal information, it is natural that professionals would
gravitate toward scientifically based conclusions. However, in the case of anabolic steroids (a
stigmatized and illegal substance), should word-of-mouth testimony from individuals closest
to the issues – those who have participated in and coached sports, those who have served
as drug-testing overseers, and journalists who relentlessly track leads and verify sources –
be set aside as irrelevant? Not if a complete picture is to emerge. In this review, hard
scientific evidence is placed on the table side-by-side with soft anecdotal evidence, without
weighting or bias. The purpose is to allow the opportunity for each to illuminate the other and,
in so doing, potentially bring us a step closer to determining the true extent of anabolic
steroid usage in athletics [04023].
Elite amateur cyclists' perspectives on drug use and professional cycling
Sports doping is condemned by sports authorities and by society at large. Cycling has a
particularly infamous relationship with sports doping, especially at professional level. Using
interviews with elite amateur cyclists, this paper examines how cyclists close to, but not
within, the ranks of professional cycling perceive the relationship between cycling and
doping. Eleven elite amateur cyclists from Melbourne were interviewed with regards to their
experiences as cyclists, use of training technologies, supplements and other substances,
and their attitudes to doping in sport, especially cycling. Interviewees described how their
training schedule is extremely demanding and frequently necessitates the use of substances
such as caffeine, anti-inflammatory medications, and energy boosters. Some distanced
themselves and their use of supplements and substances from doping and condemned such
practices as unethical and objectionable. Others appeared to empathise with professional
cyclists' use of doping substances given that they rely on cycling for their income and made
comparisons between doping and their own licit (not WADA-prohibited) substance use. It
was concluded that the perception of professional cycling as a sport intimately tied to drug
taking places those nearest to professional cycling into a practical and moral predicament.
The interviews suggest that while elite amateur cyclists do not appear supportive of drug
deregulation in sport they are not necessarily fully supportive of current anti-doping policy
[150028].
Subjective effects
The use of anabolic androgenic steroids (AAS) to increase muscle size and strength is
widespread. Information regarding self-administered AAS used nonmedically to enhance
athletic performance or improve physical appearance is sparse and poorly documented. The
purpose of one study was to identify current trends in the drug-taking habits of AAS users.
An anonymous self-administered questionnaire was posted on the message boards of
Internet Web sites popular among AAS users. Of the 500 AAS users who participated in the
survey, 78 percent (392/500) were noncompetitive bodybuilders and nonathletes; 60 percent
(298/500) of the respondents reported using at least 1000 mg of testosterone or its
equivalent per week. The majority (99 %) of AAS users (496/500) self-administer injectable
AAS formulations, and up to 13 percent (65/500) report unsafe injection practices such as
reusing needles, sharing needles, and sharing multidose vials. In addition to using AAS, 25%
of users admitted to the adjuvant use of growth hormone and insulin for anabolic effect, and
99 percent (496/500) of users reported subjective side effects from AAS use. The survey
reveals several trends in the nonmedical use of AAS. Nearly four out of five AAS users are
nonathletes who take these drugs for cosmetic reasons. AAS users in this sample are taking
519
larger doses than previously recorded, with more than half of the respondents using a weekly
AAS dose in excess of 1000 mg. The majority of steroid users self-administer AAS by
intramuscular injection, and approximately 1 in 10 users report hazardous injection
techniques. Polypharmacy is practiced by more than 95 percent of AAS users, with one in
four users taking growth hormone and insulin. Nearly 100% of AAS users reported subjective
side effects [06020].
Expectation of the doped
Long-term use of anabolic-androgenic steroids (AASs) is associated with both positive and
negative effects. The authors examined possible mechanisms by which these effects
contribute to AAS satisfaction and predict intentions for future AAS use. Five hundred male
AAS users completed an interactive Web-based instrument assessing the psychological and
physical effects of AAS use. Covariance structure modeling was used to evaluate both direct
and indirect effects of AAS consequences on satisfaction with AASs and intentions for future
AAS use. Results suggest that gain in muscle mass and psychological benefits from AAS
use uniquely contributed to both AAS satisfaction and intentions for future use. Side effects
from AAS use also uniquely contributed to AAS satisfaction, but ancillary drug use was found
to partially mediate this relationship, suggesting that the satisfaction of experienced AAS
users is enhanced by their mastery of side effects through the use of ancillary drugs. The
final model explained 29 percent of the variance in intentions for future AAS use.
Mechanisms for sustained AAS use and implications for intervention and prevention
strategies are discussed [06013].
Muscle dissatisfaction in young adult men

Appearance concerns are of increasing importance in young men's lives. It was investigated
whether muscle dissatisfaction is associated with psychological symptoms, dietary
supplement or anabolic steroid use, or physical activity in young men. As a part of a
questionnaire assessment of health-related behaviors in the population-based FinnTwin16
study, we assessed factors associated with muscle dissatisfaction in 1245 men aged 22-27
using logistic regression models. Of men, 30 percent experienced high muscle
dissatisfaction, while 12 percent used supplements/steroids. Of highly muscle-dissatisfied
men, 22 percent used supplements/steroids. Mean body mass index, waist circumference, or
leisure aerobic activity index did not differ between individuals with high/low muscle
dissatisfaction. Muscle dissatisfaction was significantly associated with a psychological and
psychosomatic problems, alcohol and drug use, lower height satisfaction, sedentary lifestyle,
poor subjective physical fitness, and lower life satisfaction. It was concluded that muscle
dissatisfaction and supplement/steroid use are relatively common, and are associated with
psychological distress and markers of sedentary lifestyle [06014].
Beliefs about the causes of success in sports and susceptibility for doping
One study set out to assess the impact of attributional beliefs about success on the
susceptibility for doping use in adolescent athletes. The sample consisted of 309
adolescent athletes participating in both team and individual sports. Participants
completed a battery of questionnaires including Beliefs about the Causes of Success
in Sport Questionnaire (BACSSQ), current and past doping use, and measures of
attitudes, norms, situational temptation and social desirability. Variance reduction
rate analysis revealed that social desirability did not act as a confounder in the
relationship between doping susceptibility and its predictors. With regard to beliefs
about the causes of success dimensions, only deception emerged as a significant
predictor of doping use susceptibility over and above the effects of well-established
520
social-cognitive predictors of doping intentions and use. These findings imply that
beliefs about the causes of success in youth sports may comprise another dimension
of risk factors for doping susceptibility and use [13051].
Rejuvenation
A search for a hormonal fountain of youth has been hotly pursued over the last century,
predominately by those who wish to market hormones to a gullible public. There is little or no
benefit of hormone replacement in persons who do not have a hormone deficiency. Overall,
the present state of the art suggests that the findings have been disappointing. In persons
who fail to get adequate sunlight, and therefore have low vitamin D levels, vitamin D
replacement appears to have positive effects, including decreasing mortality. Testosterone in
hypogonadal males has a number of positive effects such as improving libido and erectile
capacity, increasing strength and bone mineral density, and perhaps having a small effect on
cognition. These effects need to be balanced against long-term side effects, the evidence for
which studies are lacking. There is little evidence to recommend DHEA, pregnenolone,
growth hormone, ghrelin, or melatonin to older persons. Overall, exercise, adequate
exposure to sunlight, and adequate dietary protein appear to have at least as positive an
effect as any of the hormones being used to rejuvenate older persons [13052].
Interaction between athletes and coaches
The sport nutrition and doping are known to be important issues in sports, but there is
evident lack of studies which investigated those issues in swimming, especially with regard to
parallel analysis of coaches and athletes. The first aim of one study was to compare
knowledge of swimming coaches and their athletes about nutrition and doping. Also, it was
identified interrelationships between studied sociodemographic-, sport-; nutrition- and
doping-related-factors. The sample of subjects comprised 55 athletes (20 years of age; 24
females) and 22 coaches (mean age 37 years; 4 females) from Croatia (98 % of
respondents). In the first phase of the investigation we have validated specific questionnaires
to determine the knowledge of sport nutrition (KSN), and knowledge on doping (KD). The
test-retest correlation and percentage of equally responded queries revealed both
questionnaires as reliable. The discriminative validity was proven also since coaches scored
better than their athletes on both questionnaires. Athletes declared their coaches as the
primary sources of knowledge about nutrition and doping. Among coaches, formal and self-
education are equally important sources of information about doping and nutrition. The age is
negatively, while the formal education is positively correlated to KD and KSN scores among
coaches. Consequently, permanent educational programs about nutrition and doping are
emphasized, especially among older coaches and younger athletes [13053].
Athlete support personnel (ASP) failing to meet responsibilities under the World Anti-Doping
Code risk sanction. It is unclear whether the poor knowledge of responsibilities seen in sports
physicians and coaches applies to other ASP (e.g. administrators, chiropractors, family,
nutritionists, physiotherapists, psychologists, and trainers). A purposive sample of Australian
ASP (n=292) responded to a survey on knowledge of anti-doping rules (35 true/false
questions), ethical beliefs and practice, and attitudes toward performance enhancement.
Some ASP declined to participate, claiming doping was irrelevant to their practice.
Physicians were most knowledgeable (31/35), with family and trainers the least (26/35). ASP
reported that improvements were needed to support anti-doping education (e.g., basis for
anti-doping) and practice (e.g. rules). ASP also had a slightly negative attitude toward
performance enhancement. Linear regression showed that being a sports physician,
providing support at the elite level, and 15 years of experience influenced knowledge. The
521
results confirm gaps in knowledge, suggesting that stronger engagement with ASP anti-
doping education and practice is needed. Applying the principles of andragogy could help
foster active engagement through emphasis on active inquiry, rather than passive reception
of content [13054].
The purpose of one study was to examine whether the relationships between contextual
factors (i.e. autonomy-supportive vs. controlling coaching style) and person factors (i.e.
autonomous vs. controlled motivation) outlined in self-determination theory (SDT) were
related to prosocial and antisocial behaviors in sport. We also investigated moral
disengagement as a mediator of these relationships. Athletes' (n=292, median 19 years)
responses largely supported our SDT-derived hypotheses. Results indicated that an
autonomy-supportive coaching style was associated with prosocial behavior toward
teammates; this relationship was mediated by autonomous motivation. Controlled motivation
was associated with antisocial behavior toward teammates and antisocial behavior toward
opponents, and these two relationships were mediated by moral disengagement. The results
provide support for research investigating the effect of autonomy-supportive coaching
interventions on athletes' prosocial and antisocial behavior [11419].
Attitudes of coaches towards doping

Coaches are usually held to be among the main actors of doping prevention campaigns. The
aim of one study was to document certain attitudes of professional coaches faced with
doping, and to evaluate how they confronted it on an everyday basis in a prospective study
by self-reporting questionnaire. The questionnaire was mailed to the last 800 graduated
coaches (1994-1997) in the Lorraine region, Eastern France. The 260 responding coaches
comprised 77 women and 183 men, the average age being 31 + 8 years. Ten percent of
coaches consider that an athlete may use doping with no health hazard with the help of a
physician, and 30.0% that an athlete who declines doping has little chance of succeeding.
Six percent had used doping drugs in the last twelve months (1 to 6 times). Fourteen percent
of coaches mention that athletes (1 to 5 per coach on average) told them they had been
prompted to use doping drugs during the previous 12 months. Eighty-one percent consider
that the current methods of preventing doping in sport are ineffective, and 98 percent of them
consider that they have a role to play within this context, but 80 percent consider themselves
badly trained in the prevention of doping. Only 10 percent have organized a doping
prevention action during the last 12 months. In this study, professional coaches do not seem
to be efficient in the prevention of doping. Further education and training for coaches on
doping is advisable [01009].
Sports addiction
Socially valorised, sport like other forms of behaviour, can take on an addictive aspect. A
review of the English and French literatures from 1979 to 2012 was conducted, using
PubMed, Google Scholar, EMBASE, and PsycInfo, using the following key words alone or
combined :sport, dependence, exercise, addiction. Exercise dependence is defined as a
craving for physical activity that leads to extreme exercise intensity and generates
physiological and psychological symptoms. Measurement scales have been proposed to
make the diagnosis. No epidemiological studies have examined the prevalence of exercise
dependence in the general population, although some studies suggest a frequency ranging
from 10 to 80 percent. Disorders begin with a search for pleasure in physical effort, which
then gives way to an obsession for sport resulting in a need to practice a sport more and
more frequently and intensely. This addiction is more common among alcohol and illicit drug
addicts than among the general population, while the rate of eating disorders can reach 40
percent. Personality traits most often associated are perfectionism, extraversion, and
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sensation seeking, while possible links between sporting activity and intensive doping will be
discussed [13039].
Attitudes towards doping with regard to achievement goals
Understanding athletes' attitudes to doping continues to be of interest for its potential to

contribute to an international anti-doping system. However, little is known about the
relationship between elite athletes' attitudes to drug use and potential explanatory factors,
including achievement goals and the motivational climate. In addition, despite specific World
Anti-Doping Agency Code relating to team sport athletes, little is known about whether sport
type (team or individual) is a risk or protective factor in relation to doping. Elite athletes from
Scotland (n=177) completed a survey examining attitudes to performance-enhancing drug
(PED) use, achievement goal orientations and perceived motivational climate. Athletes were
generally against doping for performance enhancement. Hierarchical regression analysis
revealed that task and ego goals and mastery motivational climate were predictors of
attitudes to PED use. Compared with individual athletes, team athletes were significantly
lower in attitude to PED use and ego orientation scores and significantly higher in
perceptions of a mastery motivational climate. The study provides insight into how individual
and situational factors may act as protective and risk factors in doping in sport [14719].
There is a growing body of empirical evidence on demographic and psychosocial predictors

of doping intentions and behaviors utilizing a variety of variables and conceptual models.
However, to date there has been no attempt to quantitatively synthesize the available
evidence and identify the strongest predictors of doping. Using meta-analysis, it was aimed
to
- determine effect sizes of psychological (e.g. attitudes) and social-contextual factors

(e.g. social norms), and demographic (e.g. gender and age) variables on doping
intentions and use
- examine variables that moderate such effect sizes
- test a path analysis model, using the meta-analyzed effect sizes, based on variables
from the theory of planned behavior (TPB).
Articles were identified from online databases, by contacting experts in the field, and
searching the World Anti-Doping Agency website. Studies that measured doping behaviors
and/or doping intentions, and at least one other demographic, psychological, or social-
contextual variable were included. It was identified 63 independent datasets. Study
information was extracted by using predefined data fields and taking into account study
quality indicators. A random effects meta-analysis was carried out, correcting for sampling
and measurement error, and identifying moderator variables. Path analysis was conducted
on a subset of studies that utilized the TPB. Use of legal supplements, perceived social
norms, and positive attitudes towards doping were the strongest positive correlates of
doping intentions and behaviors. In contrast, morality and self-efficacy to refrain from
doping had the strongest negative association with doping intentions and behaviors.
Furthermore, path analysis suggested that attitudes, perceived norms, and self-efficacy to
refrain from doping predicted intentions to dope and, indirectly, doping behaviors. In the
review it was identifies a number of important correlates of doping intention and behavior,
many of which were measured via self-reports and were drawn from an extended TPB
framework. Future research might benefit from embracing other conceptual models of
doping behavior and adopting experimental methodologies that will test some of the
523
identified correlates in an effort to develop targeted anti-doping policies and programs
[14720].
Motivational and social cognitive predictors of doping
Doping use is an important issue in both competitive and non-competitive sports, and poses
potentially irreversible health consequences to users. Scholars increasingly call for theory-
driven studies on the psychosocial processes underlying doping use that will inform
subsequent policy-making and prevention interventions. The aim of the study was to
implement an integrative theoretical model to assess the direct and indirect effects of
motivational variables, moral orientations, and social cognitions on doping intentions. A
randomly selected and representative sample of 750 elite athletes anonymously completed a
battery of questionnaires on motivational and moral constructs, and social cognitions related
to doping. Hierarchical linear regression analysis and multiple mediation modeling were
used. The effects of achievement goals and moral orientations were significantly mediated by
attitudinal, normative, and self-efficacy beliefs, in both lifetime ever and never doping users.
Moral orientations indirectly predicted the doping intentions of never users, but did not predict
ever users' doping intentions. Achievement goals and sportspersonship orientations
influence doping intentions indirectly, through the effects of attitudes and self-efficacy beliefs.
Sportspersonship (moral) orientations were relevant to doping intentions among athletes with
no prior experiences with doping, while achievement goals and situational temptation were
relevant to both lifetime never and ever dopers [13040].
Life style issues

One study examined drug use patterns and perceptions of drug intervention programs
among adolescent interscholastic athletes and nonathletes. In particular, it explored the issue
of whether participation in high school athletics is related to a healthier lifestyle and
decreased use of recreational drugs and ergogenic aids. One thousand five hundred fifteen
Massachusetts high school students completed a 150-item survey that assessed illicit and
nonillicit substance use. Chi-square analyses revealed that athletes were significantly less
likely to use cocaine and psychedelics, and were less likely to smoke cigarettes, compared
with nonathletes. Conversely, nonathletes were less likely to use creatine than were athletes.
There was no difference in the use of anabolic steroids and androstenedione between
athletes and nonathletes. Descriptive analyses appear to indicate that drug interventions for
athletes are falling short of their objectives. This study suggests that athletes have a healthier
lifestyle and that the efficacy of intervention programs must be further examined [01008].
Italy
One study aimed to identify the main psychological and social correlates of doping attitudes
among Italian athletes. It is well recognized that athlete disposition and attitude towards
doping is one of the factors responsible for doping behavior. Less is known, however, about
the factors that sustain the level of athletes' attitudes towards doping. The main
psychological (i.e. perfectionism, sport motivation, self-confidence and life satisfaction) and
social correlates (i.e. social network and contact with people who use sports drugs) of
attitudes towards doping among Italian athletes are examined in this paper. Differences are
hypothesized regarding the type of sport (resistance sport vs. non-resistance sport) and
athlete participation in competitive sport (i.e. agonistics) or in non-competitive sport
(i.e.,amateurs) on the level of attitude towards doping. The research hypothesis is that each
of these constructs affects the level of athletes' attitudes toward doping. Data were collected
from a sample of athletes (n=109), aged from 15 to 45 (mean 32) recruited in a Sports
Medicine Center. Socio-demographic information, attitude towards doping, psychological and
social variables were assessed through self-report questionnaire. Hierarchical multiple
524
regression showed that both psychological (i.e. extrinsic motivation, perfectionism) and social
variables (i.e. athletes' contact with doping users) were associated with athletes' attitudes
towards doping. The results highlighted that athletes with excessive perfectionism,
extrinsically motivated and who have contact with doping users have a positive attitude
toward doping. Athletes who exhibit these characteristics should be considered at risk and
monitored to prevent possible future sports drug use [14625].
Australia
One study presents a comprehensive examination of the Sport Drug Control Model via
survey data of elite Australian athletes through a cross-sectional nationwide mail survey to
1237 elite Australian athletes. Morality (personal moral stance on performance-enhancing
substances use), reference group opinion (perceived moral stance of reference group on
performance-enhancing substances use) and legitimacy (perceptions of the drug testing and
appeals processes) evidenced significant relationships with attitude towards performance-
enhancing substances use, which in turn was positively associated with doping behaviour.
The model accounted for 81 percent and 13 percent of the variance in attitude towards
performance-enhancing substances use and doping behaviour, respectively. These findings
validate the usefulness of the Sport Drug Control Model for understanding influences on
performance-enhancing substances use. Nevertheless, there is a need to survey athletes
representing a broader range of competition levels and cross-cultural research to test the
model's applicability to other populations of athletes [14721].
Chaperons
Athlete support personnel (ASP) failing to meet responsibilities under the World Anti-Doping
Code risk sanction. It is unclear whether the poor knowledge of responsibilities seen in
sports physicians and coaches applies to other ASP (e.g. administrators, chiropractors,
family, nutritionists, physiotherapists, psychologists, and trainers). A purposive sample of
Australian ASP (n=292) responded to a survey on knowledge of anti-doping rules (35
true/false questions), ethical beliefs and practice, and attitudes toward performance
enhancement. Some ASP declined to participate, claiming doping was irrelevant to their
practice. Physicians were most knowledgeable (30.8/35), with family and trainers the least
(26.0/35). ASP reported that improvements were needed to support anti-doping education
(e.g., basis for anti-doping) and practice (e.g. rules). ASP also had a slightly negative
attitude toward performance enhancement. Linear regression showed that being a sports
physician, providing support at the elite level, and 15 years of experience influenced
knowledge. The results confirm gaps in knowledge, suggesting that stronger engagement
with ASP anti-doping education and practice is needed. Applying the principles of andragogy
could help foster active engagement through emphasis on active inquiry, rather than passive
reception of content. Future work on the context within which ASP experience anti-doping is
needed, exploring acquisition and translation of knowledge into practice [14722].
Attitudes of collegiate athletes toward doping

Doping in sport has become an increasingly prominent topic. The decision to take part in
doping practices is multifactorial and often based on many different information sources of
varying reliability. This study sought to determine the attitudes of athletes at a Canadian
Interuniversity Sport university toward doping and to discover if pharmacists are perceived to
be a valid information source on medication usage for these athletes. The university athletes
competing in at least 1 of 8 sports were asked to complete a questionnaire. Participants
were asked various questions regarding their perceptions of doping, medication use,
information available to them regarding doping and the role of pharmacists in providing
advice on medication usage. In total, 93 percent (307/331) of questionnaires were at least
525
partially completed. Generally, these athletes did not feel pressured to dope or that it was
prevalent or necessary. The fear of doping violations largely did not alter the use of
medications and supplements. The online doping education program administered by the
Canadian Centre for Ethics in Sport was the most used information source (75 %);
pharmacists were used 38 percent of the time. Pharmacists were perceived to be a good
source of information about banned substances by 76 percent (223/295) of participants,
although only 35 percent (104/297) consulted a pharmacist each time they purchased a
nonprescription medication. It thus appears that doping is neither prevalent nor worth the
risk for these university athletes. There also appears to be an opportunity for pharmacists to
play a more prominent role in providing advice on medication use to high-performance
athletes [14723].
Motivations for anabolic steroid use among bodybuilders

Steroid use is increasing, in parallel with rising concerns about body image. One study aimed
to uncover bodybuilders' motivations for using steroids using 135 questionnaires completed
by readers of two bodybuilding magazines. The analyses reveal a polarization of beliefs
about steroids between users and non-users. Steroid users were less likely to be concerned
about the physical side effects, and many believed that steroids are not harmful in
moderation, and that only “ignorant people” criticize steroid use. Their main motivations for
using steroids were: wanting to excel at competitive bodybuilding; wanting to be more
muscular; and feelings of enhanced confidence. The fact that steroid users in the sample
were 'stacking' dangerously high levels of steroids (up to 15 steroids at a time) reveals the
need for a detailed understanding of the motivations for steroid use in order to inform the
development of effective harm minimization messages [00011].
Willingness model to predict doping in sport

To enable preventive measures to be designed, it is important to identify modifiable distal
and proximal factors underlying doping behavior. This study investigated aspects of the
prototype willingness model in relation to doping. A cross-sectional study was conducted
involving 729 competitive athletes. Following ethical approval, athletes (mean
age 29 ± 10 years; 63% male) completed an online questionnaire, which assessed doping-
related attitudes, norms, prototype perceptions, outcome expectancies, and behavioral
willingness. Using hierarchical multiple regression analysis, 55 percent of the total variance
in willingness to dope was explained. Specifically, past doping, attitudes, and favorability of
performance enhancing substance user prototypes were the strongest unique predictors of
willingness to dope. Athletes appeared most willing to dope if they were to suffer an injury, a
dip in performance, or think others are doping and getting away with it. National-level
athletes displayed significantly greater willingness to dope and perceived themselves as
significantly more similar to a doper than athletes competing at any other level. The findings
highlight the importance of extending anti-doping provision beyond elite-level sport and the
need to target athletes' doping-related perceptions [14726].
One study was a preliminarily investigation into the prevention of unintentional doping on the
basis of self-determination theory (SDT). Specifically, we examined the relationship between
athletes' motives for doping avoidance and their behavior when offered an unfamiliar food
product. Participants were young Australian athletes (n=410) that were offered a free lollipop
prior to completing a questionnaire. It was noted whether participants refused to take or eat
the lollipop and whether they read the ingredients of the lollipop. The questionnaire assessed
autonomous and controlled forms of motivation, amotivation, doping intentions, and
526
adherence regarding doping avoidance behaviors. The results showed that young athletes
who adopted controlled reasons to avoid doping in sport (e.g. not getting caught) tended to
report higher adherence to behaviors related to avoiding and monitoring banned substances,
whereas those who adopted autonomous reasons (e.g. anti-doping being consistent with life
goals) appeared to be more willing to read the ingredients of the provided food. The
significant interaction effect between autonomous and controlled motivation indicated that
autonomous motivation was more predictive to doping intention for athletes with low
controlled motivation. It is concluded that SDT may help understand the motivational
processes of the prevention of unintentional doping in sport [14724].
Influence of parent-adolescent communication on willingness to try to dope

Performance-enhancing substances are used by adolescent athletes to help improve
performance. Anabolic steroids (AS) are performance-enhancing substances that pose
significant health problems when used by adolescents. Objectives were to examine the
extent to which parents and adolescents discuss AS and test whether parent-adolescent
communication about AS can generalize to, and influence, decisions to use other types of
performance-enhancing substances. Adolescent athletes (n=244) completed an anonymous
questionnaire that assessed the extent to which the adolescents discussed with their parents
the performance outcomes and protective factors associated with AS, their intentions to use
AS, and their willingness to try a newly developed, potentially illegal performance-enhancing
substance. Data were collected during 2009-2010. Adolescents reported relatively low levels
of communication with their parents about anabolic androgenic steroids (AAS).
Communication with parents about the performance outcomes associated with AS was a
positive predictor of willingness to try a newly developed performance-enhancing substance
and intentions to use AS. Communication with parents about protective factors predicted
willingness to try a new performance-enhancing substance, but not intentions. It was
concluded that parents should highlight the protective factors and avoid emphasizing the
performance outcomes associated with AS in discussions with their adolescents.
Discussions about AS may influence adolescents' decisions to use other types of
performance-enhancing substances [150032].
Knowledge and attitudes among parents of junior athletes

Strategies for doping prevention are based on prior identification of opportunities for
intervention. There is no current research focusing on the potential role in doping prevention,
which might be played by the parents of junior elite athletes. The purpose of one study was
to evaluate the knowledge and attitudes toward doping among parents of Austrian junior
athletes and to analyze factors potentially influencing these beliefs. In this study, two
questionnaires were distributed to 1818 student athletes, each with instructions that these
surveys were to be completed by their parents (n=3636). Parents filled in questionnaires at
home without observation. Responses from 883 parents were included in this analysis.
Compared to female parents, male parents demonstrated significantly better knowledge
about doping and its side effects and were more likely to be influenced by their own sporting
careers and amounts of sports activities per week. Parental sex did not demonstrate a
significant influence on responses reflecting attitudes toward doping. Additional research is
needed to compare these results with young athletes' knowledge and attitudes to determine
if and to what degree parental attitudes and beliefs influence the behavior and attitudes of
their children [150033].
Motives for use
Anabolic-androgenic steroid (AAS) use represents a major public health problem in the
United States, but the risk factors for this form of drug use are little studied. It was evaluated
527
48 men who had used AAS for at least 2 months and 45 men who had never used AAS,
using a verbal interview and a battery of questionnaires covering hypothesized demographic,
familial, and psychosocial risk factors for AAS use. All subjects in both groups were
experienced weightlifters; thus, differences between groups were likely to be associated
specifically with AAS use, rather than with weightlifting in general. The AAS users and non-
users generally described similar childhood and family experiences, but users reported
significantly poorer relationships with their fathers and greater childhood conduct disorder
than non-users. At the time that they first started lifting weights, AAS users and non-users
were similar in their perceived physical, social, and sexual status, but users were significantly
less confident about their body appearance. AAS users displayed much higher rates of other
illicit substance use, abuse, or dependence than non-users, with use of other illicit
substances almost always preceding first use of AAS. These findings suggest that AAS use
may be most likely to occur in men with high levels of antisocial traits and low levels of body
esteem [03018].
Results from large surveys sampling AAS users on Internet bodybuilding forums have
reported that the most common reason for beginning AAS use was to increase muscle mass
and decrease body fat. These users also reported feeling compelled to continue their
regimens for a fear of the withdrawal that would result in excessive hypogonadal symptoms
and the loss of muscle mass. It must be recognized that there are substantial variations in
types and amounts of AAS used. A subset of aggressive users may develop a dependence
syndrome of combined physiologic and psychologic etiology, subjecting themselves to long-
term or permanent endocrine dysfunction. Given these case reports as well as the known
AAS side effects, one might wonder whether AAS users experience regret over their decision
to use AAS. If these suspicions are correct, then determining the reasons for regret would be
a valuable tool in educating current patients previously on AAS who are seeking TRT for
hypogonadism. In stark contrast to the classic drug abusers, most AAS users show
considerable forethought in their illicit substance use. Users typically obtain all of the
necessary medications before beginning their self-determined “treatment” cycle and follow
calculated dosing schedules. AAS users are often hesitant to stop their regimens and often
present to physicians with requests for diagnostics or unwarranted therapies without the
intent of stopping illicit AAS use. It is also common for AAS users to want to cycle off all TRT,
feeling that this enhances their responsiveness and improves safety. As such, the treatment
of AAS users poses a unique challenge for physicians. It may be helpful to gauge the
patient's knowledge of AAS-associated complications while working to address
misconceptions that often stem from Internet forum trends and popular anecdotal evidence.
Central to this is the need for physicians to become more educated about the psychology
and pathophysiology underlying AAS use [14427].
Several factors that are unique to our current society may contribute to young people's using
drugs to succeed in sports. First is the message that is being sent by many sports idols
today. The steroid investigations in baseball and books by former players make it clear that
steroids and other drugs have played a part in many record-breaking performances.
However, the fame and the respect that still is garnered by these athletes sends the
message that ergogenic drugs are accepted, if not necessary, to reach such success. As
young athletes begin to model themselves after sport icons, heartbreaking stories are
beginning to unfold. One young athlete in a tear-filled confrontation with his father only
months before committing a now highly publicized suicide linked to his steroid use
confessed, “I'm on steroids, what do you think? Who do you think I am? I'm a baseball
player, baseball players take steroids. How do you think Bonds hits all his home runs? How
do you think all these guys do all this stuff? You think they do it from just working out
normal?”17 Second, society today places a huge emphasis on sports with collegiate football
stadiums seating nearly 100000 people and countless events featured on national television.
528
From high school basketball all-star games to the Little League World Series, our youths are
placed under the national spotlight at exceedingly young ages. With professional scouts now
following high school sports and collegiate coaches eyeing even younger talent, the pressure
to succeed is placed now on younger, more impressionable shoulders than ever before.
Finally, several economic factors encourage drug use to gain an edge in sports. One is the
resultant money and social stature that accompany athletic success when worthy of
garnering professional contracts. Another more subtle but increasingly wide-reaching
monetary influence is the rising cost of collegiate education. This has been shown to be a
self-reported factor for high school girls to use performance-enhancing drugs while
competing for prestigious and now very valuable athletic scholarships to college. Whether
they turn to ergogenic drugs for the competitive edge or as a means of keeping up with fellow
students who are already using these substances, it is clear that the pressure to do so is
significant today [06003].
The use of anabolic androgenic steroids (AAS) has been associated with the use of illegal
drugs. Earlier observations suggested that users of illegal drugs may use AAS for reasons
other than increasing muscle strength or size. The aim of one study was to investigate the
motives for AAS use among outpatients at a substance abuse center in Stockholm, Sweden.
All male patients under the age of 50 were asked whether they had used AAS during a 2-
month period. An AAS survey was administered to those who reported AAS experiences in
the admittance interview. Twenty of the 175 respondents (11 %) reported using AAS. The
most frequently reported motives were related to anabolic effects (i.e. for a good-looking
body, to become stronger, or to perform better in sports). However, some users reported
other motives; for example, to conceal concomitant drug use, to alleviate insecurity or low
self-esteem, to become brave, or in preparation of committing a crime. Furthermore, many
respondents reported side effects that were associated with AAS; most notably, irritability
and depression/suicidal ideation. It was concluded that some users of illicit drugs also use
AAS for reasons other than the anabolic properties of these compounds. Therefore,
considering that AAS may cause or contribute to diverse morbidity, it is important to ask
users of illicit drugs about AAS use, even when obvious external signs of AAS use are
lacking [10014].
One study presents an opportunistic examination of the theoretical tenets outlined in the
Australian Sport Drug Control Model using questionnaire items from a survey of 643 elite
Australian athletes. Items in the questionnaire that related to the concepts in the model were
identified and structural equation modelling was employed to test the hypothesised model.
Morality (cheating), benefit appraisal (performance), and threat appraisal (enforcement)
evidenced the strongest relationships with attitude to doping, which in turn was positively
associated with doping susceptibility. Self-esteem, perceptions of legitimacy and reference
group opinions showed small non-significant associations with attitude to doping. The
hypothesised model accounted for 30 percent and 11 percent of the variance in attitudes to
doping and doping susceptibility, respectively. These present findings provide support for the
model even though the questionnaire items were not constructed to specifically measure
concepts contained in it. Thus, the model appears useful for understanding influences on
doping. Nevertheless, there is a need to further explore individual and social factors that may
influence athletes' use of performance enhancing drugs [11003].
One study aimed to construct a hierarchy of motives linked to doping behaviors. Between
2000 and 2005, calls to a national antidoping phone-help service by 115 cyclists, 203
bodybuilders, and 40 footballers were analyzed. The results showed that the main motives
were preserving health for cyclists, increasing muscular strength for bodybuilders, and
personal recreation for footballers. However, in contrast to the literature, group influence was
low and health preoccupations were high for cyclists; the influence of body image was
529
relatively low for bodybuilders; and footballers cited muscular strength enhancement as a
motive. The study's limitations are noted. The prevention campaigns therefore need to be
specific [11004].
Anti-ageing and body building substances became widely accepted in the 21st century due
to modified social and economical attitudes. Such requirements are characteristic of a
visually oriented consumer society. The body is considered to be a marker of social prestige
and in the striving for a nicer appearance is becoming a part of šperaon’s identity. Medical
and pseudo-medical approaches have been developed to fulfill these desires. In the general
perception of a modern society such efforts are considered as positive, but some of such
procedures are potentially risky, while others seem to be not efficient [07013].
One paper addressed a gap in the literature of empirically derived models of performance
enhancing supplement use by developing a demographic and psychosocially based model of
athlete supplement use. Selected questions were used from a larger survey conducted by
UK Sport into British athletes' experiences, knowledge, attitudes and opinions in relation to
anti-doping activities. Forward conditional step wise logistic regression was employed on
data from 757 athletes to develop a model that discriminated supplement non-users from
users. The model identified that British athletes most likely to use supplements were younger
(under 23) males who were more likely to see doping as a problem in their sport and were
more knowledgeable about testing procedures than their non-user counterparts. Post hoc
analysis reinforced that non-users saw doping as less of a problem in their sport and were
less knowledgeable about drug testing procedures relative to those using supplements. The
pattern of results indicated gender-specific interventions on supplement use for young male
athletes may yield significant benefits. The relationship between supplement use and
perception of a doping problem suggested more work is needed to understand supplement
use culture within sports. The relationship between knowledge of testing procedures and
supplement use suggested further research is needed to see whether this is a positive or
negative effect of detection-based doping deterrence activity [07014].
The objective of this study was to use self-determination theory to analyze the relationships
of several motivational variables with exercise dependence. The study involved 531
exercisers, ranging in age from 16 to 60 years old, who responded to differentquestionnaires
assessing perception of motivational climate, satisfaction of basic psychological needs,
motivation types, and exercise dependence. The results of multiple mediation analysis
revealed that ego-involving climate and perceived competence positively predicted exercise
dependence in a directed and mediated manner through introjected and external regulation.
Gender and age did not moderate the analyzed relationships. These results allow us to
better understand the motivational process explaining exercise dependence, demonstrating
the negative influence of the ego-involving climate in the context of exercise [12020].
Stress as a reason for doping

Athletes may turn to substances to cope with numerous stressors, including pressure to
perform, injuries, physical pain, and retirement from a life of sport (which happens much
earlier than retirement from most other careers). Additionally, athletes may be significantly
less likely to receive treatment for underlying mental illnesses such as depression. Athletes
receive comprehensive treatment and rehabilitation for physical injuries, but this may be less
often the case for mental illness, because of their sometimes viewing mental illness as a sign
of weakness. Untreated mental illness is often associated with substance use, perhaps in an
effort to self-treat. Alternatively, substances of abuse may cause mental illness [14612].
Specificity of motives for doping

Most of the data to rank doping motives have been gathered from epidemiological with the
530
use of questionnaires. However, questionnaires are known to systematically underestimate
the reality (regarding the nature and relative proportion of motives) of addictive behaviors
because of common social representations of doping Qualitative studies based on
retrospective interviews or information from champions’ confessions tend to emphasize
extrinsic reasons for doping and thereby limit personal responsibility and protect self-esteem.
Moreover, the “true story” may be modified to limit the risks of sanction. Whatever the
protocol design, researchers are confronted with the approach to doping in sport, in which in
which it is never attmitted to any substance abuse. Furthermore, the motives for using
prohibited substances seem to concern a minority of athletes: generally those of a high level
and those who have tested positive in doping controls. The motives offered by these
athletes, such as enhancing performance, increasing financial gain, and making a sporting
name for one-self, are associated with common social representations. The studies to
determine and rank the motives for secretive doping behaviors have met with methodological
difficulties. Few sporting figures have a personal interest in breaking the secrecy of doping,
not individual athletes or teammates, staff or federation members, journalists, or sponsors.
Deliberately damaging the popular myth of athletic perfection is anathema to virtually all
members of the world sporting community. Thus, the power of common social
representations limits access to the real thoughts and behaviors concerning doping.
Researchers can obtain cold cognitions, with memory biases (deliberate or not),
reconstructions to protect self-esteem, and external causal attributions. But classical
interviews with athletes who have tested positive cannot deliver “the whole story.” These
athletes are under intense social pressure and at risk of condemnation; media scrutiny has a
powerful impact, and any inquiry is retrospective, with responses subject to deformation and
omission. Doping thus remains a taboo, and the biases inherent to standard retrospective
studies are too great to obtain a model with quantitative ranking of doping motives in
athletes. Moreover, many studies have focused their investigations on adolescents,
sportspersons of a low level (local, regional), or retired elite athletes. Nevertheless, the
evidence shows that the risk of doping is highest among men, young adults (20-25 years
old), and high-level practitioners [10303].
One study aimed to construct a hierarchy of motives linked to doping behaviors. Between
2000 and 2005, calls to a national antidoping phone-help service by 115 cyclists, 203
bodybuilders, and 40 footballers were analyzed. The results showed that the main motives
were preserving health for cyclists, increasing muscular strength for bodybuilders, and
personal recreation for footballers. The cyclists first cited health concerns to justify the use of
several illicit substances (rank 1). They reported trying to reduce health problems associated
with their sport. Other motives in the same vein were limiting pain during effort (rank 6),
reducing fatigue (rank 7), reducing pain in general (rank 10), treating acute injury (rank 14),
and enhancing fatigue recovery (rank 14). It was thus logical that glucocorticosteroids were
the most cited substance (35 %). Therefore, the priority of the cyclists appeared to be a
strategy to preserve fitness and prevent injuries and symptoms due to intensive training.
Their health concerns were due to the training intensity and sensitivity to pain and fatigue.
Associated with this motive, it was also observed that sport doctors were a real support in
justifying consumption (rank 4). Cyclists have been shown to be predisposed to collaborate
with team sport doctors to treat and medicate for any health concerns that may affect their
performance. It is also interesting to note that concern for health was the argument put forth
by the east Germans to defend their use of illegal substances: They argued that these
substances were used not as performance enhancers but as performance enablers. From
their viewpoint, these substances were required to maintain homeostasis during heavy and
prolonged training. This conception (i.e. medical use of substances to maintain health) is also
a current topic in American horse racing, another example that emphasizes the complexity of
understanding the “doping” activity. The second motive was performance enhancement (rank
2). The third motive was the social norms associated with the sport. The cycling subculture
531
has a powerful impact on its members. Cycling is ruled by implicit principles that create high
team cohesion. Doping is one of the principles of this cohesion, but no one refers to it. The
risks are minimized, considered as banal, so that they become normal. The first motive for
the bodybuilders was strength enhancement. Thus, the frequent phone discussions about
steroids were unsurprising (78 %), as noted by several researchers and as it has been
shown that 38 percent of amateur bodybuilders used anabolic steroids, but body image
disturbance was not the main reason. First of all, these athletes hoped to increase their
muscular strength and seemed to be in good shape. The doping substance might not be
considered by the bodybuilders to be dangerous. The substance is perceived as a “natural”
addition of exercise to rapidly increase muscular volume. The second motive was the sport's
norms. Bodybuilders justify doping as a natural means to increase muscular volume, improve
attractiveness, and decrease fatigue. Because nearly all bodybuilders consume these
substances, their use is legitimized. Moreover, the bodybuilders reported receiving
considerable advice from proximal circles such as other athletes (rank 7), family (rank 11),
dealers (rank 12), others (rank 13), and friends (rank 15). This finding supports the
observation that bodybuilders have higher scores of social dependence than other athletes
They easily follow the lead of others in denying any inappropriate behavior and controlling
their use of anabolic substances. They underestimate their own consumption compared with
other athletes who use steroids dangerously in intensive sessions, such as cyclists. The third
motive was body image. The high value that society gives to physical attractiveness
encourages bodybuilders to try to conform to an ideal image. They are fascinated by their
own image, and the walls of mirrors in fitness centers reinforce this feeling. This motive was
associated with the need to affirm masculine identity. The bodybuilders wanted to become
more “male” and attractive. They hoped to dominate and use their bodies as a power vehicle.
However, in this particular sport culture, a pervasive belief is that power must be acquired by
pain and effort, enhancing virility and courage, with bodybuilders seen as modern gladiators.
The first motive of the footballers was conformity to the social norms of their general culture
and not their sport. This motive was associated with the influence of friends (rank 4) and
suggests that football exerts a pressure on players to do as others do. The substances
massively cited were cannabinoids (52 %) as well as stimulants (13 %). Creating and
maintaining a festive atmosphere thus emerged as a predominant motivation are widely used
at social events among friends and are characterized by relaxation and a decrease in
inhibition. They are found in all social classes. Although the players reported using cannabis
for reasons other than performance enhancement, the substance is still prohibited by football
governing bodies and the WADA. The group enhances a collective dynamic to use this
substance. The second motive was the control of anxiety, probably due to the uncertainty of
the game results. The game of football is highly unpredictable because it is a team sport that
depends on the performances of many players, the performances of opponents, the weather,
the partiality/impartiality of referees, coaches’ choices, and injuries, among other. The high
number of factors that footballers cannot control generates considerable anxiety. The
efficiency of cannabis to decrease anxiety has been well demonstrated. Use of this
substance can also be a self-handicapping strategy to limit the impact of poor sporting
results and explain defeat. The third motive was strength enhancement. Football requires leg
speed, and anabolic steroid substances can enhance individual performance during
matches. The results showed that doping motives differed between cyclists, bodybuilders,
and footballers. The main motives for doping were specific: preserving health for cyclists,
increasing muscular strength for bodybuilders, and enjoyment for footballers. The callers all
knew the best substances to obtain the desired effect. Compared with the literature, the
surprising results of this study were the weak group influence and the impact of health
preoccupations for the cyclists, the relatively low influence of body image for the
bodybuilders, and the motivation to increase muscular strength in the footballers. Thus,
prevention campaigns need to be specific. Comparing motives for prohibited substance use
can lead to confusion if the individual sport is not taken into consideration. The first motive
532
cited by cyclists indicates the paradoxical strategy of current antidoping campaigns. The
campaigns focus on the dangers of illicit substances to athletes’ health. Yet these athletes
were using appropriate and efficient substances to preserve their health. Thus, a prevention
message targeting health maintenance misses the point because their main doping motive is
to combat the very real (and subjective) risk of falling ill. The real risk instead seems to be
self-medication, and a more effective message to cyclists should thus emphasize means to
maintain a good balance between high performance and optimal health. Issues of domination
and male identity need to be addressed in campaigns for bodybuilders via better health
education. Prevention should be more focused on body self-acceptance. The footballers
hoped to strengthen their team relationships. Prevention campaigns must insist on limiting
use of recreational drugs and supplements [10303].
The use of anabolic androgenic steroids (AAS) has been associated with the use of illegal
drugs. Earlier observations suggested that users of illegal drugs may use AAS for reasons
other than increasing muscle strength or size. The aim of the present study was to
investigate the motives for AAS use among outpatients at a substance abuse center in
Stockholm, Sweden. All male patients under the age of 50 were asked whether they had
used AAS during a 2-month period. An AAS survey was administered to those who reported
AAS experiences in the admittance interview. Twenty of the 175 respondents (11 %)
reported using AAS. The most frequently reported motives were related to anabolic effects
(i.e. for a good-looking body, to become stronger, or to perform better in sports). However,
some users reported other motives; for example, to conceal concomitant drug use, to
alleviate insecurity or low self-esteem, to become brave, or in preparation of committing a
crime. Furthermore, many respondents reported side effects that were associated with AAS;
most notably, irritability and depression/suicidal ideation. It was thus concluded that some
users of illicit drugs also use AAS for reasons other than the anabolic properties of these
compounds. Therefore, considering that AAS may cause or contribute to diverse morbidity, it
is important to ask users of illicit drugs about AAS use, even when obvious external signs of
AAS use are lacking [10316].
Population heterogeneity
Appearance- and performance-enhancing drugs (APEDs) constitute a wide range of
substances, including anabolic-androgenic steroids, nonsteroidal anabolics, and licit and illicit
ergo/thermogenics. A great deal of heterogeneity exists in APED use patterns among weight-
lifting men, and, consequently, little is known about how these patterns are related to side
effect profiles or risk potential. In the current study, a sample of 400 adult men who were
regular APED users completed an interactive Web-based instrument detailing information
about APED use, side effects, and related indicators of risk. To explore the heterogeneity of
APED use patterns, the authors subjected data on use patterns to latent class analysis
(LCA), latent trait analysis (LTA), and factor mixture analysis to determine the best model of
APED use. Results indicated that a 4-class factor mixture model provided a better fit than
LCA and LTA models. The authors also found that severity and latent class were uniquely
associated with negative outcomes. Each of the 4 classes was associated with unique side
effects, motivations, and participant use patterns. Implications for identifying pathological
forms of APED use are discussed [07016].
An age factor
It was attempted to qualitatively investigate why men of two age categories have chosen not
to use androgenic-anabolic steroids (AASs). Twelve men (22 years [group I] and 53 years
[group II]) were selected on the basis of specific inclusion criteria, including age and fitness
levels (i.e. "do you weight train?"). Subjects were classified in 1 of 2 categories-younger or
older precluders-and were asked to complete two survey instruments before their
participation. The Drive for Muscularity Scale (reliability 0.85) and Body Image Questionnaire
533
were used to gain a better understanding of perceptions and motivations regarding health,
fitness, and body image. A series of semistructured questions were used to enhance focus
group discussion regarding attitudes. Questions were validated by a panel of experts in
qualitative methods. Member checks were conducted to enhance trustworthiness of the data.
Data were transcribed verbatim and analyzed with thematic open-coding techniques. Various
behaviors were reported regarding body image. Emerging themes showed a clear
demarcation between age categories. Younger subjects cited power, control, body image,
and narcissism, whereas older subjects viewed AAS use as more of an athletic-based
phenomenon, such as with performance enhancement, when asked about steroids. Groups
were in agreement that media trends and perceptions of the ideal male body are becoming
"superhuman" and unattainable without chemical means. Understanding attitudinal
perspectives might help complement national data on AAS trends. Future investigations
could help coaches and allied health professionals collaborate with each other, as well as
with national groups and foundations, to devise more appropriate strategies in addressing
this growing athletic and public health concern [07017].
Despite potential health risks and penal repercussions (e.g. forfeiture of winnings, sport
banishment) associated with doping and the use of other unsanctioned products/equipment,
the use of banned performance enhancing products (PEPs) remains an issue in virtually all
adult competitive sports. In the empirical literature, the use of any substance (sanctioned or
unsanctioned) to enhance performance in the face of perceived obstacles is generally
defined as “doping behavior.” Although ample evidence illustrates the dangers of doping and
prevalence rates among athletes in different sports, research exploring the attitudes and
beliefs that may contribute to use of unsanctioned substances/products are inconsistent.
While some findings suggests that doping users and non-users may perceive health risks
differently, the contributions of proposed demographic and psychological variables (e.g. self
esteem, anxiety) are equivocal Despite uncertainty in the literature, it is generally understood
that athletes’ rationales for using PEPs likely include a desire to maximize performance/ and
succeed and perceptions of risks to health. A challenge in developing theoretically sound and
applicable PEP use models is the complexity and heterogeneity in which these behaviors
occur. It is well known in the social sciences literature that beliefs, motivations, attitudes and
environmental factors play an important role in understanding and predicting behavior. Within
the context of doping behaviors, this is perhaps best summarized in Backhouse’s WADA
manuscript on drug use attitudes and behaviors in sports (2011). In this comprehensive
literature review, the authors systematically highlight how doping appraisals and actions are
largely dependent on contextual factors, including the type of sport (e.g. body building vs.
cycling), level of competition (e.g. high school, college, professional competition), and the
characteristics of the individual (e.g. athlete, coach, general population) [14022].
Existing models of doping behaviors attempt to account for the complexity of this
phenomenon, and often incorporate key psychological and societal/environmental factors.
For example, the Drugs in Sports Deterrence model (DSDM) highlights the role of decisional
processes involved in performance enhancer use, as well as the contributions of other
factors (e.g. affect, cognition) that may influence this cost-benefit analysis. Another
conceptual framework, the Drug Compliance in Sports Model, presents eight factors believed
to influence intentions towards drug use: threat appraisal; benefit appraisal; personal
morality; legitimacy; personal self-esteem; reference group opinion; drug affordability; and
drug availability. While these and other promising theories exist further empirical derived
models are need, as existing perspectives often fail to account for considerable amounts of
variance in predicting doping behaviors and attitudes. A challenge encountered by sports
governing bodies is the reality that it is virtually impossible for laws/regulations/rules to keep
up with the technological advances of doping, particularly as newer drugs mimic natural
human processes. This challenge also falls on athletes; in pursuit of an advantage, a
534
competitor may find a category of PEPs that is not explicitly listed as banned or non-banned.
Legal/non-banned PEPs is a broad and subjective category, as an athlete may (correctly or
incorrectly) perceive virtually anything as contributing to improved performance (e.g.
equipment, dietary modifications, clothing, etc). Even among established guidelines, the
ethics of PEPs use can be unclear, confusing, and contradictory. For example, while the use
of technologically-constructed hypoxic environments are approved by many sports governing
bodies, the mechanisms underlying their efficacy are similar to those of erythropoietin (EPO),
a banned substance. Another example is the well-publicized use of AOD-9604, an analogue
of growth hormone releasing factor, by the Australian Essendon Football Club in 2012. This
relatively new compound fell under the WADA’s “S.O.” category, and thus, should have been
prohibited. Predictably, the ethics of “legal” and “illegal” performance enhancement have
become increasingly blurred as recent studies illustrate that society has become more
tolerant of doping over time. Another potential concern lies in dietary supplements -
frequently used by athletes as non-banned PEPs – which are not required to undergo testing
to confirm efficacy or safety. Unfortunately, our knowledge of effective anti-doping programs
is still in its infancy; this outcome literature is narrowly focused in the realm of anabolic
steroid use and results suggest that education alone is likely insufficient to change behaviors
[14022].
Cyclists
Use of banned performance enhancing products (PEPs) remains an issue in virtually all
competitive sports despite penal consequences and known health risks. The lines
distinguishing "fair" and "unfair" performance enhancement have become increasingly
blurred. Few studies have explored how attitudes towards legal performance enhancers
(drugs/substances, diet, and equipment modifications) may influence motivations to use
banned PEPs. In the present study, 68 competitive cyclists completed a survey examining
the importance of choosing banned and non-banned PEPs using World Anti-Doping Agency
(WADA) and Union Cycliste Internationale (UCI) criteria. Results showed that over 60
percent of cyclists used non-banned PEPs while 8 percent used banned PEPs. Health was
overall the most important factor in choosing a PEP while apprehension by a doping agency
was least important. Mixed- model ANOVA analyses revealed that motivations to use banned
PEPs were complex, as the importance of health, violating the sprit of the sport, performance
improvement, and getting caught were differentially influenced by PEP legality and whether a
cyclist endorsed non-banned PEP use. The importance of winning, sponsorship, and
maintaining competitiveness did not influence non-banned PEP use. The findings illustrate
the multifactorial nature of PEP use/doping attitudes and highlight the unique role that "legal"
performance enhancement may plays in influencing banned and/or unethical sports
behaviors. Key PointsUse of performance enhancers is high even among non-professional
athletesCyclists overall rated "risk to health" as the most important factor in choosing to use
a performance enhancing product.Motivations to use banned performance enhancer are
complex and are significantly influenced by whether an athlete utilizes "legal" performance
enhancers [14022].
Motivation for anti-doping
Motivation in sport has been frequently identified as a key factor of young athletes' intention
of doping in sport, but there has not been any attempt in scrutinizing the motivational
mechanism involved. The present study applied the trans-contextual model of motivation to
explain the relationship between motivation in a sport context and motivation and the social-
cognitive factors (attitude, subjective norm, perceived behavioral control, and intention) from
the theory of planned behavior (TPB) in an anti-doping context. Questionnaire data was
collected from 410 elite and sub-elite young athletes in Australia (Mean age 18 + 4 years, 55
535
% male). It was measured the key model variables of study in relation to sport motivation
(Behavioral Regulation in Sport Questionnaire), and the motivation (adapted version of the
Treatment Self-Regulation Questionnaire) and social cognitive patterns (the theory of
planned behavior questionnaire) of doping avoidance. The data was analyzed by variance-
based structural equation modeling with bootstrapping of 999 replications. The goodness-of-
fit of the hypothesized model was acceptable. The bootstrapped parameter estimates
revealed that autonomous motivation and amotivation in sport were positively associated
with the corresponding types of motivation for the avoidance of doping. Autonomous
motivation, subjective norm, and perceived behavioral control in doping avoidance fully
mediated the relationship between autonomous motivation in sport and intention for doping
avoidance. The findings support the tenets of the trans-contextual model, and explain how
motivation in sport is related to athletes' motivation and intention with respect to anti-doping
behaviors [14440, §50026].
Measurement of attitudes
Doping attitude is a key variable in predicting athletes' intention to use forbidden performance
enhancing drugs. Indirect reaction-time based attitude tests, such as the implicit association
test, conceal the ultimate goal of measurement from the participant better than
questionnaires. Indirect tests are especially useful when socially sensitive constructs such as
attitudes towards doping need to be described. The present study serves the development
and validation of a novel picture-based brief implicit association test (BIAT) for testing
athletes' attitudes towards doping in sport. It shall provide the basis for a transnationally
compatible research instrument able to harmonize anti-doping research efforts. Following a
known-group differences validation strategy, the doping attitudes of 43 athletes from
bodybuilding (representative for a highly doping prone sport) and handball (as a contrast
group) were compared using the picture-based doping-BIAT. The Performance
Enhancement Attitude Scale (PEAS) was employed as a corresponding direct measure in
order to additionally validate the results. As expected, in the group of bodybuilders, indirectly
measured doping attitudes as tested with the picture-based doping-BIAT were significantly
less negative. The doping-BIAT and PEAS scores correlated significantly for bodybuilders,
but not significantly for handball players. There was a low error rate (7 %) and a satisfactory
internal consistency for the picture-based doping-BIAT. It was concluded that the picture-
based doping-BIAT constitutes a psychometrically tested method, ready to be adopted by the
international research community. The test can be administered via the internet. All test
material is available "open source". The test might be implemented, for example, as a new
effect-measure in the evaluation of prevention programs [14023].
Beliefs about the causes of success in sports
One study set out to assess the impact of attributional beliefs about success on the
susceptibility for doping use in adolescent athletes. The sample consisted of 309 adolescent
athletes participating in both team and individual sports. Participants completed a battery of
questionnaires including Beliefs about the Causes of Success in Sport Questionnaire
(BACSSQ), current and past doping use, and measures of attitudes, norms, situational
temptation and social desirability. Variance reduction rate analysis revealed that social
desirability did not act as a confounder in the relationship between doping susceptibility and
its predictors. With regard to beliefs about the causes of success dimensions, only deception
emerged as a significant predictor of doping use susceptibility over and above the effects of
well-established social-cognitive predictors of doping intentions and use. These findings
imply that beliefs about the causes of success in youth sports may comprise another
dimension of risk factors for doping susceptibility and use [14024].
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Elite athletes' attitudes, beliefs, and knowledge
One paper qualitatively explores national level athletes' willingness to report doping in sport.
Following ethical approval, semi-structured interviews were conducted with nine national
level athletes from rugby league (n=5) and track and field athletics (n=4). Thematic analysis
established the main themes within the data. Contextual differences existed around the role
that athletes perceived they would play if they became aware of doping. Specifically, track
and field athletes would adopt the role of a whistle-blower and report individuals who were
doping in their sport. In comparison, the rugby league players highlighted a moral dilemma.
Despite disagreeing with their teammates' actions, the players would adhere to a code of
silence and refrain from reporting doping. Taking these findings into account, prevention
programs might focus on changing broader group and community norms around doping. In
doing so, community members' receptivity to prevention messages may increase. Moreover,
developing skills to intervene (e.g., speaking out against social norms that support doping
behavior) or increasing awareness of reporting lines could enhance community responsibility
for doping prevention. In sum, the findings highlight the need to consider the context of sport
and emphasize that a one-size-fits-all approach to anti-doping is problematic [14003].
In the absence of objective information on the use of performance-enhancing drugs (PED),

attitudes are often used as a proxy for doping behaviour, assuming that those who use
banned substances show greater leniency towards doping than those who stay clear of
doping. Consequently, researchers have identified the need to develop more sophisticated
and bespoke interventions to support the athletes with attitudes that increase their likelihood
of using banned substances, and the need to develop empirically tested models. Doping has
evolved greatly in recent years, and greater understanding of it is essential for developing
efficient prevention programmes. In the psychosocial approach, attitudes are considered an
index of doping behaviour, relating the use of banned substances to greater leniency towards
doping. The aim of one review was to gather and critically analyse the most recent
publications describing elite athletes' attitudes, beliefs and knowledge of doping in sport, to
better understand the foundations provided by the previous work, and to help develop
practical strategies to efficiently combat doping. For this purpose, we performed a literature
search using combinations of the terms "doping", "sport", "elite athletes", "attitudes",
"beliefs", "knowledge", "drugs", and "performance-enhancing substances" (PES). A total of
33 studies were subjected to comprehensive assessment using articles published between
2000 and 2011. All of the reports focused on elite athletes and described their attitudes,
beliefs and knowledge of doping in sport. It has been emphasized that athletes who use
banned substances mainly do so to improve their performance, even though most athletes
acknowledge that doping is dishonest, unhealthy and risky because of the impact of
sanctions. The ‘‘false consensus effect’’ seems to play a key role in legitimizing the use of
banned substances. Anti-doping programmes are generally considered to be ineffective and
inefficient, and the way tests are performed is often criticized, particularly WADA’s location
reporting system. Athletes consider the severity of punishment to be appropriate or not
severe enough, although there are some differences between sports. In this sense, the
advisors and stakeholders who can influence athletes should also be educated and punished
if they are found guilty of supporting doping. In this way, all interested parties would be aware
of the magnitude of the problem. The current generation of athletes is more familiar with anti-
doping rules than earlier generations, but there is still a lack of knowledge that should be
improved using well designed educational programmes. There is also a distinct lack of
information around dietary supplements and the possible side effects of PES. There is a
general belief about the inefficacy of anti-doping programmes, and athletes criticise the way
tests are carried out. Most athletes consider the severity of punishment is appropriate or not
severe enough. There are some differences between sports, as team-based sports and
537
sports requiring motor skills could be less influenced by doping practices than individual self-
paced sports. However, anti-doping controls are less exhaustive in team sports. The use of
banned substance also differs according to the demand of the specific sport. Coaches
appear to be the main influence and source of information for athletes, whereas doctors and
other specialists do not seem to act as principal advisors. Athletes are becoming increasingly
familiar with anti-doping rules, but there is still a lack of knowledge that should be remedied
using appropriate educational programmes. There is also a lack of information on dietary
supplements and the side effects of PES. Therefore, information and prevention are
necessary, and should cater to the athletes and associated stakeholders. This will allow us to
establish and maintain correct attitudes towards doping. Psychosocial programmes must be
carefully planned and developed, and should include middle- to long-term objectives (e.g.
changing attitudes towards doping and the doping culture). Some institutions have developed
or started prevention or educational programmes without the necessary resources, while the
majority of the budget is spent on anti-doping testing. To minimize the phenomenon of
doping, information and prevention programmes, starting with athletes at a young age, and
involving other stakeholders (e.g. the athletes’ doctors, coaches or family), are necessary to
establish and maintain correct attitudes and behaviours. It is also very important that the
sport institutions at all levels (from WADA to regional governments) provide more resources
to psychosocial projects in relation to the biomedical approach (i.e. anti-doping controls),
which have been the main priority of anti-doping programmes currently in use. Also, event
organizers and federations should check that sporting rules do not favour the possible
advantages of using banned substances in competitions (i.e. by reducing the distance
covered in competitions, allowing longer recovery between stages and encouraging, where
possible, the importance of technical/tactical aspects rather than physical aspects). The
programmes targeting athletes and those around them must be carefully planned and
developed as a middle- to long-term objective and, ultimately, change attitudes towards
doping and the doping culture. Current research methods are weak, especially
questionnaires. A combination of qualitative and quantitative measurements are
recommended, using interviews, questionnaires and, ideally, biomedical tests. Studies
should also examine possible geographical and cultural differences in attitudes towards
doping [13017].
Reports of illicit substance use by college athletes have become commonplace in recent
years, yet comparatively little effort has been put forth by the research community to
understand these behaviors. Data for this study came from a large, national dataset collected
by the National Collegiate Athletic Association (NCAA). This study compared substance use
behaviors of male undergraduate student athletes who reported using ergogenic
performance enhancing substances (e.g., anabolic steroids and peptide hormones) during
college (PES users) to those who did not (PES non-users).A consistent pattern of higher
substance use rates was observed among PES users compared to non-users, including
heavier drinking, higher prevalence rates of cigarettes, marijuana, amphetamines, narcotics,
and a variety of permissible and impermissible dietary supplements. An unexpected finding
was that there were large discrepancies in reported prevalence rates between similar or
overlapping survey items (e.g., past year use of "narcotics" versus "I have taken Vicodin,
Oxycontin or Percocet with/without a prescription"). These findings suggest that male college
athletes who use PES while in college demonstrate a general tendency to engage in alcohol
and drug use behaviors, regardless of whether these behaviors improve or impede athletic
performance. The results further suggest that college athletes may not fully appreciate drug
categorizations that are commonly employed to gauge substance use behaviors. Changes to
drug education and prevention programs may be needed to enhance understanding of drug
properties and actions [13041].
It was examined whether constructs outlined in self-determination theory [Deci & Ryan,
538
2002], namely, autonomy-supportive and controlling motivational climates and autonomous
and controlled motivation, were related to attitudes toward performance-enhancing drugs
(PEDs) in sport and drug-taking susceptibility. It was also investigated moral disengagement
as a potential mediator. It was surveyed a sample of 224 competitive athletes (59 % female;
medan age 20 years; median 10 years of experience participating in their sport), including 81
elite athletes. Using structural equation modeling analyses, the hypothesis proposing positive
relationships with controlling climates, controlled motivation, and PEDs attitudes and
susceptibility was largely supported, whereas our hypothesis proposing negative
relationships among autonomous climate, autonomous motivation, and PEDs attitudes and
susceptibility was not supported. Moral disengagement was a strong predictor of positive
attitudes toward PEDs, which, in turn, was a strong predictor of PEDs susceptibility. These
findings are discussed from both motivational and moral disengagement viewpoints [13797].
Although nutrition and doping are important factors in sports, neither is often investigated in
synchronized swimming (Synchro). One study aimed to define and compare Synchro
athletes and their coaches on their knowledge of sports nutrition (KSN) and knowledge of
doping (KD); and to study factors related to KSN and KD in each of these groups.
Additionally, the KSN and KD questionnaires were evaluated for their reliability and validity.
Altogether, 82 athletes (17 ± 2 years of age) and 28 coaches (31 ± 5 years of age) from
Croatia and Serbia were included in the study, with a 99 percent response rate. The test and
retest correlations were 0.94 and 0.90 for the KD and KSN, respectively. Subjects responded
equally to 91 percent queries of the KD and 89 percent queries of the KSN. Although most of
the coache sare highly educated, they declared self-education as the primary source of
information about doping and sport-nutrition. Coaches scored higher than their athletes on
both questionnaires which defined appropriate discriminative validity of the questionnaires.
Variables such as age, sports experience and formal education are positively correlated to
KSN and KD scores among athletes. The athletes who scored better on the KD are less
prone to doping behavior in the future. These data reinforce the need for systematic
educational programs on doping and sports nutrition in synchronized swimming. Special
attention should be placed on younger athletes. Although most of the synchro coaches are
highly educated, self-education is declared as the primary source of information about doping
and sportnutrition.The knowledge of doping and doping-health hazards are negatively related
to potential doping behavior in the future among synchronized swimmers. The data reinforce
the need for systematic educational programs on doping and sports nutrition in synchronized
swimming. It was advocated improving the knowledge of sports nutrition among older
coaches and the knowledge of doping among younger coaches, while among athletes,
younger swimmers should be targeted [13042].
Although athletes' beliefs and values are known to influence whether or not an athlete will
use banned drugs, little is known about the athletes' beliefs and attitudes in different sports.
The aim of this study was to clarify the beliefs and attitudes of elite athletes towards banned
substances and methods in sports. A total of 446 athletes (response rate 90 %; 446/494)
financially supported by the National Finnish Olympic Committee completed a structured
questionnaire during their national team camps in 2002. More than 90 percent of the athletes
reported to believe that banned substances and methods have performance enhancing
effects, and 30 percent reported that they personally know an athlete who uses banned
substances. Of the male athletes 35 percent, and 23 percent of females reported they
personally know an athlete using banned substances. A total of 15 percent of the athletes
reported that they had been offered banned substances: 21 percent of the speed and power
athletes, 14 percent of the team sport athletes and of the athletes in motor skills demanding
events, and 10 % of the endurance athletes. Stimulants were the most often offered
substance group (to 7 % of all the athletes) followed by anabolic steroids (4 %). Subjects
who regarded doping as a minor health risk seemed to be more often associated with doping
539
users than those regarding doping as a significant health risk. Athletes in different sports
have a different approach to doping. Risk of doping appears to be highest in speed and
power sports and lowest in motor skills demanding sports. Males are at higher risk than
females. Controlling doping only by tests is not sufficient. A profound change in the attitudes
is needed, which should be monitored repeatedly [06015].
One study aimed to investigate, among a sample of elite Australian athletes, the extent to
which this group supports drug testing as a deterrent to drug use. Data was collected from a
convenience sample of (n=974) elite Australian athletes who self-completed a questionnaire,
and semi-structured telephone surveys with key experts. The athletes surveyed endorsed
testing for banned substances as an effective way of deterring drug use; believed that the
current punishments for being caught using a banned substance was of the appropriate
severity; and indicated that there should be separate policies regarding illicit drug and
performance-enhancing drug use. A large proportion of elite athletes in Australia endorse
drug testing as an effective means of deterring drug use. However, they perceive a
difference between being detected using a performance-enhancing drug and an illicit drug
and believe that penalties should reflect this difference. Future research may wish to
investigate attitudes towards newer methods employed to detection drug use [10315].
Information-seeking behaviours among elite athletes

Many sporting organisations conduct drug information seminars for their athletes; however, it
is uncertain whether these programs provide athletes with pertinent drug information in
formats that are conducive to information retention. The aims one study were to investigate
self-reported confidence in knowledge of illicit drugs and information seeking behaviours
among elite athletes. Data were collected from two sources: quantitative surveys with elite
athletes; and qualitative interviews with key experts who come into contact with elite athletes.
Athletes were confident in their knowledge of the effects of illicit drugs such as cannabis and
meth/amphetamine, but less confident in their knowledge of the effects of illicit drugs such as
GHB and ketamine. A substantial proportion felt that athletes in their sport would benefit from
more information concerning illicit drugs. It was concluded that both athletes and key expert
believed that information on illicit drugs should be delivered to athletes in a specific and
relevant manner. There may be stigma attached to information seeking within a sports club
or organisation. Accordingly, improving the accessibility to creditable information via the
Internet may prove to be an effective means by which to educate athletes on the effects of
illicit drugs [11417].
Knowledge of anabolic steroids among gym users in Jamaica
Self-administered questionnaires were completed by 1062 gym-users in 14 gyms in Trinidad

from February 1997 to July 1997 to determine the knowledge, attitudes and practices
regarding anabolic steroids (AS). Five hundred and sixty (53 %) females and 502 (48 %)
males completed the questionnaire. Half of the total sample was individuals in the 20 to 29
year age group. From the 17 questions that tested knowledge about AS, the median number
of correct responses was 7 with a mode of 8. Increased muscle mass was correctly identified
as one of the effects of AS by 841 respondents (79 %), while 249 (24 %) of the total sample
thought asthma was treated with AS. Most (872 or 82 %) felt that their knowledge about AS
was inadequate and 700 (66 %) were of the opinion that AS should be banned from use in
competitive sports. Similarly, 733 (70 %) of the gym-users thought AS should only be
available by prescription. Thirty respondents reported having used AS (2.9 %). The
prevalence of AS use was higher among males than females. Improvement of physical
appearance and not competitive advantage in sport was the main reason cited for AS use.
Anabolic steroid users knew more about the adverse effects of AS than non-AS users but the
540
therapeutic uses of AS were comparatively less well known. The study demonstrated a
general lack of knowledge concerning AS use and that a small but significant proportion of
persons using gyms admitted to abusing AS [00012].
Associations of physical activity and sport with at-risk substance use in young men
One study aimed to measure the associations of physical activity and one of its components,
sport and exercise, with at-risk substance use in a population of young men. Baseline (2010-
2012) and follow-up (2012-2013) data of 4748 young Swiss men from the Cohort Study on
Substance Use Risk Factors (C-SURF) were used. Cross-sectional and prospective
associations between at-risk substance use and both sport and exercise and physical
activities were measured using Chi-squared tests and logistic regression models adjusting
for covariates. At baseline, logistic regression indicated that sport and exercise is negatively
associated with at-risk use of cigarettes and cannabis. A positive association was obtained
between physical activity and at-risk alcohol use. At baseline, sport and exercise was
negatively associated with at-risk use of cigarettes and cannabis at follow-up. Adjusted for
sport and exercise, physical activity was positively associated with at-risk use of cigarettes
and cannabis. It was concluded that sport and exercise is cross-sectionally and longitudinally
associated with a low prevalence of at-risk use of cigarettes and cannabis. This protective
effect was not observed for physical activity broadly defined. Taking a substance use
prevention perspective, the promotion of sport and exercise among young adults should be
encouraged [14438].
Attitude of elite cyclists on doping
The protection of the health of athletes is one of the three criteria taken into account when
registering a substance in the World Anti-Doping Agency prohibited list. Nevertheless, in
elite-level cycling, banned substance use is widespread. On study adopted a psychological
approach to examine how or whether perceived health risks influence elite-level cyclists'
decisions to use banned substances. Sixteen semi-structured interviews were conducted
with cyclists hoping to join a professional team (n=6), neo-professional cyclists (n=2), and
former professional cyclists (n=8). Although an evolution was observed in the organization of
doping and perceptions of doping over the last decade, the perceived health hazards did not
influence, most of the time, decisions to use banned substances among the sample of
cyclists. There was a systematization of exogenous substance use in the cycling
environment and a trivialization of the side effects of the banned substances. Finally,
younger cyclists were not concerned about the long-term health consequences of banned
substances; they were more focused on the short-term performance-enhancing benefits.
There is a need to implement more effective preventive programs to change athletes'
attitudes toward doping and its health risks [12021].
Age of onset of performance-enhancing drug use
There are nine studies from the US, Australia, and the United Kingdom since the year 2000
that provide at least some data on age of onset of AAS use. These included six studies that
evaluated AAS users in person, and three Internet surveys of AAS users. In the largest
Internet study, only one out of 1,955 male AAS users (0.05 %) reported starting AAS use
before age 15, and only 6 percent started before age 18. In five other studies, collectively
evaluating 801 AAS users, only 12 (1.5 %) started before age 16, and 199 (25 %) started
before age 20. Notably, the median age of onset across all studies consistently fell into the
narrow range of 22 to 24 years. However, the actual median age of onset is probably higher,
since at the time of recruitment many study candidates had not completed the age-range of
541
risk for starting [14017].
Young athlets attitudes towards doping
There is evidence of a small but significant proportion of adolescents engaging in doping

practices. Young athletes face very specific pressures to achieve results as they strive for a
career at an elite level. This study used an anonymized questionnaire to survey 403 (12-21
years old) talented young athletes' attitudes toward performance-enhancing substances and
supplements. Two-thirds of the sample comprised males. Athletes were generally against the
use of doping substances to enhance sporting performance. Within this generally
unfavorable view, males tended to express a more permissive attitude toward performance-
enhancing methods than females. Those convinced of the necessity of supplementation for
sporting success were also more likely to express permissive attitudes. When asked whether
they would take a "magic" drug that, while undetectable, would significantly enhance
performance, the overwhelming majority of athletes said "no," but many thought others would
take the substance. Interestingly, there was a significant association between the projected
use of the hypothetical drug by competitors and the individual respondent's willingness to
take the hypothetically "magic" substance. The study offers an insight into young athletes'
attitudes toward specific forms of performance enhancement, and the strength of their beliefs
in the face of a tempting hypothetical scenario [12022].
Attitudes on reporting of whereabouts
To improve anti-doping efforts in sports, the World Anti-Doping Agency (WADA) introduced
the World Anti-Doping Program, in which (among others) regulations for providing athletes'
whereabouts are described. Because the effectiveness and efficiency of this system depends
on the co-operation and compliance of athletes, the perspective of elite athletes is important.
One paper answered the following research questions: What is the perspective of Dutch elite
athletes on the current whereabouts system in general and how important is their privacy in
providing whereabouts in particular? In addition, this study explores how far the whereabouts
system can be developed in the future. Are athletes willing to accept greater invasions of
their privacy in order to reduce administrative effort and whereabouts failures? A structured
questionnaire was completed by 129 Dutch elite athletes registered in the national and/or
international testing pool. The results of this study indicate widespread dissatisfaction with
the whereabouts system. Most respondents support anti-doping testing in general, but many
athletes feel that WADA's whereabouts system is unacceptable in several respects. In terms
of physical privacy, there was a great dissatisfaction. Nearly half of the athletes felt that the
“1-hour time slot” limits their freedom, but on the other hand, most athletes disagreed with
the statement that the distinction between their sport and private life is disturbed. For almost
one in three respondents, the whereabouts system has a negative influence on the pleasure
they experience in being an elite athlete. In terms of informational privacy, almost all athletes
had confidence in the confidential treatment of their whereabouts information. Almost all
athletes would accept giving their phone number to Doping Control Officials, but only half of
the athletes would accept sharing their location on their mobile phone. Furthermore, almost
two in ten of the athletes would accept wearing a permanent wrist or ankle bracelet or accept
being implanted with a GPS chip in order to facilitate future anti-doping testing. Thus, the
results of this study indicate that a majority of the athletes are not likely to accept a greater
violation of their privacy than the current whereabouts regulations already entail [14002].
Judgments of the fairness of using performance enhancing drugs
542
Undergraduates (total n=185) were asked about performance-affecting drugs. Some drugs
supposedly affected athletic performance, others memory, and other attention. Some
improved performance for anyone who took them, others for the top 10 percent of
performers, others for the bottom 10 percent, and finally, yet other drugs worked only on the
bottom 10 percent who also showed physical abnormalities. Participants were asked about
the fairness of allowing the drug to be used, about banning it, and about whether predictions
of future performance based on testing with or without the drug were better. The study found
that participants appreciated the "interaction effect," that they felt it was less unfair to allow
the drug if it affected the bottom 10 percent than if it affected everyone, and they were more
eager to have the drug banned if it affected everyone. Participants were least tolerant of
drugs that affected athletic performance and most tolerant of those that affected attention
[05009].
Knowing and, if necessary, altering competitive athletes' real attitudes towards the use of
banned performance-enhancing substances is an important goal of worldwide doping
prevention efforts. However athletes will not always be willing to reporting their real opinions.
Reaction time-based attitude tests help conceal the ultimate goal of measurement from the
participant and impede strategic answering. One study investigated how well a reaction time-
based attitude test discriminated between athletes who were doping and those who were not.
We investigated whether athletes whose urine samples were positive for at least one banned
substance (dopers) evaluated doping more favorably than clean athletes (non-dopers). It was
approached a group of 61 male competitive bodybuilders and collected urine samples for
biochemical testing. The pictorial doping Brief Implicit Association Test (BIAT) was used for
attitude measurement. This test quantifies the difference in response latencies (in
milliseconds) to stimuli representing related concepts (i.e. doping-dislike/like-[health food]).
Prohibited substances were found in 43 percent of all tested urine samples. Dopers had
more lenient attitudes to doping than non-dopers. D-scores greater than -0.57 (CI95 = -0.72
to -0.46) might be indicative of a rather lenient attitude to doping. In urine samples evidence
of administration of combinations of substances, complementary administration of
substances to treat side effects and use of stimulants to promote loss of body fat was
common. The study thus demonstrates that athletes' attitudes to doping can be assessed
indirectly with a reaction time-based test, and that their attitudes are related to their behavior.
Although bodybuilders may be more willing to reveal their attitude to doping than other
athletes, these results still provide evidence that the pictorial doping BIAT may be useful in
athletes from other sports, perhaps as a complementary measure in evaluations of the
effectiveness of doping prevention interventions [14626].
Other athletes’ attitudes regarding doping
Review articles suggest a small but significant proportion (between 3 and 12% ) of male
adolescents have used anabolic-androgenic steroids (AAS) at some point. A total of 40
talented male and female athletes (mean average age 20 years) from 13 different sports
attended 12 focus groups held over the UK intended to investigate athletes' attitudes toward
doping. Focus group transcriptions were analysed and coded with the use of QSR NVivo 8.
Athletes in general did not report a significant national doping problem in their sport, but
exhibited sporting xenophobia with regard to both doping practices and the stringency of
testing procedures outside of the UK. Athletes often viewed doping as “unnatural” and
considered the shame associated with doping to be a significant deterrent. Athletes
perceived no external pressure to use performance enhancing drugs. In response to
hypothetical questions, however, various factors were acknowledged as potential “pressure”
points: most notably injury recovery and the economic pressures of elite sport. Finally, a
significant minority of athletes entertained the possibility of taking a banned hypothetical
543
performance enhancing drug under conditions of guaranteed success and undetectability. It
was concluded that the athletes in this study generally embraced those values promoted in
anti-doping educational programmes, although there were some notable exceptions. That the
social emotion of shame was considered a significant deterrent suggests anti-doping efforts
that cultivate a shared sense of responsibility to remain “clean” and emphasise the social
sanctions associated with being deemed a “drugs cheat”, resonate with this atypical social
group [10314].
Doping-related attitudes in soccer players

The aim of this experiment was to analyse the consequences of changing attitudes related to
doping through thoughtful versus non-thoughtful processes. Participants were young soccer
players. They received a persuasive message either against or in favour of the legalisation of
several doping behaviours in soccer (e.g. the use of anabolic androgenic steroid), and
participants' level of elaboration (i.e. deliberative thinking) was manipulated in two different
experimental (high vs low) conditions. Attitudes towards the legalisation proposal were
assessed immediately following the message and one week later. Results showed attitude
change was a function of message direction and was relatively equivalent for both high and
low elaboration participants immediately after reading the message. That is, those who
received the message against legalisation showed significantly more unfavourable attitudes
towards the proposal than did those who received the message in favour of legalisation
regardless of the extent of elaboration. However, attitude change was found to be persistent
only for high elaboration participants one week after message exposure. In the present
paper, we discuss implications of changing attitudes related to doping depending on whether
the change occurred through psychological processes that require either extensive or small
amounts of deliberative thinking and elaboration [14428].
How athletes discuss on doping

One study investigates the discursive management of taking prohibited substances in sport.
In particular, it explores how one high-profile athlete, Australian cricketer Shane Warne,
accounted for his drug-taking behavior when talking to the media. Discursive psychology
(DP) is used as the theoretical and methodological framework to study drug discourses in
sport. Emphasis in DP is on making explicit how psychological concepts (e.g. drug
explanations) are used in everyday talk to perform certain actions. The research data is
Warne's first public press statement concerning his 2003 positive test for hydrochlorothiazide
and amiloride. Analysis reveals that Warne constructed his drug taking as not being related
to performance enhancement and substantiated this with a history of negative test results.
Warne worked up his taking as the result of ignorance rather than deliberate deception.
Further, he presented this as a one-off event and not reflective of systematic drug usage. It is
argued in this study that to better understand drug taking in sport, sport researchers need to
understand how athletes talk about drugs. For it is through talk that most sporting activities
are conducted and maintained, and it is this talk that needs to be understood and analyzed
[07022].
Attitudes against dopers
It was investigated the social image of anabolic steroids (AS) users grounding our analysis
on the achievement goal theory of Nicholls. The main goal was to explore how an athlete's
acceptance of AS use would impact on the way that athlete will be perceived by others. Non-
AS-using participants reacted to one of two scenarios portraying a male athlete either
accepting or refusing to engage in drug use behavior. The results suggested that the
acceptance of anabolic steroids yielded an unfavorable social image – perceivers inferred a
predominant ego orientation to characterize the AS-user's motivation as well as weaker
544
sportspersonship and a stronger proclivity for reactive aggression than instrumental
aggression. Moreover, the analyses did not yield significant gender or interaction effects.
Finally, those findings are commented in view of methodological shortcomings and of the
perspectives that they may offer for future research concerning the motivational aspects of
the social perceptions of drug use in sport [13050].
Dissatisfaction with body imaging as a reason for doping
The most recent decade of research has revealed that body image is an issue for young
men. Around two-thirds of adolescent boys are dissatisfied with their bodies, and this is
equally split between those who desire weight loss and those who want to gain muscle. One
study, conducted in Greece, indicated that the levels of body dissatisfaction were similar for
adolescent boys and girls. An early Australian study reported that 59 percent of adolescent
boys were “currently trying to build up their body” and 74 percent believed that they should
“develop their muscles”. Interviews with adolescent boys revealed that half wanted to change
their body weight, and the majority of participants thought that body size, shape and
muscularity was important. Body dissatisfaction is known to lead to the adoption of
deleterious weight change strategies such as excessive exercise, and the use of
supplements, some of which may be drugs and/or substances that are banned in sports
competitions. Cross-sectional and longitudinal research has confirmed that body
dissatisfaction and drive for muscularity predict the consumption of nutritional supplements
such as protein powders, as well as anabolic steroids. The consumption of these substances
is a concern due to the immediate harms of use, but also the potential that they will
eventually act as a gateway to more serious substances and illegal drug use. Gateway
theory proposes that adolescents who use legal substances such as alcohol and cigarettes
are more likely to progress to using more and more serious, illegal drugs, in a predictable
pattern. There is some recent evidence to suggest that the same progression also occurs for
performance and image enhancing supplements, in that the use of protein powders and
supplements predicts the use of anabolic steroids. In a study of 212 male and female
competitive adolescent athletes, those who were taking nutritional supplements were 3.5
times more likely to report doping, and had significantly more positive attitudes towards the
use of drugs in sport. Users of anabolic-adrenergic steroids have also been found to be more
likely to engage in the consumption of alcohol and other drugs. Thus, reports of high levels of
use of protein powders and nutritional supplements among young men are a concern
because these substances may act as a gateway for the use of drugs and illegal substances
to enhance appearance or sports performance. The aim of one study was to investigate the
relationship between body dissatisfaction, weight change behaviors, supplement use, and
attitudes towards doping in sport among an adolescent male sample. Participants were 1148
male adolescents (age range 11-21 years) in Australia who completed a self-report
questionnaire that measured weight change behaviors, supplement use, body dissatisfaction
(Male Body Attitudes Scale; MBAS) and attitudes towards doping in sport (Performance
Enhancing Attitudes Survey; PEAS). There was a positive correlation between MBAS total
and PEAS scores, indicating that the young men who were more dissatisfied with their
bodies were more likely to support the use of doping in sport. Young men who were currently
attempting weight loss or weight gain, and those currently consuming energy drinks and
vitamin/mineral supplements were also significantly more supportive of doping in sport.
However, those involved in weight lifting, and using protein powders were not. These findings
suggest that body dissatisfaction, weight change behaviors, and supplement use are related
to more lenient attitudes towards doping in sport among adolescent boys. Future research
might examine whether combining educational content for the prevention of body
dissatisfaction and the use of drugs in sport may have a greater preventive impact than
current programs aimed at young men [14026].
545
The authors sought to expand on previous observations suggesting that body-image
pathology is associated with illicit use of anabolic-androgenic steroids (AAS). In particular,
the authors compared current versus past AAS users and short-term versus long-term users
in this respect. The authors assessed 89 heterosexual men who lifted weights regularly-48
AAS users and 41 nonusers-on measures of self-esteem, attitudes toward male roles, body
image, eating-related attitudes and behaviors, and muscle dysmorphia ("reverse anorexia
nervosa"). AAS users as a whole showed few differences from nonusers on most measures
but showed greater symptoms of muscle dysmorphia (e.g. not allowing their bodies to be
seen in public, giving up pleasurable activities because of body-appearance concerns). The
current and past AAS users each differed only modestly from nonusers on most measures.
Short-term AAS "experimenters" were also largely indistinguishable from nonusers, but the
long-term AAS users showed striking and significant differences from nonusers on many
measures, including marked symptoms of muscle dysmorphia and stronger endorsement of
conventional male roles. It was concluded that both body-image pathology and narrow
stereotypic views of masculinity appear to be prominent among men with long-term AAS use.
Although our cross-sectional observations cannot confirm that these factors help to cause or
perpetuate AAS use, a causal hypothesis is certainly plausible and deserving of further
testing in longitudinal studies. If these factors are indeed causal, then AAS users might
respond to cognitive behavior approaches that simultaneously take aim at both types of
maladaptive beliefs [06016].
Men's fitness goals are influenced by the lens through which they view their bodies, which is
different from the way women view their bodies. Their increased focus on a muscular,
hairless body means that they exercise to enhance their physical bulk and are more likely to
engage in depilatory behaviors. In addition, the drive for muscularity may be associated with
an increased risk anabolic-androgenic steroids and other nutritional supplements whose
utility not clearly demonstrated. In the extreme, the drive for muscularity may manifest itself
as a form of body dysmorphic disorder referred to as muscle dysmorphia. However, not all
men focus on their muscularity. Gay men are more likely than heterosexual men to
experience a desire to be thin and are at greater risk for eating and body image disorders
[07015].
Muscle dysmorphia as an addiction
Using the “addiction components model”, one main contention is that muscle dysmorphia
(MD) actually comprises a number of different actions and behaviors and that the actual
addictive activity is the maintaining of body image via a number of different activities such as
bodybuilding, exercise, eating certain foods, taking specific drugs (e.g. anabolic steroids),
shopping for certain foods, food supplements, and purchase or use of physical exercise
accessories. While the hypothesized specifics relating to each addiction component
sometimes lack empirical support itnwas still believed that the main thesis (that almost all the
thoughts and behaviors of those with MD revolve around the maintenance of body image) is
something that could be empirically tested in future research by those who already work in
the area. It was hoped that the “Addiction to Body Image” model that was proposed provides
a new framework for carrying out work in both empirical and clinical settings. The idea that
MD could potentially be classed as an addiction cannot be negated on theoretical grounds as
many people in the addiction field are turning their attention to research in new areas of
behavioral addiction [150052].
Muscle dysmorphia (MD) describes a condition characterised by a misconstrued body image

in which individuals interpret their body size as both small and weak even though they may
look normal or even be highly muscular. Those experiencing the condition typically strive for
546
maximum fat loss and maximum muscular build. MD can have potentially negative effects on
thought processes including depressive states, suicidal thoughts, and in extreme cases
suicide attempts. These negative psychological states have also been linked with concurrent
use of Appearance and Performance Enhancing Drugs (APED) including Anabolic
Androgenic Steroids (AAS). The use of these substances may not just relate to body image,
but also social or sexual aspects such as producing an enhanced libido or a sense of
physical and psychological wellbeing. MD was originally categorised as Reverse Anorexia
Nervosa, due to characteristic symptoms in relation to body size. It has been considered to
be part of the spectrum of Body Dysmorphic Disorders (BDD); one of a range of conditions
that tap into issues surrounding body image and eating behaviours. Parallels have also been
drawn with Obsessive-Compulsive Disorder (OCD) given some similarities in symptom
expression like ritualistic activity [150053].
Muscle dysmorphia (MD) describes a condition characterised by a misconstrued body image

in which individuals who interpret their body size as both small or weak even though they
may look normal or highly muscular.MD has been conceptualized as a type of body
dysmorphic disorder, an eating disorder, and obsessive–compulsive disorder
symptomatology. Through a review of the most salient literature on MD, this paper proposes
an alternative classification of MD – the “Addiction to Body Image” (ABI) model – using
Griffiths (2005) addiction components model as the framework in which to define MD as an
addiction. It is argued the addictive activity in MD is the maintaining of body image via a
number of different activities such as bodybuilding, exercise,eating certain foods, taking
specific drugs (e.g., anabolic steroids), shopping for certain foods, food supplements,and the
use or purchase of physical exercise accessories). In the ABI model, the perception of the
positive effects on the self-body image is accounted for as a critical aspect of the MD
condition (rather than addiction to exercise or certain types of eating disorder). Thus, based
on empirical evidence to date, it is proposed that MD could be re-classified as an addiction
due to the individual continuing to engage in maintenance behaviours that may cause long-
term harm [150053].
A person with an ABI may:
- have cognitive disturbances that lead to a total preoccupation with activities that
maintain body image such as physical training and eating according to a strict dietary
intake
- be able to perform other tasks such as work and shopping as these tasks will be
designed and built around being able to engage in specific body image maintenance
behaviours such as physical exercising and eating
- be able to manipulate their personal situation to ensure they can perform these
maintenance tasks
The individual with ABI may even change or forego career opportunities and other daily
activities as it may reduce their ability to train or control eating behaviour during the day
[150053].
Reverse salience
If the person with ABI cannot engage in maintenance behaviours such as training or eating
regimes, their thought processes are likely to be excessively preoccupied by the need to
carry out the desired behaviours to maintain body image. This may result in the manifestation
of physical symptoms. More specifically, the cognitive disturbance creates a negative thought
process that facilitates the manifestation of physical symptoms (e.g. shakes, sweating,
nausea, etc.) as seen in other addictions. Due to some of the dietary restrictions the person
547
with ABI places upon their body, physical symptoms such as fainting and falling unconscious
may be present due to low blood sugar levels [150053].
Mood modification
For an individual with ABI, being able to engage in the maintenance behaviours brings a
sense of reward. As a consequence, training and food intake (either restrictive or over-
eating) should facilitate the release of endorphins into the bloodstream, which would increase
positive mood. The physical act of engaging in physical exercise and training (whether
cardio- or weight-based) may produce a physical state whereby the muscles are enriched
with blood (which at their biggest is known as a “pump”). This pump brings a sense of
euphoria and happiness to the person. The ABI model proposes that engaging in the
maintenance behaviours – for example weight training – will create a chemical high created
by the body though the release of chemicals such as endorphins. A person with ABI will
desire these chemical changes and this may have the same effect (both physiologically and
psychologically) as other psychoactive substances. Once their maintenance behaviours have
been completed, the person’s mood will relax due to completion of the activity, and the
person may also have a feeling of utopia, a sense of inner peace, or an exceptional high.
This feeling has been linked to the use of AASs in gym training. The person with ABI will
need to control their food intake (i.e. less or more protein and carbohydrates). The ABI model
proposes this will become a secondary dependence due to the food intake being part of the
process to maintain the primary dependence (i.e. the sculpting of the body). This will be due
to the body adapting to the amount of calories it is being fed, but also due to requirement of
being lighter or heavier – and for longer – which in turn will allow the person to obtain the
desired body shape [150053].
Tolerance
The person with ABI may need to increase the levels and intensity of the training or the food
restriction (i.e. the maintenance behaviours) to achieve the desired physiological and/or
psychological effects. This can be achieved through different training strategies or by the
consumption of different foods. In some circumstances, this may be achieved through the
use of psychoactive substances such as AASs or other food inhibiting drugs. Record keeping
of training sessions and seeking out changes in activities may assist the individual in
combatting the effects of tolerance [150053].
Withdrawal
The person with ABI is expected to have negative physical and/or psychological effects if
they are unable to engage in the maintenance activities. This would be likely to include one
or more psychological and/or physical components such as intense moodiness and
irritability, anxiety, depression, nausea, and stomach cramps. They will not be able to just
stop the maintenance behaviours without experiencing one or more of these symptoms
[150053].
Conflict
The person with ABI becomes focused on their maintenance behaviours of training and/or
eating. These behaviours can become all consuming, and the need to train, control diet, and
exercise may conflict with their family, their work, the use of resources (e.g. money) and their
life in general. An individual quoted noted “bodybuilding is my life, so I make sacrifices
elsewhere.” In some cases of the addiction, the process is thought have healthy physical
consequences and add to life in the short-term, in the long-term, the addiction will detract
from their overall quality of life [150053].
548
Relapse
If the person with ABI manages to stop the maintenance behaviours for a period of time, they
may be susceptible to triggers to re-engage in the behaviours again. CBT approaches for
treatment of MD include aspects which address triggers or reinforcing behaviour, and
reducing stress around maintaining body image to prevent likelihood of relapse. When a
person with ABI re-engages with behaviours again, they may go straight back into previous
destructive training and eating patterns [150053].
The ABI model differs from other addiction models in relation to the primary and secondary
dependencies. For instance, in exercise addiction, the individual has the primary goal of
exercising, and the cognitive dysfunction in this condition is the act of exercising in, and of,
itself. If the person loses weight or increases their body size through their exercise, this is
seen as a secondary dependence as it is a natural consequence of the primary dependence
and is not the primary goal. In MD, the primary dependence is maintenance in behaviours
that facilitate body size change due to the cognitive dysfunction of negative perceptions of
their body image. Exercise and/or dietary controls are the secondary dependence as they
assist in achieving the primary goal of maintaining their desired body size and composition.
In addition, exercise addiction tends to relate to compulsive aerobic exercise, with the
endorphin rush from the physical exertion rather than a reward from physique change
[150053].
Use of placebo-induced performance enhancement in sports
While an increasing number of research is devoted to the understanding of placebo effects in

sports, athletes' experiences with and attitudes towards the use of placebo for performance
enhancement remain poorly understood. In one study, 79 elite athletes from different sports
were surveyed on five issues related to placebo use in sports. Results showed that 47
percent of the athletes have experienced placebo effects in the past. A majority of the
athletes (82 %) thought that placebos could affect their sports performances. A wider use of
placebos in sport settings was endorsed more by those who have experienced placebo
effects in the past than those who did not. Regardless of past experience with placebo, more
than half of the athletes (53 %) would accept an unknown but legitimate substance from the
coach, and 67 placebo of them would not mind a placebo-linked deception if that was
effective. These findings confirm that most elite athletes believe in the power of placebos in
enhancing sports performance, and those having a positive past experience exhibit slightly
more favourable attitudes in contrast to those without such experiences [14603].
Polypharmacy
A review of the literature was conducted to examine the relationship between the use of
anabolic androgenic steroid (AAS) use and the use of other drugs. Studies published
between the years of 1995 and 2010 were included in the review. The use of AAS is
positively associated with use of alcohol, illicit drugs and legal performance enhancing
substances. In contrast, the relationship between AAS and the use of tobacco and cannabis
is mixed. It was concluded that the results of the review indicate that the relationship
between AAS use and other substance use depends on the type of substance studied
[01108].
The aim of one study was to describe qualitatively and quantitatively dietary supplements
(DS) and medication use in elite athletes. Athletes (n=912; age 24 ± 6 years; 72 % male)
reported medications and DSs taken within 3 days before doping control. It was analyzed
549
data collected from 2006 to 2008, identified and classified substances. Total of 75 percent
athletes reported use of at least one substance, 61 percent took DS (3 per user) and 41
percent took medications. Among users, 21 percent reported the use of six and more
different products, and one took 17 different products at the same time. Majority of
medication users took non-steroidal anti-inflammatory drugs (NSAID) (25 %), and 22 percent
used more than one NSAID. It was found no gender differences in DS use. Individual sport
athletes used more DS. The study showed widespread use of DS and drugs by elite athletes.
Consumption of DS with no evident performance or health benefits, demonstrated the need
for specific educational programs focused on DS use. Amount, quantity and combination of
the reported products raised concern about the risk of potential side effects [11009].
Medical and nonmedical use of stimulant drugs

Smith and Farah (2011) presented a scholarly review of critical areas related to their
intriguing title "Are Prescription Stimulants 'Smart Pills'?" It was contend that they
accomplished the main goal of the article, to get the facts straight about possible cognitive
enhancement via the nonmedical use of stimulant drugs by individuals without a diagnosis of
attention-deficit/hyperactivity disorder (ADHD). At the same time, they justified their main
conclusions that (a) individuals are seeking and engaging in nonmedical use of stimulant
drugs with the expectations of cognitive enhancement despite uncertainty whether such
expectations are valid and (b) on some tasks, there are small average benefits of nonmedical
use, but the overall pattern is not clear (e.g., small beneficial effects across most individuals
or large beneficial effects only in a few individuals, both of which result in small average
effects). It was offeedr comments in 3 areas to amplify key topics mentioned but not
emphasized by Smith and Farah: (a) characterization of the cognitive effects of medical use
of stimulants to contrast with the cognitive effects of nonmedical use; (b) justification of
medical use of stimulants by placement on a normally distributed dimension of behavior
rather than categorical diagnosis of ADHD, which varies widely across countries; and (c)
evaluation of the potential risks of nonmedical use to individuals and to society (e.g., the
likelihood of addiction to stimulant drugs in a small minority of the population) rather than just
the potential benefits of cognitive enhancement [11418].
Concomittant use of other substances
Reports of illicit substance use by college athletes have become commonplace in recent
years, yet comparatively little effort has been put forth by the research community to
understand these behaviors. Data for this study came from a large, national dataset collected
by the National Collegiate Athletic Association (NCAA). The study compared substance use
behaviors of male undergraduate student athletes who reported using ergogenic
performance enhancing substances (e.g. anabolic steroids and peptide hormones) during
college (PES users) to those who did not (PES non-users). A consistent pattern of higher
substance use rates was observed among PES users compared to non-users, including
heavier drinking, higher prevalence rates of cigarettes, marijuana, amphetamines, narcotics,
and a variety of permissible and impermissible dietary supplements. An unexpected finding
was that there were large discrepancies in reported prevalence rates between similar or
overlapping survey items (e.g. past year use of "narcotics" versus "I have taken Vicodin,
Oxycontin or Percocet with/without a prescription"). The findings suggest that male college
athletes who use PES while in college demonstrate a general tendency to engage in alcohol
and drug use behaviors, regardless of whether these behaviors improve or impede athletic
performance. The results further suggest that college athletes may not fully appreciate drug
categorizations that are commonly employed to gauge substance use behaviors. Changes to
drug education and prevention programs may be needed to enhance understanding of drug
properties and actions [13037].
550
Nutritional supplement and doping: Possible underlying social cognitive processes
There is growing evidence suggesting that nutritional supplement (NS) use is strongly
associated to doping use in elite and amateur sports. However, there is a paucity of research
on the psychological processes that underlie this association. The present study investigated
the cognitive and behavioral components of the association between NS use and doping
among adolescent sub-elite athletes. Six hundred and fifty adolescent athletes completed a
questionnaire including measures of doping intentions, attitudes, norms, and beliefs about
NS use. The results showed that NS users who did not report doping use had significantly
stronger doping intentions and more positive attitudes and favorable beliefs toward doping
use, as compared with athletes who did not use NS. In support of the "shared mental
representations" hypothesis, the present findings show that NS use is associated with biased
reasoning patterns in favor of doping use. This mechanism may explain why some NS users
decide to engage in doping [150034].
Progression from anabolic-androgenic steroid use to opioid dependence

Athletes who abuse anabolic-androgenic steroids may go on to abuse opioid agonist-
antagonists such as nalbuphine or even classic opioids such as heroin. It was studied this
phenomenon among patients treated at Sunrise House, a private inpatient facility for
substance-dependence treatment in northern New Jersey. Among 227 men admitted for
dependence on heroin or other opioids in 1999, it was found that 21 (9.3 %) had a history of
anabolic-androgenic steroid use. In contrast, among 197 men admitted for opioid
dependence in 1990, only 1 (0.5 percent) reported prior use of anabolic-androgenic steroids.
None of the 21 men in 1999 reported any form of substance abuse or dependence before
their use of anabolic-androgenic steroids. The information they provided strongly suggests
that they were introduced to opioids through anabolic-androgenic steroid use and the
bodybuilding subculture: 17 of the 21 men (81 %) first purchased opioids from the same drug
dealer who had sold them anabolic-androgenic steroids; 14 (67 %) were introduced to
opioids by a fellow bodybuilder; 18 (86 %) claimed that they first used opioids to counteract
insomnia and irritability induced by anabolic-androgenic steroids; and 14 (67 %) had used
opioids to counteract depression associated with withdrawal from anabolic-androgenic
steroids. All 21 of the men reported at least one of these four attributes. Demographically,
these men appeared atypical for opioid users; they all lived in suburban New Jersey and
reported a mean household income of USD 69,800 (range, USD 38,000 to USD 145,000).
They reported serious associated morbidity. Since the time of their first use of opioids, 15 (71
%) had been charged with possession of a controlled substance or prescription fraud; 5 (24
%) had served time in prison, including 1 for attempted murder; and 7 (33 %) had made at
least one suicide attempt. In the 1 to 11 months since their discharge from Sunrise House,
17 (81 %) have relapsed into opioid use, and 2 (10 %) have committed suicide. These
findings suggest an alarming trend: that anabolic-androgenic steroids may serve as
“gateway” drugs to opioid dependence, with substantial associated morbidity and even
mortality. Although the study cannot establish that anabolic-androgenic steroid use per se led
to opioid dependence in these men, the data we report strongly suggest this interpretation.
Progression from anabolic-androgenic steroid use to opioid dependence deserves further
exploration as a public health problem [00014].
Neuropharmacy addiction
The use of psychoactive substances to neuroenhance cognitive performance is prevalent.

Neuroenhancement (NE) in everyday life and doping in sport might rest on similar attitudinal
representations, and both behaviors can be theoretically modeled by comparable means-to-
end relations (substance-performance). A behavioral (not substance-based) definition of NE
551
is proposed, with assumed functionality as its core component. It is empirically tested
whether different NE variants (lifestyle drug, prescription drug, and illicit substance) can be
regressed to school stressors. Participants were 519 students (26 years old, 73 % female).
Logistic regressions indicate that a modified doping attitude scale can predict all three NE
variants. Multiple NE substance abuse was frequent. Overwhelming demands in school were
associated with lifestyle and prescription drug NE. Researchers should be sensitive for
probable structural similarities between enhancement in everyday life and sport and
systematically explore where findings from one domain can be adapted for the other. Policy
makers should be aware that students might misperceive NE as an acceptable means of
coping with stress in school, and help to form societal sensitivity for the topic of NE among
our younger ones in general [13038].
Connexion to use of dietary supplement
Recently published case reports, coupled with a large observational study of 1017 deployed
servicemen to Iraq (January 2009), has highlighted the issue and potential concerns
regarding the unregulated use of dietary and exercise supplements within the British military.
Consequently, an exploratory pilot study was undertaken to assess whether the findings of
the previous Iraq study were applicable to current deployed British servicemen in
Afghanistan. From 150 questionnaires handed out there were 87 completed questionnaires
(58 % return). The mean age was 28 with 90 percent being male. From the total of 87
persons 46 percent were self-declared current smokers with 38 percent admitting to drinking
>6 caffeinated drinks per day. Forty nine persons (56 %) admitted to a history of supplement
use with 35 (40 % compared with 32 % in 2009 in Iraq) declaring current use. The average
duration of supplement use among current users was 3 (2-9) months. The main sources of
supplement supply were via local NAAFI purchase (57 %), internet purchase (40 %) and via
their local chemist (3 %). The main types of supplement used were proteins/amino acids (86
%), creatine (34 %), chromium (31 %), stimulants (17 %), hydroxycut (6 %), and testosterone
boosters (1 %) with no persons admitting to the use of ephedra or anabolic steroids. It was
concluded that a significant proportion of the British servicemen employed on operations in
Afghanistan who were sampled, admitted to current dietary and exercise supplement use
whilst on deployment. The results of this small study suggest that their use on operations
may be increasing. Smoking rates and caffeine consumption, on deployment, remain high in
the British military. A larger detailed study with greater representation among soldiers
deployed to forward operating bases would be helpful to fully appreciate the scale of
supplement use [11423].
The aim of one study was to describe the prevalence, trends and associated factors of
dietary supplements (DS) and anabolic-androgenic steroids (AAS) use among Finnish
adolescents. The sample comprised 30 511 adolescents aged 12-18 years, of which 22 519
(74 %) answered a questionnaire. It was also studied associations between 14
socioeconomic, health and health behavioural variables and DS and AAS use by logistic
regression. The proportion of respondents using DS was 45 percent during the past year and
it increased linearly by age. Vitamins (37 %) and herbal products (13 %) were the most
common DSs. In 1991, 9 percent of the boys aged 16-18 years reported protein use, while
the frequency in 2005 was 17 percent, which was a significant increase. AAS use was
uncommon; only 53 boys (0.5 %) and 20 girls (0.2 %) reported AAS use. The strongest
factors associated with DS use in multivariate model were physical exercise outside sports
clubs (odds ratio 1.9; 95 % confidence interval 1.6 to 2.2), and in sports clubs (odds ratio 1.7;
95 % confidence interval 1.5 to 1.9). Recurrent drunkenness (odds ratio 5.8; 95 %
confidence interval 1.5 to 21.6) and peer drug use in boys (odds ratio 2.1; 95 % confidence
interval 1.2 to 3.7) were the risk factors for AAS use, whereas physical exercise outside
sports clubs (odds ratio 0.3; 95 % confidence interval 0.1 to 0.5) was a protecting factor. It
552
was concluded that although the overall use of DS remained at the same level during the
study period, there was a slight trend towards increasing use of vitamin and protein
supplements. Dietary supplements use is associated with frequent sports participation and
poorer than average health, while anabolic-androgenic steroids use is associated with
health-compromising behaviours [10015].
Nutritional supplement (NS) use is widespread in sport. This study applied an integrated
social cognitive approach to examine doping attitudes, beliefs, and self-reported doping use
behavior across NS users (n=96) and nonusers (n=116). Following ethical approval, 212
competitive athletes (age mean 21; 137 males) completed self-reported measures of doping-
related social cognitions and behaviors, presented in an online format where completion
implied consent. Significantly more NS users (23 %) reported doping compared with
nonusers (6 %). NS users presented significantly more positive attitudes toward doping and
expressed a significantly greater belief that doping is effective. When presented with the
scenario that performance-enhancing substances are effective and increase the possibility of
winning, NS users were significantly more in favor of competing in situations that allow
doping. In sum, doping use is three-and-a-half times more prevalent in NS users compared
with nonusers. This finding is accompanied by significant differences in doping attitudes,
norms, and beliefs. Thus, this article offers support for the gateway hypothesis; athletes who
engage in legal performance enhancement practices appear to embody an "at-risk" group for
transition toward doping. Education should be appropriately targeted [13035].
Attitudes against doping among users of nutritional supplements

There is growing evidence suggesting that nutritional supplement (NS) use is strongly
associated to doping use in elite and amateur sports. However, there is a paucity of research
on the psychological processes that underlie this association. The present study investigated
the cognitive and behavioral components of the association between NS use and doping
among adolescent sub-elite athletes. Six hundred and fifty adolescent athletes completed a
questionnaire including measures of doping intentions, attitudes, norms, and beliefs about
NS use. The results showed that NS users who did not report doping use had significantly
stronger doping intentions and more positive attitudes and favorable beliefs toward doping
use, as compared with athletes who did not use NS. In support of the "shared mental
representations" hypothesis, the present findings show that NS use is associated with biased
reasoning patterns in favor of doping use. This mechanism may explain why some NS users
decide to engage in doping [14725].
Fitness supplements as a gateway substance for AAS use

Approximately 3 percent of young Americans have used anabolic-androgenic steroids (AAS).
A traditional model of adolescent substance use, the gateway hypothesis, suggests that drug
use follows a chronological, causal sequence, whereby initial use of a specific drug leads to
an increased likelihood of future drug use. Therefore, the use of illicit appearance and
performance enhancing drugs (APED), such as AASs, also follows an analogous
progression, whereby legal APEDs, (e.g. nutritional supplements) precedes illicit APED use.
It was examined the relationship between nutritional supplement use, beliefs about APEDs,
and APED use in 201 male (n=100) and female (n=101) undergraduates. Participants
completed measures of muscle dysmorphia (MDDI), body checking (BCQ, MBCQ), eating
disorder symptoms (EDE-Q), perfectionism (FMPS), positive beliefs about the efficacy-safety
of AAS use and APED use patterns. A series of covariance structure models (CSM) showed
body image disturbance, compulsive exercise, illicit drug use, and perfectionism,
independent of gender, were significant predictors of positive beliefs about AAS. Those who
used both fat burning and muscle building supplements reported the strongest beliefs in AAS
efficacy-safety, which was associated with higher likelihood of current illicit APED use. There
was evidence of significant indirect relationships between supplement use and illicit APED
553
use through contact with other AAS users and beliefs about AAS. The potential role for
nutritional supplement use in the initiation of illegal APED use is discussed. Future
prevention efforts may benefit from targeting legal APED users in youth. (PsycINFO
Database Record) [12024].
Complementary and alternative medicine (CAM)
Athletes are high achievers who may seek creative or unconventional methods to improve
performance. Western medicine as is practiced in many industrialised countries is generally
regarded as conventional, or orthodox, and its use has a long-established history in these
societies. Interest and use of complementary and alternative medicine (CAM) has, however,
been growing in recent times in Western countries, as reflected by the increasing number of
research papers in medical and scientific journals. The literature indicates that athletes are
among the heaviest users of complementary and alternative medicine (CAM) and thus may
pioneer population trends in CAM use. Surveys in many countries have suggested a high
use of CAM: in the United States, about a third of adults aged 18 years or older use CAM.
While nonathletes may use CAM for prevention, treatment or rehabilitation from
illness/injuries, athletes may possibly also use CAM for performance enhancement. If links
between sport motivation and doping exist, and athletes' sport motivation and CAM use are
related, a connection between athletes' CAM use and doping may also occur. Despite this
growing interest, the definition of what is complementary or alternative remains very
subjective and is certainly not universally accepted. While individual organisations have their
own definitions, the perceptions of the general population or the end users and even the
practitioners of the various forms of medicine of what constitutes CAM vary tremendously. In
addition, More physicians are also seeking training in CAM: there is an estimated 3,000
American physicians who integrate acupuncture into their practice and an estimated one-
third of homeopaths who are physicians or osteopaths. Unlike non-athletes, athletes may use
CAM not just for prevention, treatment or rehabilitation from illness or injuries, but also for
performance enhancement. Assuming that athletes' creative use of anything unconventional
is aimed at "legally" improving performance, CAM may be used because it is perceived as
more "natural" and erroneously assumed as not potentially doping. This failure to recognise
CAMs as pharmacological agents puts athletes at risk of inadvertent doping. The general
position of the World Anti-Doping Authority (WADA) is one of strict liability, an application of
the legal proposition that ignorance is no excuse and the ultimate responsibility is on the
athlete to ensure at all times whatever is swallowed, injected or applied to the athlete is both
safe and legal for use. This means that a violation occurs whether or not the athlete
intentionally or unintentionally, knowingly or unknowingly, used a prohibited substance/
method or was negligent or otherwise at fault. Athletes are therefore expected to understand
not only what is prohibited, but also what might potentially cause an inadvertent doping
violation. Yet, as will be discussed, athlete knowledge on doping is deficient and WADA itself
sometimes changes its position on prohibited methods or substances. The situation is further
confounded by the conflicting stance of anti-doping experts in the media. These highly
publised disagreements may further portray inconsistencies in anti-doping guidelines and
suggest to athletes that what is considered doping is dependent on the dominant political
zeitgeist. Taken together, athletes may believe that unless a specific and explicit ruling is
made, guidelines are open to interpretation. Therefore doping risk-taking behaviours may
occur because of the potential financial, social and performance gains and the optimistically
biased interpretation (that trying alternatives is part of the "spirit of sport") and doping risk-
taking behaviours may occur. This discussion paper seeks to situate the reader in a world
where elite level sports and CAM intersects. It posits that an understanding of the underlying
motivation for CAM use and doping is currently lacking and that anti-doping rules need to be
repositioned in the context of the emerging phenomenon and prevalence of CAM use. Data
from non-athlete patients suggests that ingested CAM substances are not viewed as
554
medications because they are perceived as “natural”. Thus athletes may use CAM in the
belief that it is more natural and, erroneously, not potentially doping (either as a “method” or
“substance” under the anti-doping Code). The lack of understanding of various forms of
medicine by patients and athletes may result in their not informing doctors (or coaches)
about non-conventional treatment use because it is not viewed as important or relevant to
their medical management. Patients thus risk complications from CAM drugs and their
interaction with prescribed medications. The additional consequence from failure to
recognise CAMs as pharmacological agents puts athletes at risk of inadvertent doping
[12015].
Connexion to eating habits
Health professionals concerned about the risks of adolescent obesity and disordered eating
practices need greater understanding of how families with adolescents manage food in
today's fast paced environment. One paper sought to gain conceptual understanding of the
food and eating routines of families with a female adolescent athlete from the perspectives of
mothers and daughters. Ten white, non-Hispanic mothers and their daughters were
purposively sampled from high school track and cross country teams in Upstate New York.
Informants completed in-depth, qualitative interviews. Researchers used the constant
comparative method to analyze transcripts for emergent themes and to build a conceptual
framework that represented the many factors and processes involved in the construction of
family food routines. Families varied in forms and patterns of family eating activities with
mothers playing a pivotal role in these routines. Family members' individual needs and
values were negotiated in constructing these routines. In this sample the daughters'
involvement in sports influenced family eating routines, but mothers' employment, ethnicity,
social support, income, and areas of residence also played a role. The model describes how
individual participants' food choice processes interact to produce family food routines. The
conceptual model can inform research and practice related to the family environments in
which adolescents experience food and eating [10016].
Eating disorders
Eating disorders do occur in male athletes. They are less prominent than in female athletes,
and therefore in danger of being missed. The high-risk sports fall into the same categories as
with females: aesthetic sports, sports in which low body fat is advantageous, such as cross-
country and marathon running, and sports in which there is a need to "make weight",
including wrestling and horse racing. Athletic involvement may foster the development of an
eating disorder. Some male athletes, in their preoccupation with body image, will abuse
anabolic steroids. While sports participation may contribute to the aetiology of an eating
disorder, the converse is also true. Exercise may be used as therapy for some cases of
eating disorder. In order to adequately treat eating disorders in the male athlete, it is first
essential to identify cases. Psychoeducation of athletes, their families, coaches and trainers
is an important first step. Counselling an athlete to pursue a sport appropriate to his body
type, or to leave his sport behind altogether (an unpopular recommendation from a coach's
perspective) can be important to treatment. Treatment of co-morbid psychiatric conditions is
essential. Treatment can be structured using a biopsychosocial approach, and all appropriate
modalities of therapy, including individual, family and group, as well as psychopharmaco-
therapy, where appropriate, should be applied [06017].
Eating disorders and anabolic steroids

Dissatisfaction with the body is very common in the population, in females in all ages as well
as among males. Studies on female and male body image show the role of the media in
defining and perpetuating body ideals, e.g. a muscular ideal male body type, or a thin female
555
ideal. A meta-analysis of the effects of the media on male body image concerns, yielded
similar effect sizes as those found with women. As a result of internalization of cultural
norms, females become dissatisfied with the lower part of their bodies from the waist down
and try to lose weight while males primarily want to change the shape of upper part of their
bodies (stomach and chest) and are more likely to desire an increase in weight. Body
dissatisfaction has been reported as a risk factor, and one of the strongest predictors for
onset of an eating disorder (ED) and is also associated with low self-esteem and depression.
Dissatisfaction with the body seems to be the common and prominent denominator, not only
between the sexes, but also between males with ED and males using anabolic androgenic
steroids (AAS). Both ED and the use of AAS may seriously affect physical health and the
psychological and social wellbeing of those who suffer from those problems. Comparisons
between males with ED, male bodybuilders and normal controls revealed that bodybuilders
more closely resembled the ED group than normal controls regarding body dissatisfaction
and loss of sexual desire. Other similarities found between males with ED and body-builders
including AAS users were characteristics such as perfectionism, ineffectiveness and low self-
esteem. An essential question is whether more similarities can be found between males with
ED and AAS users or if these groups differ in some essential respects. It is for example
unclear whether there is a distinction between males with ED and males using AAS
regarding the occurrence of underlying interpersonal profiles like negative self-image and the
severity of psychiatric symptoms. Based on earlier studies showing several similarities
between these groups, we anticipated that negative self-image and psychiatric symptoms
would be similar between males with eating disorders and males who recently used AAS.
Few studies have however investigated if there are other similarities in respect to self-image
or psychiatric symptoms between clinical samples of eating disordered males and males in
treatment for negative effects of AAS use. The aim of one study was to compare two clinical
samples, one of males with ED and one of males who used AAS, regarding self-image and
psychiatric symptoms. The study compared males with eating disorders (n=13) and males
who recently stopped AAS use (n=29) on self-image and psychiatric symptoms, using The
Structural Analysis of Social Behavior self-questionnaire and a shortened version of The
Symptom Check List. The eating disorder group reported significantly lower scores for Self-
emancipation and Active self-love and higher scores for Self-blame and Self-hate. Both
groups reported serious psychiatric symptoms. The common denominator between groups
was serious psychiatric symptomatology rather than negative self-image. It was concluded
that the negative self-image profile, especially self-hate, found among males with Eating
Disorders may indicate that the studied groups differ in aetiology of the underlying problems.
The serious psychiatric symptoms in both groups call staff to pay attention to any thoughts of
suicide due to severe depressive symptoms where by specialized psychiatric treatment may
be needed [13036].
Concomittant symptoms and signs
To determine whether adolescents who participate in a weight-related sport are at increased

risk for unhealthful weight-control behaviors and steroid use a study was performed. Subjects
were 4,746 adolescents (50 % males, 50 % females) from 31 public middle and high schools
in the Minneapolis/St Paul area of Minnesota. More males (20 %) than females (16 %)
reported participation in a weight-related sport. Males who reported participation in a weight-
related sport had an increased risk of past-week vomiting (odds ratio 5.7), laxative use (OR
6.8), as well as past-year vomiting (OR 4.9), laxative use (OR 3.4), diuretic use (OR 6.0), and
steroid use (OR 3.7), compared with those males who did not report participation. Females
who reported participation in a weight-related sport had an increased risk of past week
vomiting (OR 2.1), as well as past year vomiting (OR 2.0), laxative use (OR 2.6), and steroid
use (OR 2.6), compared with those who did not report participation in a weight-related sport.
The current study shows that participation in a sport that adolescents perceive as
556
emphasizing weight is strongly associated with unhealthful weight-control behaviors and
steroid use. Preventive efforts, targeting parents, coaches, and adolescents are needed to
decrease this risk [07020].
Adolescents with learning difficulties

One study investigated the relationships among sleep problems, learning difficulties and
substance use in adolescence. Previous research suggests that these variables share an
association with executive functioning deficits, and are intertwined. The sample comprised
427 adolescents (medain age 16 years) attending remedial schools and 276 adolescents
(median age 15 years) attending a mainstream school. Participants completed anonymous
self-report questionnaires. Results indicated that adolescents without learning difficulties
were more likely to use tobacco, methamphetamine and cannabis, whereas those with
learning difficulties engaged in more inhalant use. Adolescents who had more sleep
problems were more likely to use tobacco, alcohol, methamphetamine, cannabis, inhalants,
cocaine, ecstasy and any other illegal drug. Adolescents with learning difficulties had more
sleep problems than those without learning difficulties. However, sleep problems remained
independently associated with tobacco, cannabis and inhalant use when learning difficulties
were taken into account [11422].
Female adolescents
During the 1990s, 3 different national surveys of US adolescents documented a 2-fold to 4-
fold increase in the prevalence of anabolic steroid use among adolescent girls. Public
awareness concerning escalating female anabolic steroid use further heightened in 2004
when the Centers for Disease Control and Prevention reported that more than 7 percent of
ninth-grade girls indicated current or prior anabolic steroid use, a level exceeding that of
some young male subgroups. National attention focused on steroid use in adolescent girls
when it became a topic discussed during the 2005 congressional hearings on drug use in
sports. Previous associations with female anabolic steroid use have been limited to older
women, and most reports of mature women taking anabolic steroids have related the use to
competitive athletics and to bodybuilding. Using the nationally representative 2003 Youth
Risk Behavior Surveillance System data set, it was examined the characteristics of girls
reporting anabolic steroid use. Because of the association between steroid use and sports
participation among older women, we particularly explored that relationship among girls
reporting prior or ongoing anabolic steroid use. Female students in grades 9 through 12
(n=7544) self-reported anabolic steroid use was compared with other health-related
behaviors and with sports participation. Prior or ongoing anabolic steroid use was reported
by 5.3 percent of female high school students. Those adolescent girls had a marked increase
in other health-compromising behaviors, including past 30-day use of alcohol (odds ratio, 8.8;
95 % confidence interval 5.5 to 14.2), cigarettes (OR, 5.1), marijuana (OR, 7.9), cocaine
(OR, 10.8), and diet pills (OR, 4.9). They were more likely to carry a weapon (OR, 7.5), have
had sexual intercourse before age 13 years (OR, 2.9), and have had feelings of sadness or
hopelessness almost every day for at least 2 consecutive weeks (OR, 4.1). They were less
likely to play school-sponsored team sports (OR, 0.52). Those who reported anabolic steroid
use were more likely to use extreme measures to lose weight, including greater 30-day use
of vomiting and laxative use (OR 5.0) and diet pills, powders, or liquids taken for weight loss
(OR, 4.9). Last, psychological problems were prominent among these adolescents. Anabolic
steroid users were more likely to indicate having had feelings of sadness or hopelessness for
2 consecutive weeks or more (OR, 4) and were more likely to have attempted suicide (OR,
7.3). Overall, 52 percent of female students indicated participation on team sports. Team
sports participants were less likely to be steroid users compared with team sports
nonparticipants (OR, 0.52). In addition, being in sports did not seem to identify a unique
subgroup of steroid users. Anabolic steroid users in team sports were more likely to use
seatbelts (OR 3.1) and condoms or birth control pills (OR, 5.2), compared with anabolic
557
steroid users who were not in team sports. Steroid users participating in sports shared the
same problem behaviors as steroid users not participating in team athletics. It was concluded
that self-reported anabolic steroid use is not confined to adolescent girls in competitive
athletics and is an indicator of adolescent girls with a marked increase in a cluster of other
health-harming behaviors. Adolescent female anabolic steroid users are characterized by
polysubstance abuse and by a marked increase in other health-harming behaviors.
Compared with nonusers, odds are higher that they became sexually active at a younger age
and have had more sexual partners; they are more likely to carry weapons and to have
experience with violence. Along with greater controlled and illicit drug use, they are more
likely to resort to harmful weight loss practices. These adolescent girls' mental health is more
likely to be impaired, with more than two thirds having had feelings of depression and with
almost half having planned or attempted suicide [07021].
Performance versus recreational drugs
The relationships between projected use, self-reported behavior and attitudes to

performance-enhancing (PED) and recreational (RD) drugs were investigated among 82
competitive Hungarian athletes, with 15 percent admitting using PED and 32 percent using
RD. Both the observed doping estimations (even those made by non-users) and self-
admitted use were considerably higher than the average rate of positive doping tests (2 % of
all tests). The notable overestimation by PED users (35 % vs 17 %) was in keeping with the
false consensus effect. A prediction model with attitude and projection to the likelihood of
PED use suggested at least a 70 percent chance of self-involvement of athletes, with
responses at or above the median scores (Performance Enhancement Attitude Scale ≥ 60
and estimation ≥ 50%) on the two independent measures. Users overestimated the
prevalence of doping in their sport but not RD use, with the converse holding for RD users'
views of doping. PED users also showed a significantly more lenient attitude toward doping.
This domain-specific characteristic adds new information to the ongoing research effort in
understanding drug-doping co-morbidity. The reasons for elevated in-group projection are
discussed, along with the potential application of this phenomenon in doping epidemiology
studies [11011].
Health promotning effects of sports
To study whether participation in organized sports during adolescence predicts increased

smoking of tobacco, alcohol intoxication and cannabis use from late adolescence to
adulthood when controlling for potential confounders by a survey of national sample of
Norwegian high school students (aged 13-19 years) in 1992 (T1) followed-up in 1994 (T2),
1999 (T3) and 2006 (T4) (n=3251). Outcome measures included smoking of tobacco and 12-
month prevalences of alcohol intoxication and cannabis use, respectively. Confounders
included pubertal timing, friends' drug use, perceived social acceptance, grades and parental
socio-economic status. Latent growth curve analyses showed that initial level of participation
in organized sports predicted growth in alcohol intoxication. Those involved initially in team
sports had greater growth in alcohol intoxication, but lower growth in tobacco use and
cannabis use, during the adolescent and early adult years compared to those involved in
technical or strength sports. Practising endurance sports, as opposed to technical or strength
sports, predicted reduced growth in alcohol intoxication and tobacco use. Sports participation
in adolescence, and participation in team sports in particular, may increase the growth in
alcohol intoxication during late adolescent and early adult years, whereas participation in
team sports and endurance sports may reduce later increase in tobacco and cannabis use
[09007].
558
Lifestyle alteration
Lifestyle's alterations are hazardous for health. On one hand they produce a high rate of
mortality and disease, on the other hand they cause a reduction of work outcomes and an
increase of occupational accidents with important consequences for both worker's health and
his/her financial status. The aim of one study was to review the scientific literature for
possible relationships between mental health and lifestyle alterations of young workers. It
was considered as lifestyle factors the attitudes towards: smoking, alcohol consumption,
eating, use of medications and doping substances, physical activity and sleeping. From the
study it clearly emerges the existence of correlation between lifestyle habits and mental
health; in fact behavioral alterations can produce problems of mental health and vice versa.
Furthermore, some work peculiarities can lead to psychic disturbances and/or to unhealthy
habits which can themselves cause negative effects on working activity. It is very important
for young workers to understand that unhealthy behaviors, which can be corrected, are
hazardous in terms of health and safety for both the single worker and the collectivity and
that those behaviors can enhances the other working risks. Because there is a close
interaction between mental health and lifestyles, it would be necessary a careful promotion of
mental health on workplaces and to take all the preventive measures, with particular regard
for those related to the work organization, in order to reduce the onset, exacerbation and
unmasking of mental disorders and psychological difficulties. In working environment, the
occupational health physician and his relationship with the patient are of fundamental
importance. During preventive and periodic medical examinations, the occupational health
physician should take detailed information on young worker's habits regarding smoking,
alcohol consumption, eating, physical activity, sleeping, pharmacological abuse and possible
presence of mental disorders and furthermore he/she should actively take part in the
information and education process of the worker [07012].
Prediction of later life-style
The aim of one study was to examine the associations between self-rated health (SRH),
physical activity and other lifestyle habits among former athletes and referents in late
adulthood. Male athletes (n=514) who represented Finland from 1920 through 1965 and
referents (n=368) who were classified healthy at the age of 20 years participated in this
population-based cohort study. The present analysis was based on a questionnaire study in
2001. SRH was assessed by a single question. Univariate binary and multivariate logistic
regression analyses were used to examine the associations of health-related behaviours with
SRH. The majority of former athletes (64 %) rated their health better than referents (48 %). A
higher percentage of the athletes (54 %) compared to the referents (44 %) belonged to the
most physically active groups (MET quintiles IV-V). A high percentage of the athletes (77 %)
and referents (79 %) were occasional or moderate alcohol users. The proportion of never
smokers among athletes was 59 percent and among referents 37 percent. Among current
smokers there were no differences in nicotine dependence between athletes and referents.
In the univariate analysis the odds of reporting good SRH was 2 times higher for athletes
than for referents. In multivariate logistic regression analysis, former participation in team and
power athletic groups had significantly higher SRH than the referents even after adjusting for
age, level of physical activity, alcohol and smoking habit, and occupation. People, who
participated in very active physical exercise in their youth, as indexed by participation in
competitive sports by elite athletes, continue a physically active lifestyle, and maintained
healthier lifestyle. They had significantly higher SRH than the referents in their senior years,
which were not totally explained by their physically active and healthier lifestyles [10437].
Risk factors for doping

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Grounded conceptually in social cognitive theory, this research examines how personal,
behavioral, and environmental factors are associated with risk perceptions of anabolic-
androgenic steroids. Ordinal logistic regression and logit log-linear models applied to data
gathered from high-school seniors (n=2,160) in the 2005 Monitoring the Future study showed
significant explanatory effects for gender, race, exposure to drug spots, steroid availability,
peer use of steroids, sensation-seeking, depression, and self-esteem. Females, African
Americans, and those who had seen drug spots the most frequently estimated higher levels
of risk associated with steroid use, while those who indicated ease in obtaining steroids and
those with close friends who had used the drugs estimated lower risk. Also estimating lower
levels of risk were sensation seekers, those who appeared depressed, and those with low
levels of self-esteem. Analyses reveal how steroid risk determinants may differ from those
related to methylenedioxy-methamphetamine (i.e., MDMA, ecstasy) and marijuana use
[09008].
Illicit anabolic-androgenic steroid (AAS) abuse, though an important public health problem,
remains inadequately studied. Almost all AAS abusers are male and lift weights, but the risk
factors for AAS use among male weightlifters remain poorly understood. It was recruited 233
experienced male weightlifters, of whom 102 (44 %) reported lifetime AAS use, and
assessed their childhood and adolescent attributes retrospectively, using structured clinical
interviews and computerized questionnaires. This cross-sectional cohort approach-a design
that we have formally presented in the recent methodological literature-utilizes a study
cohort, not selected for outcomes of interest, and assesses exposures and outcomes
retrospectively. It was hypothesized that conduct disorder and body-image concerns would
be major risk factors for subsequent AAS use among male weightlifters. Within the study
population, many attributes showed little association with AAS use, but conduct disorder and
body-image concerns showed strong associations. For individuals with prior conduct disorder
versus those without, the hazard ratio (95 % confidence interval) for subsequent AAS use
was 2.2 (1.5 to 3.4). For individuals in the middle versus lowest tertile of scores on a
retrospective adolescent muscle-dysmorphia scale, the hazard ratio was 1.5 (0.84 to 2.6); for
the highest versus lowest tertile, the hazard ratio was 3.3 (2.0 to 5.3). It was concluded that
conduct disorder and body-image concerns represent important risk factors for AAS use
among male weightlifters. Thus, assessment of these attributes may help to identify
individuals most likely to require interventions to discourage this form of substance abuse
[12029].
Much of the literature investigating the relationship between sports participation and
substance use has focused upon student populations, with little focus being given to athletes
who participate at elite levels. Identifying why some athletes may be at a greater risk for
substance use can help in the design and implementation of prevention initiatives. Data for
one study was from 1684 self-complete surveys with elite Australian athletes. Eight percent
(n=134) of the sample reported the use of at least one of the six illicit drugs under
investigation (ecstasy, cannabis, cocaine, meth/amphetamine, ketamine and GHB) in the
past year. Having been offered or having had the opportunity to use illicit drugs in the past
year, knowing other athletes who use drugs and identifying as a 'full-time athlete' were
significant predictors of past-year illicit drug use, while having completed secondary
education or a post-school qualification was associated with a lower likelihood of past-year
illicit drug use. Athletes are part of a sportsnet that includes family, coaches, support staff
and other athletes, and these relationships may encourage the use, supply and demand for
drugs. The current findings suggest that relationships with some of those in the sportsnet
may play an important role when understanding illicit drug use among elite athletes. As
education appears to be associated with a lower likelihood of illicit drug use among this
group, initiatives should encourage athletes to engage in off-field pursuits which may also
560
help prepare them for life after sport [12030].
Association of PED use with other high-risk behaviors

Athletes and nonathlete weightlifters that use PEDs often engage in other high-risk health
behaviors. In addition to the risks associated with concomitant use of other drugs such as
alcohol and opiates with AASs, users of high doses of AAS may be more susceptible to rage,
antisocial and violent behaviors, and suicidality. Sharing of needles and other paraphernalia
and unprotected sex may increase the risk of infections such as hepatitis and HIV. The use
of PEDs, especially in conjunction with analgesics or stimulants, may allow athletes to
engage in extremely high-intensity exercise, increasing the risk of musculoskeletal injuries
[14426].
Parenteral divorce
Several studies have reported an increase in risk behaviors among adolescents after
experience of parental divorce. The aim of one study was to investigate whether parental
divorce is associated with risk behavior among adolescents independent of mental health
problems, first when early divorce was experienced, and second after experience of late
parental divorce. One prospective (n=1861) and one cross-sectional study (n=2422) were
conducted using data from two Young-HUBRO surveys in Oslo, Norway. All 15/16 year-old
10(th) grade students who participated in the first survey in the school year 2000/01 were
followed-up in 2004 when they were 18/19 year-olds. The follow-up rate was 68 percent. The
prospective study investigated the influence of late parental divorce that occurred between
the age of 15/16 and 18/19. In the cross-sectional study we focused on early parental divorce
that occurred before the participants were 15/16 year-old. In the prospective study we could
not discern a significant association between experiencing late parental divorce and an
increase in risk behaviors among 18/19 year-old adolescents. In the cross-sectional study
parental divorce was significantly associated with cigarette smoking and using doping
agents. Parental divorce that occurs when the children of divorced parents are 15/16 year-
old or younger is associated with an increase in cigarette smoking and use of doping agents.
However, no evidence of significant association is found between experience of late parental
divorce and risk behaviors in late adolescence [14441].
Advanced cosmetic doping

Doping is considered to be a major sports problem. One article described a new threat and
challenge to the sport of bodybuilding; the nonmedical use of a chemical in order to mimic
muscle hypertrophy. Although muscle fillers are not new, being used for cosmetic purposes
in medicine for a long time, the illegal use of muscle fillers has been increasing during the
last few years and decades. The history of cosmetic doping, with particular attention to the
Brazilian case, is discussed. Limitations are noted and future needed research is suggested
[14442].
Psychiatric comorbidity diseases
"Comorbidity phenomenon" defines the not univocal interrelation between medical illnesses
and psychiatric disorders, each other negatively influencing morbidity and mortality. Most
severe psychiatric disorders, such as schizophrenia, bipolar disorder and depression, show
increased prevalence of cardiovascular disease, related to poverty, use of psychotropic
medication, and higher rate of preventable risk factors such as smoking, addiction, poor diet
and lack of exercise. Moreover, psychiatric and organic disorders can develop together in
different conditions of toxic substance and prescription drug use or abuse, especially in the
emergency setting population. Different combinations with mutual interaction of psychiatric
disorders and substance use disorders are defined by the so called "dual diagnosis". The
561
hypotheses that attempt to explain the psychiatric disorders and substance abuse
relationship are examined as common risk factors, psychiatric disorders precipitated by
substance use, psychiatric disorders precipitating substance use (self-medication
hypothesis), and synergistic interaction [13033]
Diagnostic and therapeutic difficulty concerning the problem of dual diagnosis, and legal
implications, are also discussed. Substance induced psychiatric and organic symptoms can
occur both in the intoxication and withdrawal state. Since ancient history, humans selected
indigene psychotropic plants for recreational, medicinal, doping or spiritual purpose. After the
isolation of active principles or their chemical synthesis, higher blood concentrations reached
predispose to substance use, abuse and dependence. Abuse substances have specific
molecular targets and very different acute mechanisms of action, mainly involving
dopaminergic and serotoninergic systems, but finally converging on the brain's reward
pathways, increasing dopamine in nucleus accumbens. The most common substances
producing an addiction status may be assembled in depressants (alcohol, benzodiazepines,
opiates), stimulants (cocaine, amphetamines, nicotine, caffeine, modafinil), hallucinogens
(mescaline, LSD, ecstasy) and other substances (cannabis, dissociatives, inhalants). Anxiety
disorders can occur in intoxication by stimulants, as well as in withdrawal syndrome, both by
stimulants and sedatives. Substance induced mood disorders and psychotic symptoms are
as much frequent conditions in ED, and the recognition of associated organic symptoms may
allow to achieve diagnosis. Finally, psychiatric and organic symptoms may be caused by
prescription and doping medications, either as a direct effect or after withdrawal. Adverse
drug reactions can be divided in type A, dose dependent and predictable, including
psychotropic drugs and hormones; and type B, dose independent and unpredictable, usually
including non psychotropic drugs, more commonly included being cardiovascular, antibiotics,
anti-inflammatory and antineoplastic medications [13033].
Sponsoring of sports
Organised sport provides an important setting for health promotion. Peak sporting
organisations have a role in assisting and overseeing sports clubs, including providing
funding opportunities. As such, sponsorship of these organisations may influence the funding
of community sport. One study aimed to describe the nature and scope of peak sporting
organisations' sponsorship, and particularly food and beverage company sponsors. An
analysis of national and state sporting organisations'websites for the nine most popular
sports for children and from four Australian states and territories was conducted using a
structured survey tool. Information collected included the number and type of sponsors and
sponsorship policies.The nature of food and beverage sponsors was defined as more healthy
or less healthy using criteria from a Delphi survey. 443 sponsors were identified across 55
websites. Overall, 9 percent of sponsors were food companies and 3 percent were alcohol
manufacturers. The majority of food companies (63 %) and alcohol manufacturers (100 %)
did not meet criteria as healthy sponsors. It was thus concluded that sponsorship of peak
sporting organisations is widespread and consists of a relatively high proportion of alcohol
manufacturers and food companies, some of which produce products considered to be
unhealthy. This sponsorship may influence community sport through sponsored sporting
programs or by indicating sponsors' acceptability [11005].
To examine the relationship between direct alcohol and non-alcohol sponsorship and
drinking in Australian sportspeople 652 (51 % female) individuals completed questionnaires
on alcohol and non-alcohol industry sponsorship (from bars, cafes etc.), drinking behaviour
(Alcohol Use Disorders Identification Test (AUDIT)) and known confounders. Thirty-one
percent reported sponsorship (30 % alcohol industry; 4 % both alcohol and non-alcohol
industry and 2 % non-alcohol industry only.) Multivariate regression showed that receipt of
562
alcohol industry sponsorship was predictive of higher AUDIT scores, but non-alcohol industry
sponsorship and combinations of both were not. Governments should consider alternatives
to alcohol industry sponsorship of sport. Hypothecated taxes on tobacco have been used
successfully for replacing tobacco sponsorship of sport in some countries, and may show
equal utility for the alcohol industry's funding of sport [11006].
Determining children's exposure to food and beverage company sponsorship, and the effect
of this exposure, is important in establishing the extent to which there may be health and
societal consequences. One paper aimed to provide preliminary evidence on the scope and
potential effects on children of unhealthy food and beverage sponsorship. A review of
published literature and media and marketing reports was conducted to determine the types
of food and beverage sponsorship campaigns that children are exposed to, and the effect of
corporate sponsorship (including tobacco and alcohol) on children and adolescents. A large
range of food and beverage sponsorship activities, in Australia and internationally, were
identified for both school and sport settings. In particular, food and beverage companies
have attempted to develop a marketing presence at all levels of professional and community
sport. No information was identified measuring the effect of food and beverage company
sponsorship on children and adolescents. However, empirical evidence from consumer
studies relating to tobacco and alcohol sponsorship has repeatedly demonstrated that
sponsorship has an impact on children's product recall and product-related attitudes and
behavioural intentions. It was concluded that while there is no available research on the
direct effect of food and beverage sponsorship, the demonstrated effects of tobacco and
alcohol sponsorship on children's product awareness, preferences and consumption are
likely to be applicable to food companies [11420].
Information to dopers
One study was designed to investigate anabolic steroid users' experiences of, and
motivations for, use. Five men and six women users took part in in-depth interviews. Four
themes emerged: Steroid Use vs Abuse; Side-effects; Trusted Information Sources; and
Social Pressure. Many users believed that steroids used in moderation were safe. Serious
side-effects (liver and kidney damage, hypertension) were not significant disincentives.
Information from health professionals tended to be mistrusted because it was not based on
first-hand experience of use. Social support, especially from within the body building
community, was an important motivator. It is concluded that intervention programmes need
the support of the body building community in order to be effective [06018].
Medical practitioners' knowledge, attitudes and beliefs
Central to the work of many medical practitioners is the provision of pharmaceutical support
for patients. Patients can include athletes who are subject to anti-doping rules and
regulations which prohibit the use of certain substances in and out of competition. One paper
examined the evidence on medical practitioners' knowledge, attitudes and beliefs towards
doping in sport. A systematic search strategy was followed. Research questions and
relevance criteria were developed a priori. Potentially relevant studies were located through
electronic and hand searches limited to English language articles published between 1990
and 2010. Articles were assessed for relevance by two independent assessors and the
results of selected studies were abstracted and synthesised. Outcomes of interest were
knowledge, attitudes and beliefs in relation to doping in sport. Six studies met the inclusion
criteria and were examined in detail. Samples reflected a range of medical practitioners
drawn from the UK, France, Greece, Italy and Ireland. The investigations varied with respect
to outcome focus and quality of evidence presented. It was concluded that whilst the extant
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empirical research posits a negative attitude towards illegal performance enhancement
combined with a positive inclination towards doping prevention, it also exposes a limited
knowledge of anti-doping rules and regulations. Insufficient education, leading to a lack of
awareness and understanding, could render this professional group at risk of doping
offences considering Article 2.8 of the World Anti-Doping Agency Code (WADC). Moreover,
in light of the incongruence between professional medical codes and WADC Article 2.8,
medical professionals may face doping dilemmas and therefore further discourse is required.
At present, the current evidence-base makes it difficult to plan developmentally appropriate
education to span the exposure spectrum. Addressing this situation appears warranted
[11007].
Ireland
One study examined General Practitioner's (GP) knowledge, practice and training
requirements in relation to doping in sport in Ireland. All 2083 GPs on the Irish College of
General Practitioners register received a postal questionnaire, yielding a 37 percent
response rate (n=771, 63 % male, average age 46 years). Results revealed that 14 percent
deemed their knowledge of doping agents to be good or very good, 12 percent had
completed specific training modules in doping or sport, and 24 percent were connected with
a specific sport as a team doctor or advisor. Over one in four (28 %) had been consulted for
advice on doping in sport, 33 percent possessed the current list of prohibited substances,
and 25 percent knew of the Irish Sports Council's drug-testing procedures. The current
initiatives to discourage doping in sport were felt to be ineffective, and although 92 percent
indicated that GPs had a role to play in the prevention of doping in sport, only 9 percent felt
adequately trained for such a role. There was overwhelming support for further training
among GPs, although the most appropriate method of providing training is complex and
requires strategic planning [09009].
France
To examine the attitudes to, and knowledge of, doping in sport of French general
practitioners (GPs), and their contact with drug taking athletes on an everyday basis a total of
402 GPs were randomly selected from all over France and interviewed by telephone, using a
prepared script. The response rate was 51 percent (153 men and 49 women; mean (SD) age
45.6 (5.6) years). Of the respondents, 73 percent confirmed that they had the list of banned
products, but only 35 percent stated that they were aware of the latest French law, brought
into effect in March 1999, concerning the fight against doping. Some 11 percent had directly
encountered a request for prescription of doping agents over the preceding 12 months (the
requested substances were mainly anabolic steroids, stimulants, and corticosteroids), and
10% had been consulted by an athlete who was using doping drugs and was frightened of
the health risks (the substances used were mainly anabolic steroids). Over half (52 %) of the
GPs favoured the prescription of drug substitutions to athletes who used doping agents.
According to 88 percent of respondents, doping is a public health problem, and 80 percent
stated that doping is a form of drug addiction. Most (89 %) said that a GP has a role to play
in doping prevention, but 77 percent considered themselves poorly prepared to participate in
its prevention [03019].
Anabolic steroid users' attitudes towards physicians
To assess anabolic-androgenic steroid (AAS) users' trust in the knowledge and advice of
physicians it was interviewed. AAS users and non-users. Eighty weight-lifters (43 AAS users,
37 non-users) were recruited by advertisement in Massachusetts and Florida, USA. Personal
interviews and questionnaire responses, including subjects' ratings of physicians' knowledge
regarding various health- and drug-related topics. AAS users also rated their level of trust in
various sources of information about AAS. Both groups of subjects gave physicians high
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ratings on knowledge about general health, cigarette smoking, alcohol, and conventional
illicit drugs, but gave physicians markedly and significantly lower ratings on knowledge about
AAS. When rating sources of information on AAS, users scored physicians as no more
reliable than their friends, Internet sites, or the person(s) who sold them the steroids. Forty
percent of users trusted information on AAS from their drug dealers at least as much as
information from any physician that they had seen, and 56% had never revealed their AAS
use to any physician. It was concluded that AAS users show little trust in physicians'
knowledge about AAS, and often do not disclose their AAS use to physicians. These
attitudes compromise physicians' ability to educate or treat AAS users. Physicians can
respond to these problems by learning more about AAS and by maintaining a high index of
suspicion when evaluating athletic male patients [04019].
Contagiousness of doping
Using a psychosociological approach, the purpose of one study was to identify and
understand the use of doping substances by young elite cyclists in Swizerland. Semi-
structured interviews were conducted with young cyclists who were hoping to find a
professional team and cyclists who had recently become professional. All of the young
cyclists interviewed took nutritional supplements and believed that they improved their
performance, which has been shown by other scholars to be a risk factor for doping. These
cyclists believed that doping at the professional level in cycling was acceptable but did not
approve of it at the amateur level. They were attracted to doping; they were open to using
doping substances themselves if it was the key to continuing their cycling career, but only
after they became professional. Team staff, doctors, parents and friends helped to create a
"clean" environment that prevented the young cyclists from doping before becoming
professional. The more experienced cyclists, who doped or used to dope, transmitted the
culture of doping to the young cyclists, teaching them doping methods and which substances
to use. This study could help to improve prevention and help to detect doping, as it is clear
that doping behaviours begin at the amateur level [09010].
Economical aspects
In 1990, the illicit steroid market was estimated to be USD 400 million. Steroid cycles,
typically lasting 6 to 14 weeks, can cost hundreds of dollars. A cycle consists of daily oral
doses plus weekly or monthly intramuscular depot injections. Some users take multiple
cycles per year [07031].
Illegal steroids entering the United States and distributed to athletic and at-risk populations
has increased dramatically. It is now estimated to be an over 100 million US dollar black
market for steroids in the US alone, with more than 80 percent manufactured in Mexico.
Today performance-enhancing programs and drugs are not the exclusive province of elite
athletes, but have spread to health clubs, high schools and other at-risk populations, creating
an over $1.4 billion US dollar industry that is growing daily as new compounds are
synthesized and marketed. Projecting these figures internationally suggests that the illegal
steroid market alone approaches a billion US dollars annually [08006].
It is estimated that an eight weeks performance enhancement regime of pharmaceutical

grade recombinant human growth hormone will cost about USD 2000, well out of the range
of an adolescent and the majority of weekend athletes [08006].
Economic incentives for doping

To date, the sports community also may not have sufficiently taken into consideration the
565
typical calculus of an athlete (or supervisor) who potentially considers employing a doping
strategy. This calculus, of the advantages and disadvantages of actively doping, typically
includes a variety of factors such as the athlete's expected benefits of doping, including the
additional honor derived from additional sporting success and extra pecuniary income.
“Expected” means that the athlete implicitly or explicitly forms an expectation value which
combines his or her probability of no detection/conviction, the probability of “success” of the
illicit behaviour (i.e. the technical efficiency of the treatment), and the gross pecuniary and
nonpecuniary income from the doping strategy. The expected disutility (in economic terms:
“costs”) which has to be subtracted from the gross utility includes the direct costs, for
example, for the procurement and application of the drugs, the expectation value for loss of
honor upon discovery, and other nonpecuniary costs, such as health risks and expected
financial losses resulting from financial penalties or from being barred from sporting
competitions as a result of being found guilty of doping. It also includes the pecuniary and
nonpecuniary opportunity costs, that is, the net utility of a legal behavior which has to be
abandoned on the occasion of illicit behavior. Doping athletes expect a positive net benefit
from the doping strategy due to such a rational assessment. If this net benefit is higher than
their losses from the violation of their own moral values, they opt for the doping strategy. It is
now time to check whether – beside or instead of – ever enhancing the probability of
detection by increasing the quantity (and the quality!) of laboratory controls – if other anti-
doping policies would be more effective [14416].
The technical efficiency of doping strategies could be reduced by optimization of the design
of competitions in order to reduce expected benefits from doping in the above mentioned
athletes’ calculus. The Tour de France, for example, with its usual daily distances, the
kilometers covered at altitude, and short recovery periods, leads to extreme states of
exhaustion, which make the technical efficiency of certain doping practices, from the
perspective of athletes, seem high. But, in cases like the 100-m sprint in track and field, it is
difficult to image a different design of competition. What about the most radical change of
design: canceling the competition (from the Olympic Program) for a certain period, if a certain
number of doping offenses were registered? In addition, the pecuniary incentives for doping
are generally high in the doping affected sports. All of them are “media” sports, with few
exceptions such as weightlifting, where the technical efficiency of doping may be extreme.
The high incomes in these sports are due to the fact that the athletes produce a high number
of spectator contacts for the sponsoring corporations. The large television audience figures
lead to a strong willingness to pay for the sponsorship and/or television commercials by the
companies. The audience responds to doping only to a small extent with less television
consumption. There is also little evidence of consumer boycotts against the products of
companies that intentionally or negligently allow doping among “their” teams. In fact,
sometimes the opposite is true. The watchmaker Festina, sponsor of a cycling team, which
provoked the Tour de France scandal in 1998, claimed that it reached its main goal – to
increase the level of brand awareness—precisely because of the scandal. Therefore, in
many countries public television has a special responsibility. Its financing, through quasi-tax
contributions, is usually justified by the fact that it secures the broadcasting of “merit” goods,
that is, cultural and sports events with a particular social value, which commercial TV would
not broadcast, because the events do not attract sufficient public attention. Implicitly, the
obligation of – at least public TV – should include the nontransmission of “demerit” events –
such as sports events that have fostered systematic doping and imply that cheating may be
worthwhile [14416].
Importance of sports medicine as a medical speciality
In Europe, participation in physical activity has been growing among people of all ages.
Thus, there is an increasing demand for care relating to sports medicine and this has
566
promoted the development of specialised sports physicians. Sports medicine involves a wide
range of professionals with functions of taking care of active population, recreational and
competitive athletes upon different aspects: curative, rehabilitative and preventive. In the light
of an higher demand of expertise and sport-specific burden of knowledge, such as a further
development of the phenomenon doping with all the related moral, legal and health
implications, the sport physician has to deal with a complex picture. As a result, the need to
provide prevention at all levels has become one of the most important objectives of sports
medicine. One article aimed to give a brief overview of the state of the art of this specialty in
Europe and to describe definitions, scopes and educational perspectives of the Sport
Medicine Specialty Training Core Curriculum to be adopted in the EU [09013].
The participation of sports physicians in the "doping" of athletes with banned drugs can be
documented as far back as the 1890s. Concern about the ethics and safety of doping elite
athletes appeared during the 1920s and 1930s as sport became an increasingly important
form of popular culture. While organized medicine has opposed doping as a matter of policy
at least since the 1950s, sports physicians have never adequately confronted the conflicts of
interest that arise when they choose to work with elite athletes whose first priority is
performance rather than with healing in the traditional sense. Confronted with the demands
of their athlete-clients, sports physicians have divided into two factions regarding the wisdom
and propriety of administering doping drugs to athletes. While most physicians are, in all
likelihood, unwilling to violate laws, regulations, and medical standards by doping athletes, a
significant minority of doctors has used one or more arguments to justify doping athletes:
drugs are necessary to compete effectively; athletes should be free to medicate themselves
as they please; drugs do not differ essentially from other performance-enhancing techniques
or equipment; and medically supervised doping is safer than self-medication by athletes.
Physicians can also rationalize doping as an occupational requirement of some professional
athletes. In summary, physicians have played a significant, and largely unacknowledged, role
in the doping of many elite athletes over the past 50 years [02010].
Impact of national programs
Physical activity and sports are considered as one of the determinants of health. The aim of
pne study was to review the rationale for the formulation of this public health issue and its
integration in national action plans. The study shows that fourteen national programmes were
drafted and implemented between 2001 and 2006 by seven institutions. The research
methodology was based on crossing data obtained from semi-directed interviews and
documents regarding the design, implementation and follow-up of these programmes. For
the conditions of the success, the fourteen actions scored an average of 175 + 67 out of 300
percent. Public health actors and professionals must be given more opportunities to involve
themselves and engage in developing stronger relationships and linkages, in particular with
the institutional and community settings. In general, the most invested parts of a programme
are the structural and operational aspects of activities. Six significant points surfaced from
the study: consideration of drug use as an addictive behaviour; recognition of the
psychological stress of professional athletes; acknowledgment of youth as being at high risk
for doping behaviour; integration of the concept that physical activity and sports must take
the benefit/risk perspective into account; and the necessity to promote health. Through the
exchange of numerous local and regional experiences, an optimisation of their synergistic
connections was made possible on a continuum extending from "health promotion through
physical activity and sports" to "prevention of drug-use and doping behaviours".
Professionals have been able to develop actions in the above-mentioned domains across
this continuum that have, to date, remained isolated. Proposals are made to strengthen
these dynamics. Other health determinants and public health priorities could be investigated
with the same methodology [09014].
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Staffing protocols in high-performance sport
A common feature of the current doping scandals appears to be that those organisations
under scrutiny have allowed external “experts” or “gurus” to drift in and out of their
organization and control supplementation practices in the organisation, without actually being
a bona fide part of the organisation’s staffing structure. There is no point having antidoping
policies and codes of conduct if individuals are allowed to drift into the organisation and
influence the supplementation policy, without those individuals being legally bound to the
anti-doping policy and code of conduct. No individual other than those on the
supplementation panel should be able to influence or administer supplements. The issue of
doping and the challenges for anti-doping organisations in staying ahead of scientific
developments and remain [13027].
Law issues
Detection of performance-enhancing drugs in sports has received increasing visibility.

Athletic drug testing uses sophisticated technology and both interindividual (population) and
intraindividual reference ranges to interpret data. An effective program must incorporate
educational and adjudication components in addition to testing. The difficult interface
between science and the law is evident in many recent sports arbitration decisions [00024].
The field of science and technology that proposes to apply enhanced understanding of the
human genetic code to reshaping our individual and collective destinies has generated more
interest among the general public, as well as in the athletic community, than the potential for
physical enhancement of the human body and its performance. Genometric experiments
have produced physically enhanced mice, and the production of similarly enhanced humans
may not be far off. Although it is not the objective of most genometric research, the day will
come when gene-based "treatments" will enable individuals to build muscle or increase
endurance faster than is possible through conventional methods. One article described
developments in the area of physical enhancement that may find application in the "gene
doping" of athletes. For example, human performance-related genes may be delivered to
athletes using tools developed for research in gene therapy; the protein products of these
genes may be administered in recombinant form; and recently discovered small-molecule
activators of the major genetic regulatory pathways of physical prowess may be taken orally,
providing "exercise in a pill". The article also described the attempts to regulate and punish
the use of prohibited techniques for performance enhancement among athletes. As science
advances, defining and detecting "gene doping" becomes increasingly complex. Thus, the
study of physical enhancement provides an ideal starting point for the interdisciplinary
examination of the intersection between law and science [09015].
The French law

To investigate the medico-legal aspects of national and international procedures for
monitoring prescription drug use by competing athletes it was studied the French law No. 99-
223 of March 23, 1999, relating to the protection of the health of athletes? It was also studied
annual statistics from the Ministry of Sports concerning anti-doping controls, substances
detected by the National Doping Control Laboratory and penalties applied since 2000, as
well as the World Anti-Doping Code, which came into effect on January 1, 2004, and should
be universally applied by 2006. Athletes registered with a federation or unregistered athletes
taking part in competitions approved by sporting federations can use prescription drugs but
must follow strict rules. Athletes under investigation for drug use must declare all drugs or
products recently taken. The use of prescription drugs not on the list of the prohibited
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substances is allowed, but evidence of the use of such drugs is the responsibility of the
prescriber. A medical practitioner in France who considers it essential to prescribe prohibited
drugs or drugs under certain restrictions must systematically inform the athlete about the
regulations by providing various certificates and forms. For international athletes, a form
authorizing therapeutic use must be submitted to the validation committee of the applicable
international federation. Disciplinary, ordinal and penal sanctions are also described. It was
concluded that prescription drug use by an athlete is never a light matter and always
engages the responsibility of the doctor. Anti-doping controls and sanctions encourage
physicians to comply scrupulously with the medico-legal rules set forth by the public health
code and the world anti-doping code [04024].
American law on ephedrine

On February 6, 2004, the US Food and Drug Administration banned dietary supplements
containing ephedrine alkaloids (ephedra) pending Congressional review. The ban culminates
a 7-year regulatory process, the first of its kind under the Dietary Supplement Health and
Education Act (DSHEA). One paper reviewed that process, and the governing rules of
DSHEA, within the contexts of modern science and the history of food and drug legislation.
The example of ephedra reflects a longstanding conflict between trade and safety and
suggests inherent weaknesses within DSHEA that place the public at risk [04025].
Doping + violence
Within the context of problem-behaviour theory, one study investigated the intra-relationship
between attitudes and behaviours towards exercise, sport involvement, violence in sport-
related events, eating fruits, smoking and hashish or ecstasy use in a sample of Greek
adolescents. Participants were 5991 Greek school pupils who responded to questionnaires
assessing behaviour and attitudes towards health-related behaviours. Positive associations
were found between pupils' reports of violence in sport-related events, smoking and hashish
or ecstasy use on the one hand, and eating fruits and participation in sport and exercise on
the other. In contrast, small positive association was observed between sport involvement
and violence in sport-related events. Attitudes towards health risk behaviours were inversely
related to attitudes towards health-promoting behaviours, and attitudes were positively
related to corresponding behaviours. Sport involvement and regular exercise decreased but
smoking and use of hashish or ecstasy increased with age. More males than females
participated in organized sport and violent acts in sport-related events. Males' involvement in
sport violence increased with age. Sport is a suitable context for the promotion of several
health-related behaviours apart from exercise. Nevertheless, the present sport structure
excludes most young people and is positively linked with sport violence. A less demanding
sport context should be provided for the majority of young people, particularly for females.
Sport programmes designed to promote health behaviours should be encouraged. More
concentrated actions to combat sport violence are required [10439].
Anabolic androgenic steroids and violent offending

Anabolic androgenic steroid (AAS) use is associated with aggressive and violent behavior,
but it remains uncertain if this relationship is causal in humans. It was examined the link
between AAS use and violent crime while controlling for polysubstance abuse and additional
suggested risk factors for violence in a cross-sectional study of a population-based sample.
In 2005, all Swedish-born male twins aged 20-47 years were invited to participate in the
Swedish Twin Adults: Genes and Environment (STAGE) survey of the Swedish Twin
Register (response rate 60 %). 10,365 male survey participants with information on AAS use.
Data on self-reported use of AAS, alcohol and other substances, attention deficit
hyperactivity disorder (ADHD) and personality disorder symptoms were linked to nationwide,
569
longitudinal register information on criminal convictions, IQ, psychological functioning and
childhood socioeconomic status (SES) covariates. Any lifetime use of AAS was strongly
associated with conviction for a violent crime (2.7 % vs 0.6 % in convicted and non-convicted
men, respectively; OR=5.0, 95 % confidence interval 2.7 to 9.3). However, this link was
substantially reduced and no longer significant when controlling for other substance abuse
(OR=1.6, 95 % confidence interval 0.8-3.3). Controlling for IQ, psychological functioning,
ADHD, personality disorder symptoms and childhood SES did not reduce the risk further.It
was concludet that in the general population, co-occurring polysubstance abuse, but not IQ,
other neuropsychological risks or socioeconomic status, explains most of the relatively strong
association between any anabolic androgenic steroid use and conviction for a violent crime
[14656].
Knowledge in different countries
Austria
Strategies for doping prevention are based on prior identification of opportunities for
intervention. There is no current research focusing on the potential role in doping prevention,
which might be played by the parents of junior elite athletes. The purpose of this study was to
evaluate the knowledge and attitudes toward doping among parents of Austrian junior
athletes and to analyze factors potentially influencing these beliefs. In ome study, two
questionnaires were distributed to 1818 student athletes, each with instructions that these
surveys were to be completed by their parents (ntotal 3636). Parents filled in questionnaires at
home without observation. Responses from 883 parents were included in this analysis.
Compared to female parents, male parents demonstrated significantly better knowledge
about doping and its side effects and were more likely to be influenced by their own sporting
careers and amounts of sports activities per week. Parental sex did not demonstrate a
significant influence on responses reflecting attitudes toward doping. Additional research is
needed to compare these results with young athletes' knowledge and attitudes to determine
if and to what degree parental attitudes and beliefs influence the behavior and attitudes of
their children [13046].
Austrian juniors
An important factor while developing efficient doping prevention strategies is to identify
relevant target groups, to evaluate the state of knowledge about this topic as well as to
evaluate motivations behind using prohibited substances. Measures to prevent doping
substances abuse have to be supported in early stages of childhood. The aim of one
prospective study was to evaluate the knowledge of Tyrolean junior athletes about doping in
sport. Next to the knowledge, their attitudes in regard to doping practices have also been a
focus of this project. Within a prospective cross-sectional study, Tyrolean junior athletes
aged between 14 and 19 years (n=408) were anonymously questioned by distributing
questionnaires in three Tyrolean sport schools as well as two Tyrolean sport-training centers.
To collect the data, an anonymous questionnaire with close-ended questions was used. Next
to sociodemographic data, questions also evaluated the knowledge about prohibited
substances as well as attitudes and behaviors towards doping. The concept was set up
based on contents of comparable studies and publications. The knowledge about doping
among junior athletes was moderate. The consumer behavior of the young athletes on the
other hand has turned out to be satisfactory. Nevertheless, the overall knowledge especially
regarding potential negative side effects of doping agents is poor. Thus, to incorporate an
effective doping-prevention strategy, improved education, particularly in terms of side effects,
is clearly needed. To achieve sustainable doping-prevention effects, focus has to be
generally set on education within the frame of junior competitive sport [13047].
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Australia
One study presents a comprehensive examination of the Sport Drug Control Model via
survey data of elite Australian athletes. A cross-sectional nationwide mail survey of 1237 elite
Australian athletes was conducted. Structural equation modelling was employed to test the
model. Morality (personal moral stance on performance-enhancing substances use),
reference group opinion (perceived moral stance of reference group on performance-
enhancing substances use) and legitimacy (perceptions of the drug testing and appeals
processes) evidenced significant relationships with attitude towards performance-enhancing
substances use, which in turn was positively associated with doping behaviour. The model
accounted for 81 and 13 percent of the variance in attitude towards performance-enhancing
substances use and doping behaviour, respectively. These findings validate the usefulness
of the Sport Drug Control Model for understanding influences on performance-enhancing
substances use. Nevertheless, there is a need to survey athletes representing a broader
range of competition levels and cross-cultural research to test the model's applicability to
other populations of athletes [13048].
Italy
It was explored use and attitudes toward drugs and dietary supplements (DS) and knowledge
concerning doping in cycling retrospective cross-sectional study. Forty cyclists aged 19 to 23
years and practicing for 14 to 30 h/wk were interviewed. Previous use (last 3 months) of
drugs or DS occurred in 33 of 40 (83 %) and 39 of 40 (98 %) cyclists, respectively. Almost all
the subjects named at least 1 doping agent (range, 1-10). Within a fixed list of 18 substances
(among which only 14 were doping agents), participants recognized 3 to 18 of them as
doping agents. They recognized tramadol and sildenafil as doping agents, which are not
doping agents, and failed to recognize probenecid and albumin, which actually are. Doping
knowledge correlated with drug use. Participants deemed doping prevalence high among
cyclists in general but not in their own team. It was concluded that the use of prescription
drugs and DS was a common occurrence. Doping knowledge was poor and biased, and its
relationship with drug use deserves consideration. Educational interventions are needed to
improve knowledge and awareness about prescription drugs and DS use, as well as about
doping [13049].
Illicit drugs around an Olympic game
It was aimed to describe presentations to emergency departments during the Sydney 2000
Olympic Games for conditions related to the use of illicit drugs; to discuss the implications of
such presentations for surveillance and public health action at similar events in the future.
Fifteen sentinel emergency departments in the greater Sydney metropolitan area for a 38-
day period spanning the Sydney 2000 Olympic Games had 424 presentations to sentinel
emergency departments with conditions related to illicit drug use. The mean daily number of
presentations for adverse events due to illicit drug use was significantly higher (13.3 versus
8.8 presentations) in the 2-week Olympic Games period than in the lead-up to the Games,
culminating in a large peak following the closing ceremony. There was also a significant
increase (5.1 versus 1.7 presentations) in the mean daily number of presentations related to
use of ecstasy or amphetamines, whereas no change was noted in presentations related to
heroin use. Over half (52 %) of presentations occurred at two emergency departments in
areas known as being hot-spots for illicit drug use. It was concluded that enhanced
surveillance of adverse events following illicit drug use, possibly targeting known 'hot-spots',
should be considered for future mass events. Advance preparation of preventive strategies,
such as 'party-safe' messages, will enable rapid response to unusual patterns of illicit drug-
related harm during future mass events [10440].
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US position stand on androgen and human growth hormone use
Perceived yet often misunderstood demands of a sport, overt benefits of anabolic drugs, and
the inability to be offered any effective alternatives has fueled anabolic drug abuse despite
any consequences. Motivational interactions with many situational demands including the
desire for improved body image, sport performance, physical function, and body size
influence and fuel such negative decisions. Positive countermeasures to deter the abuse of
anabolic drugs are complex and yet unclear. Furthermore, anabolic drugs work and the
optimized training and nutritional programs needed to cut into the magnitude of improvement
mediated by drug abuse require more work, dedication, and preparation on the part of both
athletes and coaches alike. Few shortcuts are available to the athlete who desires to train
naturally. Historically, the NSCA has placed an emphasis on education to help athletes,
coaches, and strength and conditioning professionals become more knowledgeable, highly
skilled, and technically trained in their approach to exercise program design and
implementation. Optimizing nutritional strategies are a vital interface to help cope with
exercise and sport demands. In addition, research-based supplements will also have to be
acknowledged as a strategic set of tools (e.g. protein supplements before and after
resistance exercise workout) that can be used in conjunction with optimized nutrition to allow
more effective adaptation and recovery from exercise. Resistance exercise is the most
effective anabolic form of exercise, and over the past 20 years, the research base for
resistance exercise has just started to develop to a significant volume of work to help in the
decision-making process in program design. The interface with nutritional strategies has
been less studied, yet may yield even greater benefits to the individual athlete in their
attempt to train naturally. Nevertheless, these are the 2 domains that require the most
attention when trying to optimize the physical adaptations to exercise training without drug
use. Recent surveys indicate that the prevalence of androgen use among adolescents has
decreased over the past 10-15 years. The decrease in androgen use among these students
may be attributed to several factors related to education and viable alternatives (i.e. sport
supplements) to substitute for illegal drug use. Although success has been achieved in using
peer pressure to educate high school athletes on behaviors designed to reduce the intent to
use androgens, it has not had the far-reaching effect desired. It would appear that using the
people who have the greatest influence on adolescents (coaches and teachers) be the
primary focus of the educational program. It becomes imperative that coaches provide
realistic training goals for their athletes and understand the difference between normal
physiological adaptation to training or that is pharmaceutically enhanced. Only through a
stringent coaching certification program will academic institutions be ensured that coaches
that they hire will have the minimal knowledge to provide support to their athletes in helping
them make the correct choices regarding sport supplements and performance-enhancing
drugs.The NSCA rejects the use of androgens and hGH or any performance-enhancing
drugs on the basis of ethics, the ideals of fair play in competition, and concerns for the
athlete's health. The NSCA has based this position stand on a critical analysis of the
scientific literature evaluating the effects of androgens and human growth hormone on
human physiology and performance. The use of anabolic drugs to enhance athletic
performance has become a major concern for professional sport organizations, sport
governing bodies, and the federal government. It is the belief of the NSCA that through
education and research we can mitigate the abuse of androgens and hGH by athletes. Due
to the diversity of testosterone-related drugs and molecules, the term androgens is believed
to be a more appropriate term for anabolic steroids [09016].
Characteristics and behaviors of older male anabolic steroid users
To compare and contrast the characteristics of 2 groups of men ≥40 years old: reported
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anabolic-androgenic steroid (AAS) users and nonusers 38 online fitness, weight lifting,
bodybuilding, and steroid Web sites were investigated. A total of 67 male AAS users and 76
male nonusers ≥40 years old were reached. Demographics, utilization of AAS and other
performance-enhancing agents (PEAs), exercise patterns, history of illicit drugs and alcohol
use, and psychiatric traits/diagnoses were noted. The majority of AAS users ≥40 years old
were caucasian (93 %), heterosexual (97 %), and classified themselves as recreational
exercisers (79 %). AAS users took more PEAs (11.5 ± 5.6 vs 4.6 ± 2.7), were more likely to
binge drink (47.8 % vs 29.0 %), report heavy alcohol use (21.0 % vs 7.9 %), meet criteria for
substance dependence disorder (27.4 % vs 4.0 %), and report an anxiety disorder diagnosis
(12.0 % vs 2.6 %) than nonusers. It was concluded that AAS misuse is prevalent among
older men and is associated with polypharmacy, more aggressive alcohol use, and a higher
incidence of substance dependence and anxiety disorders compared to nonusers. This
information may help clinicians and researchers identify and develop appropriate intervention
strategies for AAS abuse among older men [150038].
Body builders
During bodybuilding competitions individuals are assessed on their physical or esthetic

appearance and are usually required to demonstrate a high degree of muscularity and
symmetry, as well as low levels of body fat. Careful attention to nutrition and exercise
conditioning is undoubtedly important in facilitating the process of becoming competition
ready. Frequently used methods by those preparing for contest include chronic energy
restriction, dehydration (water manipulation), sporadic eating and inappropriate use of
diuretics and supplements of anabolic steroids and “fat burners”. These methods pose the
risk of adverse health consequences that can be physiological (i.e, decreased bone mineral
density, metabolic disruption, increased cardiovascular strain), hormonal and/or
psychological (i.e. anger, anxiety, loss of eating control/binge eating, pre-occupation with
food, short temper, mood disturbance, in nature. Competitors may also suffer a reduction in
their muscular function, strength and power during the preparation phase of competition, as
physique-oriented objectives are often placed above exercise performance and health goals.
A case study documented a structured nutrition and conditioning intervention followed by a
21 year-old amateur bodybuilding competitor to improve body composition, resting and
exercise fat oxidation, and muscular strength that does not involve use of any of the above
mentioned methods. Over a 14-week period, the Athlete was provided with a scientifically
designed nutrition and conditioning plan that encouraged him to (i) consume a variety of
foods; (ii) not neglect any macronutrient groups; (iii) exercise regularly but not excessively
and; (iv) incorporate rest days into his conditioning regime. This strategy resulted in a body
mass loss of 11.7 kg's, corresponding to a 6.7 kg reduction in fat mass and a 5.0 kg
reduction in fat-free mass. Resting metabolic rate decreased from 1993 kcal/d to 1814 kcal/d,
whereas resting fat oxidation increased from 0.04 g/min to 0.06 g/min. His capacity to oxidize
fat during exercise increased more than two-fold from 0.24 g/min to 0.59 g/min, while there
was a near 3-fold increase in the corresponding exercise intensity that elicited the maximal
rate of fat oxidation; 21 percent V̇O2max to 60 percent V̇O2max. Hamstring concentric peak
torque decreased (1.7 to 1.5 Nm/kg), whereas hamstring eccentric (2.0 Nm/kg to 2.9 Nm/kg),
quadriceps concentric (3.4 Nm/kg to 3.7 Nm/kg) and quadriceps eccentric (4.9 Nm/kg to 5.7
Nm/kg) peak torque all increased. Psychological mood-state (BRUMS scale) was not
negatively influenced by the intervention and all values relating to the Athlete's mood-state
remained below average over the course of study. This intervention shows that a structured
and scientifically supported nutrition strategy can be implemented to improve parameters
relevant to bodybuilding competition and importantly the health of competitors, therefore
questioning the conventional practices of bodybuilding preparation [150039].
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Influence of religion on use of doping
Strength of religious faith (SRF) is rarely studied as a protective factor against substance use
and misuse in sports. Herein, we studied the potential buffering effect of the complex socio-
educational, sports, and religiousness factors in the protection against substance use and
misuse, including cigarettes, analgesics, appetite suppressants, potential doping behavior,
and binge drinking. The sample of subjects included 40 high-class female athletes (22-26
years of age). Using a strictly anonymous questionnaire, we investigated different social,
educational, and sports factors (including SRF measured by the Santa Clara Strength of
Religious Faith Questionnaire) in relation to substance use and misuse. Following the
calculation of simple correlations, multiple regression analysis revealed that in combination
with low sports experience, SRF has a significant buffering effect against binge alcohol
drinking and consumption of appetite suppressants. The data are discussed in comparison
with previous findings and theoretical background. Future studies should study the topic
while observing samples of recreational and competitive athletes of both genders [12023].
Strength of religious faith is rarely studied as a protective factor against substance use and
misuse in sports. Herein, it was studied the potential buffering effect of the complex socio-
educational, sports, and religiousness factors in the protection against substance use and
misuse, including cigarettes, analgesics, appetite suppressants, potential doping behavior,
and binge drinking. The sample of subjects included 40 high-class female athletes (22-26
years of age). Using a strictly anonymous questionnaire, we investigated different social,
educational, and sports factors (including strength of religious faith measured by the Santa
Clara Strength of Religious Faith Questionnaire) in relation to substance use and misuse.
Following the calculation of simple correlations, multiple regression analysis revealed that in
combination with low sports experience, strength of religious faith has a significant buffering
effect against binge alcohol drinking and consumption of appetite suppressants [10026].
Religiousness is rarely studied as protective factor against substance use and misuse in
sport. The aim of one study was to identify gender-specific protective effects of the
religiousness (measured by Santa Clara Questionnaire) and other social, educational, and
sport variables as a potential factors of hesitation against doping behaviors in sport-science-
students from Mostar, Bosnia, and Herzegovina (51 women and 111 men; age range, 18-
26). The gender differences for the non-parametric variables were established by Kruskall-
Wallis test, while for the parametric variables the t-test for independent samples was used.
Multiple regression calculations revealed religiousness as the most significant predictor of
the social, health, sport and legal factors of hesitation against doping behaviors in both
genders. However, the differential influence of the social, educational, sport and religious
factors in relation to negative consequences of the doping behaviors is found for men and
women. Such differential influence must be emphasized in tailoring the anti-doping policy
and interventions [11014].
Religiousness is rarely studied as protective factor against substance use and misuse in
sport. Further, it was found no investigation where college-age athletes were sampled and
studied accordingly. The aim of the present study was to identify gender-specific protective
effects of the religiousness (measured by Santa Clara Questionnaire) and other social,
educational, and sport variables as a potential factors of hesitation against doping behaviors
in sport-science-students from Mostar, Bosnia, and Herzegovina (51 women and 111 men;
age range, 18-26). The gender differences for the non-parametric variables were established
by Kruskall-Wallis test, while for the parametric variables the t-test for independent samples
was used. Multiple regression calculations revealed religiousness as the most significant
574
predictor of the social, health, sport and legal factors of hesitation against doping behaviors
in both genders. However, the differential influence of the social, educational, sport and
religious factors in relation to negative consequences of the doping behaviors is found for
men and women. Such differential influence must be emphasized in tailoring the anti-doping
policy and interventions [13067].
The adolescent athlete
The use of doping compounds is less likely in adolescent athletes, but the detection that
much more difficult given that the baseline secretion of the endogenous hormone is shifting
during pubertal development with the greatest rise in testosterone in boys occuring about the
time of peak height velocity and maximal secretion of hGH and IGF-I. Can one grow more
rapidly during childhood and early adolescence to a taller than genetically programmed adult
height or to a more robust body composition given anabolic-androgenic steroids (AAS),
human growth hormone (rhGH), insulin-like growth factor (rhIGF-I), insulin or erythropoietin
[10001]?
The more successful late childhood and early adolescent age athletes are often more
physically mature than their age-peers. Early adolescent development seemingly permits
them to use the strength and power in sport and performance that their average (or even
slowly) developing age-peers have not yet attained. The exceptions might be those athletes
in the more aesthetic sports in which flexibility and a lower center of gravity are more
important than size and strength. These are virtually exclusively in females. Most boys'
sports require high levels of strength and power; however, at elite levels of competition
technique is a strong measure of success; for the athlete must produce, control and
efficiently use the energy in a fashion that maximizes athletic performance; for example,
explosive power in some jumping sports or overall technical skill in the pole vault. Earlier
developing children are taller and stronger than their age-peers. That may confer an
advantage at younger ages; however, sport-specific skills are important. It is because of
these that some of the "later blooming" adolescents catch-up in performance with their earlier
developing peers and likely overtake them; for they have had the discipline to attain the
requisite skills and are perhaps at lesser risk to "burn-out" and cease participation in that
sport [10017].
The rationale for taking ergogenic "effectors", such as rhGH, rhIGF-I, anabolic steroids,
insulin or erythropoietin, is that by becoming bigger and stronger the athlete will perform
better. Some boys who are not athletes take anabolic steroids and perhaps other ergogenic
aides to "look better". However, there are no definitive data for rhGH or insulin in young
adults and none at all for any of these five agents in adolescents who are not deficient in
them. In any experimental setting the study design is such that one studies the effects of a
single agent with all other things being equal. For a drug one decides on a dose, or range of
doses and the subjects are allocated randomly to receive or not receive a particular dose.
The subjects should be selected from a common pool and all should have identical
"requirements", for example, in studies of athletes, prescribed diet and exercise regimens. It
is apparent that many athletes take a "cocktail" of drugs making it virtually impossible to
denote any single agent as causing a specific outcome. The difficulty in adolescents is that
increased secretion of testosterone (and growth hormone) is a natural part of human pubertal
development. Thus it would be very difficult to ascribe any individual changes noted in
strength and body composition to any pharmacological agent at this time. The ethics of
human experimentation in adolescents precludes any serious objective study using anabolic
steroids [10017].
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Persons influencing adolescents’ doping
Amateur and professional athletes use numerous ergogenic aids that claim to enhance
sports performance. Although some studies have indicated a performance benefit in
particular athletic situations, there are few available data regarding efficacy or safety in
competitive or noncompetitive adolescents. Common ergogenic aids include nutritional
supplements, anabolic steroids and HGH. Physicians can evaluate these products by
examining four factors that can help them counsel their adolescent patients: method of
action, available research, adverse effects and legality. However, athletes report that their
most common sources of dietary and supplement information to be (in rank order) their
trainer or coach, a family member or friend, magazines and books, a physician and a
nutritionist. While most adolescents may not seek information about ergogenic benefits from
medical professionals, they may seek information about side effects or contraindications if
the physician is open and nonjudgemental. An honest discussion of the limitations of most
supplements, and acknowledgement that some supplements may work some of the time,
may allow the physician to be more credible and useful in providing medical care and
guidance to the adolescent seeking to improve body image or athletic performance [05008].
Attitudes towards doping of high school athletes
healthy, while paradoxically, they seem less anxious and they are more self-confident. Our
Attitudes and behaviors among male adolescents on doping
One study investigated attitudes toward androgenic anabolic steroids among male
adolescents who have used anabolics compared with those who have not. A cross-sectional
survey was performed in the year 2000 in all secondary schools in the county of Halland on
the west coast of Sweden. An anonymous multiple-choice questionnaire was distributed to all
classes with 14-, 16-, and 18-year-old male adolescents. The response rate was 93 percent
(n=4049). Those who admitted having used androgenic anabolic steroids differed in several
ways from those who had not. Fewer believed androgenic anabolic steroids to be harmful
(OR=0.15) and more believed that girls preferred boys with large muscles (OR=6.1). They
trained more often at gyms (OR=5.6), drank more alcohol (OR=4.2), and had used narcotic
drugs more often (OR=15.3) than the other male adolescents. More immigrants than native-
576
born adolescents had used anabolics (OR=4.2). Attitudes toward anabolics differ between
users and nonusers. These aspects may be beneficial to focus on as one part of a more
complex intervention program in order to change these attitudes and decrease the misuse of
androgenic anabolic steroids [04020].
One study was a preliminarily investigation into the prevention of unintentional doping on the
basis of self-determination theory (SDT). Specifically, we examined the relationship between
athletes' motives for doping avoidance and their behavior when offered an unfamiliar food
product. Participants were young Australian athletes (n=410) that were offered a free lollipop
prior to completing a questionnaire. It was noted whether participants refused to take or eat
the lollipop and whether they read the ingredients of the lollipop. The questionnaire assessed
autonomous and controlled forms of motivation, amotivation, doping intentions, and
adherence regarding doping avoidance behaviors. The results showed that young athletes
who adopted controlled reasons to avoid doping in sport (e.g. not getting caught) tended to
report higher adherence to behaviors related to avoiding and monitoring banned substances,
whereas those who adopted autonomous reasons (e.g. anti-doping being consistent with life
goals) appeared to be more willing to read the ingredients of the provided food. The
significant interaction effect between autonomous and controlled motivation indicated that
autonomous motivation was more predictive to doping intention for athletes with low
controlled motivation. It is concluded that SDT may help understand the motivational
processes of the prevention of unintentional doping in sport [150040].
Perfectionism versus vulnerably

Recent theory and research suggest that perfectionism is a personal factor contributing to
athletes' vulnerability to doping (using banned substances/drugs to enhance sporting
performance). So far, however, no study has examined what aspects of perfectionism
suggest a vulnerability in junior athletes. Employing a cross-sectional design, one study
examined perfectionism and attitudes towards doping in 129 male junior athletes (mean age
17 years) differentiating four aspects of perfectionism: perfectionistic strivings, perfectionistic
concerns, parental pressure to be perfect, and coach pressure to be perfect. In the bivariate
correlations, only parental pressure showed a positive relationship with positive doping
attitudes. In a multiple regression analysis controlling for the overlap between the four
aspects, perfectionistic strivings additionally showed a negative relationship. Moreover, a
structural equation model examining the relationships between all variables suggested that
coach pressure had a negative indirect effect on attitudes towards doping via perfectionistic
strivings. The findings indicate that perceived parental pressure to be perfect may be a factor
contributing to junior athletes' vulnerability to doping, whereas perfectionistic strivings may be
a protective factor [150041].
Availability of illegitimate drugs in the society
Since WADA was established, the number of doping controls performed has constantly
increased both during competition and also out of competition; this increase in testing has
not prevented an unknown proportion of athletes taking performance-enhancing drugs prior
to and/or during major competitions. Banned and harmful substances being available over
the counter without prescription compound the situation further. Drugs such as nandrolone,
stanozolol, rHuEPO, testosterone and cortisol can be purchased without difficulty over the
Internet or accessed in most fitness centres worldwide. Importantly, the issue of drugs in
sport is not confined to professional athletes but is increasingly becoming a problem among
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recreational athletes. Recreational athletes are being encouraged to increase muscle mass
and strength for aesthetic reasons through readily available anabolic steroids. This practice
has detrimental effects on health [14429].
Norway 2011-2014
Doping agents are widely and illicitly distributed through the Internet. Analysis of these
preparations is useful in order to monitor the availability of prohibited substances on the
market, and more importantly to predict which substances are expected to be found in urine
samples collected from athletes and to aid clinical and forensic investigations. Based on a
close collaboration with the Norwegian police and the Norwegian custom authorities, the
Norwegian Doping Control Laboratory has performed analyses of confiscated material
suspected of containing doping agents. The analyses were performed using gas
chromatography (GC) and liquid chromatography (LC) combined with mass spectrometry
(MS). The majority (67 %) of the analyzed black market products contained anabolic-
androgenic steroids (AAS) as expected, whereas peptide- and protein-based doping
substances were identified in 28 percent of the preparations. The Norwegian Doping Control
Laboratory receives samples collected from recreational and elite athletes in addition to
samples collected in clinical and forensic investigations. The findings in the seized material
reflected the findings in the urine samples analyzed regarding the anabolic steroids. Thus,
analyzing material seized in Norway may give a good indication of doping agents available
on the local market [150044].
Denmark 2002-2003
A description of the illicit drug market in Denmark's second largest city was provided based
upon the prevalence of narcotics and illicitly sold medicals during the years 2002 and 2003.
The changes on the illicit drug market were described by comparing the results to a similar
study conducted ten years earlier. The study comprised of 469 cases of seized material by
Aarhus Police during 2002-2003. Additional information relating to the 341 persons charged
is also included in the study. Heroine, cocaine and amphetamine were seized in 31 percent,
30 percent, and 28 percent of the cases, respectively, and comprise the most frequently
encountered hard drugs on the market. The prevalence of cocaine in Aarhus Police District
has increased more than tenfold during the past ten years. The purity of the three drugs
decreased significantly during the same period, although large variations in the quality of
drugs were observed. Medicals were found in 16 percent of the seizures (containing 32
different active substances). The most frequent group of medicals was benzodiazepines,
which made up a total of 74 percent of the medicals in the study. Anabolic steroids, ecstasy
and methamphetamine were each found in 4 percent of the seizures. Men with an average
age of 29 years comprised 92 percent of the persons charged in the study. Persons with a
foreign nationality comprised 15 percent of the charged, while 25 percent had a birthplace
outside Denmark. This means that the prevalence of stimulants especially cocaine have
increased significantly during the past ten years. Meanwhile the purity of the drugs has
decreased. The benzodiazepines are still the most frequent group of medicals on the illicit
market [08034].
Germany 2012-2013
The desire to increase the athletic performance, to “optimize” an individual's appearance,

and to complement but also to arguably substitute exercise by means of drugs and drug
candidates has generated a considerable (illicit) market for compounds such as anabolic-
androgenic steroids, stimulants, growth promoting peptide hormones, and so on. Genuinely
578
developed for therapeutic use, their abuse/misuse generates enormous health risks, which
has necessitated comprehensive controls of compound trafficking by customs and anti-
doping authorities. From 2012 to 2013, the Bureau of Customs Investigation confiscated
products containing anabolic-androgenic steroids (AAS; 259 kg), stimulants (13 kg),
selective estrogen receptor modulators (SERMs; 24 kg), and human growth hormone (hGH;
3500 ampules). In cooperation with the Bureau and under the umbrella of the European
Monitoring Center for Emerging Doping Agents (EuMoCEDA), the Cologne Anti-Doping
Laboratory analyzed an additional 337 (black market) products between 2010 and 2013,
allowing to monitor developments in drug use and, hence, the anticipation of new challenges
in sports drug testing. Main tools utilized in characterizing confiscated materials were liquid
chromatography-high resolution mass spectrometry (LC-HRMS), gas chromatography-high
resolution mass spectrometry (GC-HRMS), and polyacrylamide gel electrophoresis (PAGE)
with subsequent bottom-up identification of peptidic compounds using nano liquid
chromatography-tandem mass spectrometry (nanoLC-MS/MS). Among the 337 substances
analyzed in the doping control laboratory in Cologne, 67 active ingredients were found, 49 of
which being categorized as doping agents by the World Anti-Doping Agency (WADA). A
total of 84 percent accounted for steroidal substances (predominantly testosterone,
trenbolone, and nandrolone and corresponding esters), 13 percent accounted for peptide
hormones and growth factors (predominantly hGH and growth hormone releasing peptides
(GHRPs)), 3.2 percent of the products contained hormones and metabolic modulators, and
0.3 percent accounted for diuretic agents. Outstanding findings were the detection of the
selective androgen receptor modulator (SARM) LGD-4033, the thymic hormone thymosin
beta4, and a fusion protein of unknown biological activity. It was concluded that trafficking of
considerable amounts of arguably performance and/or body-enhancing compounds has
been observed during the past 4 years, the majority of which is categorized as relevant to
sports drug testing. Several substances are of fake/non-approved nature and represent
enormous health risks to the “customer [14702].
Switzerland 2013-2014
One retrospective study evaluates the content, the destination and the source of 960 postal
items seized by the Swiss customs authorities at the Swiss border between 2013 and 2014.
The packages were seized because they contained at least one prohibited doping product as
identified by the Swiss law on encouraging sports and physical activity. A total number of
1825 different doping products were confiscated from these parcels, accounting for an
average of 1.9 doping products per seized item. In 74 percent of the cases, where seizures
were made, anabolic androgenic steroids, mostly testosterone esters, were discovered. An
obvious trading channel for doping products was identified in this study. The seized
compounds were predominately manufactured in Asian countries, but sent to Switzerland
mostly via South Eastern Europe countries. Due to the unique collaboration between the
Swiss customs authorities and the national anti-doping agency, this study uncovered an
alarming trend of illegal doping product trafficked to Switzerland [150043].
Spain
Anabolic androgenic steroids (AAS) can cause serious adverse effects when used without a
therapeutic purpose. One article aims to show that the AAS are susceptible to being sold on
the black market. It also aims to describe how certain limitations on the health inspection
services of the Galician health service to pursue these illegal actions prompted a regulatory
initiative demanding that additional actions be granted to community pharmacies when
dispensing AAS. Four pharmacy inspections detected the diversion of a total of 3118
packages of AAS, which led to the opening of four disciplinary proceedings. In two of these,
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specialized police forces were called in as there was sufficient evidence of possible diversion
to gymnasiums, resulting in a police operation called Operation Fitness [150042].
Brazil 2007-2014
The objectives of one work were to evaluate current legislation on dietary supplements in the
United States, the European Union and Brazil, and the profile of adulterated and/or irregular
products on these markets. Due to a less restrictive legal framework, a supplement product
that is freely available in the US may be considered a drug or even be proscribed in the EU
and Brazil, thus giving rise to a clandestine market based on smuggling. From 2007 to 2014,
the United States Food and Drug Administration reported 572 cases of supplement
adulterations in the country, mainly products for sexual enhancement (42 %). Data from the
European Union Rapid Alert System for Food and Feed showed 929 adulterations during the
same period, over 40 percent due to unauthorized ingredients or undeclared medicines.
From 2007 to 2013, the Brazilian Federal Police Department seized 5470 supplement
products, 92.2 percent with an American-declared origin. Qualitative chemical analyses
performed on 2898 products found 180 adulterations, 41.1 percent due to undeclared drugs,
mainly anabolic steroids, anorectics and products for erectile dysfunction, all considered
medicines in Brazil. Educating the public regarding the potential risks they are taking when
consuming adulterated or irregular products is necessary to protect the health of consumers
[150045].
Internet drug availability
It has previously been suggested that the Internet is the most common source for men to
obtain AAS as well as ancillary drugs. Access to these suppliers can vary from open access
to special invitations offered by Internet forum members or via word of mouth at local
gymnasiums. Internet suppliers offer bundled packages that commonly include testosterone
and synthetic androgens as well as selective estrogen receptor modulators (SERMs),
aromatase inhibitors (AIs), human chorionic gonadotropin (hCG), and phosphodiesterase-5
inhibitors (PDE5i). Beyond nonphysician sources, nutritional supplements sold legally online
or in retail stores have been found to contain AAS or other ancillary drugs that may or may
not be listed as ingredients on the product label [14427].
Today's Internet provides extensive "underground" guidelines for obtaining and using illicit
substances, including especially anabolic-androgenic steroids (AAS) and other appearance-
and performance-enhancing drugs (APEDs). It was attempted to qualitatively characterize
APED-related Internet sites. It was used relevant Internet search terms (e.g. "steroids
bodybuilding" and "buy steroids online") to assess (i) the numbers of site visitors; (ii) offers of
drugs for sale; and (iii) the quality of online medical information. We also chose the examples
of (iv) "site-enhancing oils" and (v) "cattle implants" to illustrate the volume of available
Internet information as compared with that in the medical literature. It was found thousands
of sites involving AAS and other APEDs. Most sites presented an unabashedly pro-drug
position, often openly questioning the qualifications and motivations of mainstream medical
practitioners. Offers of AAS and other APEDs for sale, together with medical advice of
varying legitimacy, were widespread across sites. Importantly, many sites provided detailed
guidelines for exotic forms of APED use, some likely associated with serious health risks,
which are probably unknown to most practicing clinicians. It seems important for practitioners
to be aware of the extent of this "underground literature," which may strongly influence their
patients' decisions about use and abuse of APEDs [13058].
Psychoactive drugs in the society

580
Analysis of urine samples collected across a city centre, for the detection of novel
psychoactive substances (NPS) was performed with a cross-sectional study of anonymized
urine samples used for the analysis of classical recreational drugs, NPS and metabolites.
Pooled urine samples collected from portable stand-alone four-person urinals across a city
centre were analysed using full-scan accurate-mass high-resolution liquid chromatography
coupled to tandem mass spectrometry. Data were processed against compound databases
containing >1700 drug compounds and metabolites. Seven established recreational drugs
(3,4-methylenedioxyamphetamine, cocaine, cannabis, ketamine, 3,4-methylenedioxy-N-
methylamphetamine, methamphetamine and amphetamine) and six potential NPS
[hordenine (all 12 urinals), cathine (11), methylhexaneamine (9), 4-methylmethcathinone (6),
methiopropamine and metabolites (2) and methoxetamine and metabolites (1)] were
detected. Methylhexaneamine, methiopropamine and hordenine are currently uncontrolled in
the UK, whereas methoxetamine is currently subject to a Temporary Class Drug Order.
Metabolites of the anabolic steroid nandrolone were found in two urinals and trenbolone
metabolites and clenbuterol in one urinal. It was concluded that analysis of pooled urine
samples collected anonymously from stand-alone urinals in a large inner city can detect the
use of recreational drugs, NPS and anabolic steroids. Metabolite detection indicates actual
drug use, metabolism and elimination rather than simply discarded drugs in the urinals. This
technique by confirming the actual drug(s) used has the potential to be additive to currently
used datasets/key indicators providing more robust information for healthcare authorities,
legislative and law enforcement on the drugs actually being used [13059].
Organised crime and drugs in sport
A report was from the Australian Crime Commission and entitled “Organised Crime and
Drugs in the Sport” had the main following conclusions:
- Australian professional athletes, facilitated by sports scientists, coaches and support

staff are using prohibited substances including peptides and hormones
- This behaviour is occurring in a number of professional sporting codes in Australia
- The use of illicit drugs within some sporting codes is higher than previously recorded
- Organised crime is involved in the domestic distribution of peptides and hormones
- There are significant integrity concerns within professional sports in Australia
- The use of prohibited substances by athletes is leading to an association between
professional athletes and criminal identities
- There is a culture in some professional sports of administering untested and

experimental substances to athletes
- Some sports scientists and medical practitioners are involved in supplying
peptides and hormones to athletes.
These revelations rocked the Australian sporting community and have caused
significant soul-searching and reflection within sporting organisations, particularly at
high-performance level. A number of organisations conducted or are conducting
internal reviews of their processes and protocols around the administration of
supplements and medications. What was initially perceived as a widespread issue
across elite sport in Australia now appears to be a more focal issue, concentrated in
small pockets of professional sport. There are assertions, yet to be validated, that in
some professional clubs there were systemic injection programmes as part of the
581
supplementation strategy. It is almost inconceivable that such practices did not cause
alarm bells to ring for club officials. If the claims are proved to be correct, then one
can only conclude that those organisations do not have robust internal governance
processes or have drifted away from such processes [13027].
Legitimate use of drugs in sports
It was assessed and compared the prevalence of declared medication, such as

corticosteroids, nonsteroidal anti-inflammatory drugs (NSAIDs), beta2-agonists, narcotic
analgesics, anaesthetics, and antidepressant drugs, in time and between different sports
among athletes tested for doping control in a 4-year period in Belgium. Data were obtained
from 18,645 doping control forms gathered between 2002 and 2005 from national doping
organisations in Belgium and The Netherlands, the International Cycling Union (UCI), and
the Belgian Cycling Federation. All athletes were asked by doping control officers to declare
the medication taken in the last three days before competition after which the doping control
forms were double blinded and handed over to the laboratory. The overall declared use of
medication belonging to one of the monitored categories increased from 20 percent in 2002
to 25 percent in 2005. Differences in use of medication were observed between sports with a
higher prevalence of use of NSAIDs in ball sports compared to other sports and a higher use
of beta-agonists and corticosteroids in cycling with percentages of declared corticosteroid
use in samples from the UCI exceeding 36 percent in 2005. These results indicate that the
current granting of therapeutic use exemption for corticosteroids and beta-agonists needs to
be revised and that threshold levels for beta-agonists should be implemented [08035].
Enquiries made in the Drug Information Database (DIDTM) were retrospectively analysed to
develop a better understanding of athletes' interests and concerns regarding the prohibited
status of available substances regarding anonymous enquiries recorded in the DID in 2006
and 2007. The DID recorded 223,717 enquiries with 200 of the >6000 UK Licensed
Pharmaceutical products receiving over 100 enquiries each. The majority (79 %) of these
enquiries were in the pharmaceutical product category, followed by recreational drugs (10
%). A variety of common medications was subject to enquiry with anti-inflammatory agents,
decongestants and broncodilators being most common; a trend in keeping with reported
medication use by athletes. Of all enquiries, 42 percent were not found owing to misspelled
words or enquiries about unregulated substances. The proportion of enquiries about
substances not listed in the database is relatively high and has increased over the 24-month
observation period. The DID is a well used information resource with some 10,000 enquiries
being made each month. With some 73 percent of enquiries being made by the athletes
themselves, further investigations are warranted to explore enquiry patterns in relation to
specific sports. Of the unsuccessful enquiries, a large number were related to nutritional
supplements which warrants further investigation. The DID database appears to be a valid
mirror of athletes' chemically assisted practices and may be successfully used to inform
health professionals as well as anti-doping prevention programmes [08036].
Over-the-counter medicine
A questionnaire was administered to elite athletes from Australia, Canada, the UK, and the
USA representing 10 Olympic sports in order to explore knowledge and understanding of
over-the-counter (OTC) medication since the removal of many of these substances from the
World Anti-Doping Agency Prohibited List, in 2004. Athletes demonstrated limited knowledge
and understanding. Around half (51 %) knew the penalty incurred following a doping violation
involving a banned OTC stimulant. The terms Monitoring Program and Specified Substance
582
List were understood by 43 and 68 percent of respondents, respectively. Overall, the status
of substances in relation to the Prohibited List was correctly identified in just 35 percent of
cases. As a whole, athletes were of the opinion that OTC stimulants posed a risk to health,
were performance enhancing and that their use was against the spirit of sport. They were
undecided as to whether these drugs should be returned to the Prohibited List. Elite athletes
require targeted education programmes that will enable them to make informed decisions on
the potential of OTC medications for therapeutic or performance enhancing purposes
[08037].
Many over-the-counter (OTC) drugs used in the symptomatic relief of upper respiratory tract
(URT) conditions are banned by sports governing bodies. It would appear therefore that
athletes are being penalised for practising conventional pharmacological methods in the
management of common ailments. The aim was to identify any differences between athletes
and non-athletes and amongst athletic groups, with respect to the prevalence of URT
conditions and the use of OTC drugs to treat such conditions. Questionnaires were
distributed at domestic and international athletics meetings and at university lectures and
tutorials. Respondents (n=401) represented both track and field athletes (n=199) and non-
athletes (n=202). No differences were found between athletes and non-athletes and between
elite and non-elite athletes in terms of the frequency of episodes of URT conditions reported
in the previous year. A higher proportion of elite, as opposed to non-elite athletes did not take
OTC medicines and of those that did take OTC medicines a higher proportion of elite
athletes (68 %) as opposed to non-elite (32 %) took those not containing sympathomimetics,
banned by the International Olympic Committee (IOC). Athletes were found to have greater
knowledge of IOC banned OTC drugs (p=0.002) and within this group, elite athletes were
most knowledgeable. Although most respondents (81 %) believed that OTC drugs should not
be prohibited in sport, athletes made up the greatest proportion in support of prohibition (24
% as opposed to 14 % of non-athletes) with elite as opposed to non-elite most in favour.
These results suggest that URT conditions are no more prevalent between athletes and non-
athletes or between endurance and power athletes. Athletes competing at the highest level
tended to avoid OTC medicines or those containing IOC banned drugs and were most
knowledgeable in terms of banned OTC drugs and most in favour of their prohibition
suggesting that the control mechanisms in place are only reaching elite athletes [03020].
Doping agents as medical treatment
Many performance-enhancing supplements and/or drugs are increasing in popularity among

professional and amateur athletes alike. Although the uncontrolled use of these agents can
pose health risks in the general population, their clearly demonstrated benefits could prove
helpful to the critically ill population in whom preservation and restoration of lean body mass
and neuromuscular function are crucial. Post-intensive care unit weakness not only impairs
post-intensive care unit quality of life but also correlates with intensive care unit mortality. A
review covered a number of the agents known to enhance athletic performance, and their
possible role in preservation of muscle function and prevention/treatment of post-intensive
care unit weakness in critically ill patients. These agents include testosterone analogues,
growth hormone, branched chain amino acid, glutamine, arginine, creatine, and beta-
hydryoxy-beta-methylbutyrate. Three of the safest and most effective agents in enhancing
athletic performance in this group are creatine, branched-chain amino acid, and beta-
hydryoxy-beta-methylbutyrate. However, these agents have received very little study in the
recovering critically ill patient suffering from post-intensive care unit weakness. More
placebo-controlled studies are needed in this area to determine efficacy and optimal dosing.
It is very possible that, under the supervision of a physician, many of these agents may prove
beneficial in the prevention and treatment of post-intensive care unit weakness [09031].
583
Pharmacists
When athletes consult sports outpatient or orthopedic clinics it is possible to undergo drug
treatment with the medical staff having prior knowledge of that patient being an athlete.
However, if athletes seek any other diagnosis and treatment as an ordinary patient, the
possibility of medical staff realizing the potential for imposing a doping issue on the athlete is
extremely low. As a result, if the athlete fails to provide medical staff with information
regarding anti-doping regulations when receiving clinical treatment, drug treatment
administered as part of medical practices could be viewed as doping, resulting in the athlete
being disciplined. In order to avoid this, pharmacist should participate in training in order to
be able to provide information for anti-doping purposes. There is an opinion that knowledge
regarding anti-doping is something that should be shared by all pharmacists, as pharmacists
are educated in the fields of pharmacology and pharmacokinetics during the pharmacy
education process, and sports pharmacology is a part of this. However, in order for
pharmacists to understand sports pharmacology, it is necessary to provide education not
only on the benefits and adverse effects of pharmaceutical products, but also on the concept
of banned substances. It can be considered one of the pharmacist's duties to protect athletes
who purchase drugs at a pharmacy or consult medical institutions as patients. With this, it
may be proposed considering the potential for introducing sports pharmacology to
pharmaceutical education, and specialist pharmacist training in the sports spectrum [12038].
To describe opportunities and obligations for pharmacists regarding doping control in sports,
and to present information and resources on drugs and dietary supplements that are popular
among athletes for performance enhancement. Sports medicine journals and articles in
English obtained from Medline (1966 through June 2003) using the search terms doping in
sports, drugs in sports, dietary supplements, sports, amphetamine, stimulants, ephedrine,
ephedra, caffeine, anabolic steroids, human growth hormone, erythropoietin, darbepoetin,
androstenedione, dehydroepiandrosterone, and creatine. Information was also obtained from
sports-governing agencies, such as the National Collegiate Athletic Association and the
International Olympic Committee. Studies and reports that were credible and scientifically
sound that evaluated the ergogenic effects of drugs and dietary supplements. Pharmacists
can participate in doping control programs in a number of ways. Pharmacists also have an
obligation when counseling, advising, and treating athletes to help them avoid banned
substances. Athletes use a host of drugs for their performance-enhancing effects, many of
which are banned by major sports-governing bodies. Myriad dietary supplements are
marketed to athletes, claiming to have ergogenic effects. Some of these popular
supplements have proven performance-enhancing effects, while others do not. Adverse
effects of these drugs and dietary supplements are discussed. It was concluded that a variety
of drugs and dietary supplements have proven performance-enhancing effects in athletes.
However, many of these substances have adverse effects and are banned by various sports-
governing organizations. Pharmacists can play a key role in participating in doping control
programs, and can prevent athletes from inadvertently consuming a banned substance
[04026].
Compared to general practitioners
Information about doping awareness among medical professionals is scarce. It was

evaluated the attitudes, level of knowledge and experience among general practitioners
(GPs) and pharmacists (Ps) with regard to doping. In a cross-sectional national survey a 59-
item self-administered questionnaire was sent to a representative random sample of 645
584
GPs and 330 Ps. Overall, 204/975 (133 GPs, 71 Ps) questionnaires were returned and
available for analysis. Fewer than half (39 % GPs vs 48 % Ps) of respondents were familiar
with the formal definition of doping. The abbreviation WADA was correctly interpreted by 42
percent (33% vs 59 %), and 65 percent knew that the European Commission has the
legislation to fight against doping. More GPs (69 % vs 31 %) agreed to have a role to play in
doping prevention, similar proportions considering themselves to have sufficient knowledge
of prevention initiatives (65 % vs 35 %). Overall, 12 percent of respondents (9 % GPs, 19 %
Ps) reported being directly confronted with a request for prescription of doping agents in the
previous 12 months (mainly stimulants, anabolic agents, hormones, corticosteroids). It was
concluded that GPs and Ps are frequently exposed to questions about and requests for
doping agents. They have acceptable level of general knowledge but are in need for more
specific information on prohibited substances and legislature [12039].
Pharmacy students
Doping is one of the most serious problems for the sport community, and it is important that
pharmacists have more interaction with athletes to ensure safer drug usage. Education is
one of the most important roles of sports pharmacists, who are specialists regarding drug
usage for athletes. It was investigated pharmacy students' interests and comprehension
regarding drug usage, doping and supplement intake by using the form of a questionnaire,
since it is important to know how they understand these subjects as part of their greater
educational program. The subjects were sophomore and junior pharmacy students at three
universities. It was revealed that most of the students have negative images regarding
doping violation, and they answered that they are familiar with doping. However, only sixteen
percent of the students had attended lectures by specialists on doping. In addition, one third
of pharmacy students did not know that some over-the-counter (OTC) drugs might contain
doping substances. With regard to supplement intake, approximately two thirds of the
respondents had an interest in and positive image of supplement intake. However, it was
revealed that only one third of them recognized supplements as food, and their information
regarding supplements was obtained from uncertain media. It was suggested that it is
important for pharmacy students to have more opportunities to learn about what doping is.
More education and enlightenment by sports pharmacists would be effective for pharmacy
students as well as athletes, and it would help us to broaden the scope of what we can do for
athletes and society [13045].
Qatar
The aim of one study was to evaluate the current knowledge and perceptions of pharmacists
in Qatar with regard to the use of drugs in sport and to explore their views on the introduction
of education and training in the area of sports pharmacy. A cross-sectional survey was
conducted targeting both hospital and community pharmacists in Qatar. A questionnaire
consisting of three domains pertaining to participants' knowledge, perceived role of
healthcare professionals, and attitudes towards educational needs on the use of drugs in
sports was developed and validated. The online survey link and paper-based questionnaires
were distributed to the target population. A total of 300 pharmacists responded to the survey.
Respondents had a limited awareness of doping and anti-doping and achieved an average
knowledge score of 53 percent regarding the prohibited status of drugs that may be used by
athletes, particularly with respect to over-the-counter medicines and supplements. The
majority (82 %) of the pharmacists expressed an interest in receiving education and training
on sports pharmacy. Specialised training programmes are warranted to ensure that
pharmacists have the knowledge and skills required to provide athletes with accurate
information about anti-doping issues and the safe and effective use of medicines in sport.
585
The development of these programmes should be supported by national pharmacy policy
makers and designed in collaboration with anti-doping agencies and sports pharmacy
experts and educators [150029].
To assess pharmacy students' knowledge and perceptions of doping and anti-doping in

sports and to explore the curricular needs for undergraduate pharmacy in the field of sports
pharmacy a cross-sectional, descriptive, web-based survey of pharmacy students was
conducted at Qatar University College of Pharmacy from March to May 2014. Data were
analyzed using descriptive and inferential statistics. Results. Eighty respondents completed
the online survey (80 % response rate). Sixty percent were unaware of the World Anti-
Doping Agency, and 85 percent were unaware of the International Pharmaceutical
Federation's statement on the pharmacist's role in anti-doping. Students' knowledge score
regarding the prohibited status of drugs that may be used by athletes was around 50 percent.
Fourth-year pharmacy students had significantly higher knowledge scores than the other
groups of students. Respondents acknowledged the important role of health care
professionals, including pharmacists, as advisors on the safe and effective use of drugs in
sports. Ninety percent of the students supported the inclusion of sports pharmacy in the
curriculum. It was concluded that pharmacy students indicated a strong desire to play a role
in doping prevention and ensure safe and rational use of drugs among athletes. They
suggested requiring an education and training strategy for sports pharmacy in undergraduate
pharmacy curricula [150030].
A cancer risk of doping?
Anabolic steroid and peptide hormones or growth factors are utilized to increase the
performance of athletes of professional or amateur sports. Despite their well-documented
adverse effects, the use of some of these agents has significantly grown and has been
extended also to non-athletes with the aim to improve appearance or to counteract ageing.
Pre-clinical studies and epidemiological observations in patients with an excess of hormone
production or in patients chronically treated with hormones/growth factors for various
pathologies have warned about the potential risk of cancer development and progression
which may be also associated to the use of certain doping agents. Anabolic steroids have
been described to provoke liver tumors; growth hormone or high levels of its mediator insulin-
like growth factor-1 (IGF-1) have been associated with colon, breast, and prostate cancers.
Actually, IGF-1 promotes cell cycle progression and inhibits apoptosis either by triggering
other growth factors or by interacting with pathways which have an established role in
carcinogenesis and cancer promotion. More recently, the finding that erythropoietin (Epo)
may promote angiogenesis and inhibit apoptosis or modulate chemo- or radiosensitivity in
cancer cells expressing the Epo receptor, raised the concern that the use of recombinant
Epo to increase tissue oxygenation might favor tumor survival and aggressiveness. Cancer
risk associated to doping might be higher than that of patients using hormones or growth
factors as replacement therapy, since enormous doses are taken by the athletes often for a
long period of time. Moreover, these substances are often used in combination with other licit
or illicit drugs and this renders almost unpredictable all the possible adverse effects including
cancer. Anyway, athletes should be made aware that long-term treatment with doping agents
might increase the risk of developing cancer [07032].
Dependence in clinical practice
The nonmedical use of anabolic-androgenic steroids (AAS) appeals to athletes across

586
several sports, particularly those whose activity makes muscle size and strength
advantageous, and in individuals (usually men) with body dysmorphic disorder. Patterns of
nonmedical use, including supratherapeutic doses of illicitly obtained drugs, increase the risk
for adverse psychiatric and other medical consequences. Although AAS users may be more
likely to consult physicians for nonpsychiatric medical consequences than changes in their
mental status, it is argued that the motivation for persistent use despite adverse
consequences is sustained in large part by psychological variables. Therefore, all physicians
who treat nonmedical AAS users will benefit from an understanding of these psychological
variables, including the potential for AAS to cause dependence [09032].
Treatment of drug addicts
The objective of this review is to describe self-administration procedures for modeling

addiction to cocaine, cannabis and heroin in the human laboratory, the benefits and pitfalls of
the approach, and the methodological issues unique to each drug. In addition, the predictive
validity of the model for testing treatment medications will be addressed. The results show
that all three drugs of abuse are reliably and robustly self-administered by non-treatment-
seeking research volunteers. In terms of pharmacotherapies, cocaine use is extraordinarily
difficult to disrupt either in the laboratory or in the clinic. A range of medications has been
shown to significantly decrease cocaine's subjective effects and craving without decreasing
either cocaine self-administration or cocaine abuse by patients. These negative data
combined with recent positive findings with modafinil suggest that self-administration
procedures are an important intermediary step between pre-clinical and clinical studies. In
terms of cannabis, a recent study suggests that medications that improve sleep and mood
during cannabis withdrawal decrease the resumption of marijuana self-administration in
abstinent volunteers. Clinical data on patients seeking treatment for their marijuana use are
needed to validate these laboratory findings. Finally, in contrast to cannabis or cocaine
dependence, there are three efficacious Food and Drug Administration-approved
medications to treat opioid dependence, all of which decrease both heroin self-administration
and subjective effects in the human laboratory. In summary, self-administration procedures
provide meaningful behavioral data in a small number of individuals. These studies
contribute to our understanding of the variables maintaining cocaine, marijuana and heroin
intake, and are important in guiding the development of more effective drug treatment
programs [08038].
Educational programs
Continuing educational programs developed for these at-risk populations by national olympic
organizations and athletic federations are important first steps to curb these doping behavior
Medical professionals, teachers, coaches and sports organizations must all be made aware
of this continuing problem in our adolescent and at-risk populations and contribute to its
solution by open, honest discussion. Most importantly, professional athletes must serve as
role models and spokesmen for drug-free sport and lifestyle. This position must be actively
supported by the media, owners of teams and international sports federations by providing
consistent leadership and advocacy of anti-doping programs in sport, regardless of costs and
consequences. Accepting the magnitude of doping in at-risk populations and developing
education, prevention and treatment programs is the only way we can prevent the continuing
spread of the abuse of doping in sport and its spread into the most fragile groups in our
society, our youth and at-risk populations [08006].
587
Social perceptions
It was explored social perceptions related to the use of anabolic steroids in sports. More
specifically, 78 women and 102 men read one of two scenarios depicting a male athlete
facing either a drug use or non-drug use situation. Then, participants reported their
perceptions of the scenario protagonist in terms of self-determined sport motivation,
sportspersonship orientations, and athletic aggression. Results of a multivariate analysis of
covariance indicated that, in comparison with a non-using protagonist, the anabolic steroid-
using athlete was regarded as less self-determined in one's motivation (i.e. sport participation
based on predominant feelings of pressure to obtain external rewards or avoid punishment)
and as displaying weaker sportspersonship orientations (i.e. lesser concerns for opponents,
the social conventions of sport, and for one's own athletic commitment). In addition, the
steroid-using athlete was perceived as resorting more readily to reactive aggression than to
instrumental aggression (i.e. intent to injure one's opponent vs merely hinder his
performance). Finally, the analyses did not disclose significant gender or interaction effects
[08040].
The aim of one study was to improve understanding of the development of multiple drug use
in patients seeking treatment at an addiction clinic for steroid-related problems. It was
interviewed six patients (four men and two women) with experience of AAS use who were
attending an addiction clinic for what they believed were AAS-related problems. The patients
were interviewed in-depth about their life stories, with special emphasis on social
background, substance use, the development of total drug use and subjective experienced
psychological and physical side effects. There was significant variation in the development of
drug use in relation to social background, onset of drug use, relationship to AAS use and
experience of steroid effects. All patients had initially experienced positive effects from AAS
but, over time, the negative experiences had outweighed the positive effects. All patients
were dedicated to excess training and took AAS in combination with gym training, indicating
that the use of these drugs is closely related to this form of training. Use of multiple drugs
was common either in parallel with AAS use or serially. The study shows the importance of
understanding how AAS use can develop either with or without the concomitant use of other
drugs of abuse. The use of anabolic steroids can, however, progress to the use of other
drugs. The study also indicates the importance of obtaining accurate, comprehensive
information about the development of AAS use in designing treatment programmes and
prevention strategies in this area [08041].
Medical risks with illegal drugs
Unfortunately, cadaver extracts of pituitary human growth hormone may still be in circulation.
It has been reported that a Russian coach was arrested and, upon searching his apartment
in Moscow, over 1000 cadaver pituitary glands were found preserved in a large container
[08042]. Moreover, the problem of counterfeit drugs also exists with hGH: illegal
pharmaceutical manufacturers are now flooding the black market with hGH vials of unknown
quality and safety [08006].
Human growth hormone is marketed on the internet in many forms: pills, drops and aerosol
formulations; most are ineffective and shams. The normal route of administration of hGH is
injection, posing an additional health risk of infection from non-sterile counterfeit drugs and
the risk of HIV and hepatitis transmission caused by shared needles. However, also using
high quality human growth hormone may lead to life-threatening health conditions, especially
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since some estimates report that athletes who use growth hormone to enhance performance
are taking 10 times the therapeutic dosage. Some reported side effects of hGH are abnormal
bone growth, hypertension, cardiovascular disease, cardiomyopathy, glucose intolerance,
colonic polyps, decreased life span, and cancer [08043].
As with steroids and growth hormone, doping with erythropoietin is often injected in
supernormal doses that could cause increased blood viscosity, deep vein and coronary
thromboses, cerebral thromboses, pulmonary embolism, arrhythmias, stroke and death. It
has been estimated that 20 European cyclists have died since 1987 due to abuse of
erythropoietin, making it one of the most deadly doping agents [08006].
Increased mortality in former dopers
Physical training has been shown to reduce mortality in normal subjects, and athletes have a
healthier lifestyle after their active career as compared with normal subjects. Since the
1950s, the use of anabolic androgenic steroids (AAS) has been frequent, especially in
power sports. The aim of the present study was to investigate mortality, including causes of
death, in former Swedish male elite athletes, active 1960-1979, in wrestling, powerlifting,
Olympic lifting, and the throwing events in track and field when the suspicion of former AAS
use was high. Results indicate that, during the age period of 20-50 years, there was an
excess mortality of around 45 percent. However, when analyzing the total study period, the
mortality was not increased. Mortality from suicide was increased 2-4 times among the
former athletes during the period of 30-50 years of age compared with the general population
of men. Mortality rate from malignancy was lower among the athletes. As the use of AAS
was marked between 1960 and 1979 and was not doping-listed until 1975, it seems
probable that the effect of AAS use might play a part in the observed increased mortality and
suicide rate. The otherwise healthy lifestyle among the athletes might explain the low
malignancy rates [14703].
Increased premature mortality of competitive powerlifters

Misuse of supraphysiological doses of anabolic steroids is claimed to have serious side
effects. The aim of the study was to determine the mortality, and the cause of premature
deaths among a group of subjects who are strongly suspected to have used anabolic
steroids for a non-medical purpose over several years. The mortality of 62 male powerlifters
placed 1st-5th in weight series 82.5-125 kg in Finnish championships during 1977-1982 was
compared with the mortality of population controls. The mortality during the 12-year follow-up
was 13 percent for the powerlifters compared to 3 percent in the control population. By 1993
eight of 62 powerlifters and 34 of 1094 population controls had died, thus the risk of death
among the powerlifters was 4.6 times higher (95 % confidence interval 2.04 to10.45), a
statistically highly significant difference. The causes of premature death among the
powerlifters were suicide (3), acute myocardial infarction (3), hepatic coma (1) and non-
Hodgkin's lymphoma (1). These findings add to the growing amount of evidence of an
association between anabolic steroid abuse and premature death, and support the view that
measures to decrease AAS misuse among both competitive and amateur athletes are
justified [00013].
Doping and the respiratory system
Historically many different drugs have been used to enhance sporting performances. The
magic elixir is still elusive and the drugs are still used despite the heavy adverse effects. The
respiratory system is regularly involved in this research probably because of its central
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location in the body with several connections to the cardiovascular system. Moreover people
are aware that O2 consumption and its delivery to mitochondria firstly depend on ventilation
and on the respiratory exchanges. The second step consists in the tendency to increase
VO2max and to prolong its availability with the aim of improving the endurance time and to
relieve the fatigue. Many methods and substances had been used in order to gain an artificial
success. Additional oxygen, autologous and homologous transfusion and erythropoietin,
mainly the synthetic type, have been administered with the aim of increasing the amount of
oxygen being delivered to the tissues. Some compounds like stimulants and caffeine are
endowed of excitatory activity on the CNS and stimulate pulmonary ventilation. They did not
prove to have any real activity in supporting the athletic performances. Beta-adrenergic
drugs, particularly clenbuterol, when administered orally or parenterally develop a clear illicit
activity on the myosin fibres and on the muscles as a whole. Salbutamol, terbutaline,
salmeterol and formoterol are legally admitted when administrated by MDI in the treatment of
asthma. The prevalence of asthma and bronchial hyperactivity is higher in athletes than
amongst the general population. This implies that clear rules must be provided to set a
correct diagnosis of asthma in the athletes and a correct therapy to align with the actual
guidelines according to the same rights of the "other" asthmatic patients [07132].
The purpose of one study was to investigate the effects of resistance training and long-term
anabolic androgenic steroids (AASs) administration on respiratory function. Subject groups
consisted of AAS users (n=9) who were still using AAS at time of testing (SU); AAS users (n=
6) who had been abstinent for > 3 months (SA), bodybuilding controls (n=8) (BC), and (n=8)
sedentary male controls (SC). FEV1, FVC, and PEF were measured. The results found that
all subjects were within normal range, and there were no differences between groups.
Maximum inspiratory pressure (MIP), and grip strength were both significantly greater in SU
compared with SC; no significant difference was found between the other groups. Their MIP
and grip strength was significantly correlated. The data from this study suggest that the
combination of resistance training and AAS administration produce a significant increase in
MIP in a cohort of long-term AAS users [11569].
Information on doping
Many sporting organisations in Australia conduct drug information seminars for their athletes;
however, it is uncertain whether these programs provide athletes with pertinent drug
information in formats that are conducive to information retention. The aims of one study
were to investigate self-reported confidence in knowledge of illicit drugs and information
seeking behaviours among elite athletes. Data were collected from two sources: quantitative
surveys with elite Australian athletes; and qualitative interviews with key experts who come
into contact with elite athletes. Athletes were confident in their knowledge of the effects of
illicit drugs such as cannabis and meth/amphetamine, but less confident in their knowledge
of the effects of illicit drugs such as GHB and ketamine. A substantial proportion felt that
athletes in their sport would benefit from more information concerning illicit drugs. Both
athletes and key expert believed that information on illicit drugs should be delivered to
athletes in a specific and relevant manner. There may be stigma attached to information
seeking within a sports club or organisation. Accordingly, improving the accessibility to
creditable information via the Internet may prove to be an effective means by which to
educate athletes on the effects of illicit drugs [11022].
Internet
Identifying the use of non-approved drugs by cheating athletes has been a great challenge
590
for doping control laboratories. This is due to the additional complexities associated with
identifying relatively unknown and uncharacterized compounds and their metabolites as
opposed to known and well-studied therapeutics. In 2010, the prohibited drug candidates and
gene doping substances AICAR and GW1516, together with the selective androgen receptor
modulator (SARM) MK-2866 were obtained by the Cologne Doping Control Laboratory from
Internet suppliers and their structure, quantity, and formulation elucidated. All three
compounds proved authentic as determined by liquid chromatography-high resolution/high
accuracy (tandem) mass spectrometry and comparison to reference material. While AICAR
was provided as a colourless powder in 100 mg aliquots, GW1516 was obtained as an
orange/yellow suspension in water/glycerol (150 mg/mL), and MK-2866 (25 mg/mlL was
shipped dissolved in polyethylene glycol (PEG) 300. In all cases, the quantified amounts
were considerably lower than indicated on the label. The substances were delivered via
courier, with packaging identifying them as containing “amino acids” and “green tea extract”,
arguably to circumvent customs control. Although all of the substances were declared “for
research only”, their potential misuse in illicit performance-enhancement cannot be excluded;
moreover sports drug testing authorities should be aware of the facile availability of black
market copies of these drug candidates [11023].
Internet websites offering androgenic anabolic steroids (AAS) were identified and available
products were examined. Keywords for the website search were: "anabolic steroids,"
"anabolic steroids buy,""anabolic steroid purchase." The first 10 websites offering AAS in the
first 10 pages of results were considered. At least two AAS-containing products per website
were selected. Thirty AAS-selling websites were identified, mainly located in the United
States (47 %) and Europe (30 %). Most websites sold other anabolic/ergogenic products
(clenbuterol, 77 %; GH/IGF, 60 %; thyroid hormones, 47 %; erythropoietin, 30 %; insulin, 20
%) or products for AAS-related adverse effects (mainly: estrogen antagonists, 63 %;
products for erectile dysfunction, 57 %; 5alpha-reductase inhibitors, 33 %; anti-acne
products, 33.3%). AAS were sold as medicines (70 %) or as dietary supplements (30 %).
AAS in medicines were mainly: nandronole (20 %), methandrostenolone (18 %), and
testosterone (12 %). Dietary supplements contained mainly DHEA and included several fake
compounds. Manufacturers were declared for 98 percent of medicines and 67 percent of
dietary supplements; however, several manufacturers were not found on the Internet.
Described benefits were usually few adverse effects and no estrogenicity. Toxicity was
seldom reported and presented as mild. Recommended doses were two-fourfold higher than
current medical recommendations. In conclusion, misleading information and deceiving
practices were common findings on AAS-selling websites, indicating their deleterious
potential for public health [11024].
This study examined whether different types of media affect the use of dietary proteins and
amino acid supplements, and intent to use anabolic-androgenic steroids. A random sample
of 618 boys aged 11-18 years from eight schools in the Flemish part of Belgium completed
standardized questionnaires as part of the Media and Adolescent Health Study. The survey
measured exposure to sports media, appearance-focused media, fitness media, use of
dietary supplements, and intent to use anabolic-androgenic steroids. Data were analyzed
using logistic regressions and are presented as adjusted odds ratios (OR); 9 percent
indicated to have used dietary proteins, 4 percent indicated to have used amino acid
supplements, and 12 percent would consider using anabolic-androgenic steroids. After
adjusting for fitness activity, exposure to fitness media was associated with the use of dietary
proteins (OR 7.2) and amino acid supplements (5.2). Intent to use anabolic-androgenic
steroids was associated with exposure to fitness media (2.4) and appearance-focused media
(6.0). Sports media did not correlate with the use of dietary supplements and intent to use
anabolic-androgenic steroids. Specific types of media are strong predictors of the use of
supplements in adolescent boys. This provides an opportunity for intervention and prevention
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through the selection of fitness media as a communication channel. Health practitioners
should also be aware that the contemporary body culture exerts pressure not only on girls
but also on boys [13044].
Users' sources of information and medical advice of doping substances
When developing a self-designed treatment plan, AAS users spend considerable time
researching and seeking advice from more experienced associates. Historically, AAS use
was developed by a gym subculture whereby novice bodybuilders interested in performance-
enhancing substance use would obtain the drugs and information from more experienced
users at the gym, often establishing a mentor-mentee relationship. Now the most easily
accessible source for information regarding the details of illicit AAS use is the Internet.
Numerous blogs and forums exist (e.g. www.steroid.com, www.steroidology.com) where
AAS users around the world can anonymously offer or request advice, share drug sources,
chronicle results, and collaborate on dosing schedules. Another source of information
involves specific “nutritionists” who, for a fee, usually yearly, give advice to these men about
AAS, dietary supplements, and nutritional plans. The AAS user's rationale for choosing
various drugs and protocols is typically based on anecdotal evidence and interpretations of
quasiscientific literature propagated via Internet forums. It is also apparent that some users
achieve popular authority within the Internet bodybuilding community and are often consulted
for medical advice via forums. Indeed, given the minimal exposure that physicians have to
AAS, coupled with the fact that many “expert” users have experimented with the majority of
available AAS and their companion medications, it is not too far-fetched to consider first-
hand experience of achievable gains, side effects, and “optimal” self-treatment regiments by
seasoned AAS users to be of perceived “greater value” than an average physician's
recommendation. Indeed, it is the general consensus within the AAS community that
experienced AAS users are more educated than their physicians on AAS use, a sentiment
that may contribute to the AAS user's hesitancy to approach his physician for advice when
adverse symptoms occur [14427].
Telephone hot-line
With the support of the Swedish National Institute of Health a national information service
was started in 1993 aiming to capture the abuse of doping agents in the general public. It
was organized as a telephone service, called the Anti-Doping Hot-Line, from our department
and managed by trained nurses co-operating with clinical pharmacologists. Important
information collected about all callers (anonymous) was: date of call, its origin, category of
caller, doping experience and main question being asked. Abusers were asked about their
age, sex, affiliation, abused drug(s), duration of abuse, habit of administration and adverse
reactions (ADRs). Between October 1993 and December 2000 25,835 calls were received
with a peak during spring and autumn. Most calls (12,400) came from non-abusers, 60
percent being males. Callers connected with gyms represented the largest group (30 %).
Most calls about specific drugs concerned anabolic androgenic steroids (AAS). Other drugs
or products included ephedrine, clenbuterol and creatine. The most commonly abused
anabolic steroids were testosterone, nandrolone-decanoate, methandienone and stanozolol.
The ten most commonly reported ADRs of AAS were aggressiveness (835), depression
(829), acne (770), gynecomastia (637), anxiousness (637), potency problems (413),
testicular atrophy (404), sleep disorders (328), fluid retention (318) and mood disturbances
(302). Female side effects included menstruation disturbances, hair growth in the face, lower
voice and enlarged clitoris. During the period 1996-200, totally 4339 persons reported about
10,800 side effects. This figure should be compared with the very low number of ADRs (27)
reported by prescribers to the Swedish ADR committee during the same period. Abuse of
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doping agents appears to be a new public health problem that needs detection, medical care
and prevention [03025].
The aim of one retrospective study was to analyze the calls concerning anabolic products
(AP), received at Écoute Dopage, a French anti-doping hot-line. It was reviewed all phone
calls handled between 2000 and 2008, among them 214 concerned AP. Information
collected include demographic data, reason for the phone call, name of AP, characteristic of
consumption, adverse reactions. Fifteen different AP (mainly testosterone) were reported.
Calls concerned information about side-effects (42 % of calls), risk for doping (28 %), and
risk for health (10 %), psychological assistance (10 %), and legislation (2 %). Most calls
came from fitness practitioners or bodybuilders (85 %). The reason for use was documented
in 137 subjects: to increase muscular strength (76 %), improve social life ability (15 %),
improve sporting ability (6 %), and losing weight (3 %). Eighty subjects (37 %) reported at
least one side-effect mainly uro-genital (40 cases) or psychic disorders (25 cases), both 15
cases. Among these 80 patients, 17 patients (21.25%) presented signs of AP dependence.
The abuse of AP in sport is a public health problem well known, but data on the dependence
on AAS are sparser. Information and education should be emphasise to fight against doping.
[13055].
Media medicine
One paper uses UK media coverage of the sleep drug modafinil to investigate the
medicalisation of sleep at a conceptual level. Using metaphorical frame analysis it was
investigated the conceptual links created in media discourse between sleep and health, and
the body and technology in the UK. Using this novel analytical tool it was explored under
what circumstances modafinil is constructed as a necessary medical treatment or a
(il)legitimate performance enhancement and, how in this process, various images of the body
are constructed. It was found that media discourse on modafinil was structured through four
types of sleep discourse: patient, sports, recreational, and occupational. Each discourse was
built up around the specific deployment of three central metaphorical frames “war“,
“commodity” and “competition” that acted to construct the biological body in a particular way.
How the body was framed in each discourse impacted upon how modafinil use was
portrayed in terms of therapy or enhancement and the level of engagement with a medical
rhetoric. This had distinct normative implications strongly influencing the legitimacy afforded
to modafinil use in each domain. It was argued that medical authority acts to legitimise
modafinil use for repair, restoration and relief of suffering, whilst being deployed to pass
judgment on its use in bodies already perceived as functioning normally. This led the authors
to conclude that conceptually, the acceptability of “enhancement” is strongly tied to context of
use and intricately related to medical social control [08044].
On doping in daily newspapers

To study the coverage by French newspapers of doping in sports, it was performed a
systematic review of articles appearing between January and March 2003 on the following
French websites: L'Equipe, Le Monde, Le Figaro, Libération, La Dépêche du Midi and
Agence France-Presse (AFP). It was recorded a total of 58 articles about doping. Among
them, 48 (83%) were collected from the AFP news. L'Equipe, a French sports newspaper,
published seven articles (12 %). Most of the recorded data reported results of worldwide
antidoping control (71 %). No information about new drugs was found. The analysis of the
selected articles pointed out the following: (i) the seriousness of observations related to
doping since, during this 3-month period, it was noted two deaths of athletes; (ii) the risks
associated with the use of dietary supplements, particularly products including amphetamine
derivatives; (iii) the interest in judicial investigation as an information source about doping in
sports (investigation of suspicious deaths of Italian football players); and (iv) identification of
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the sports involved in doping (cycling, but also athletics, football, rugby). Systematic analysis
of newspaper reports can be considered as a relevant method for monitoring the
pharmacovigilance and pharmacoepidemiology of doping in sports [04022].
Factors contributing to the limited appreciation of the adverse effects of

doping
Given the high prevalence of PED (performance-enhancing drug) use, and in particular the
high prevalence of AAS use (the largest category of illicit PEDs), one might ask why their
adverse effects are not better understood and why policymakers have not allocated more
resources to investigate and mitigate the public health impact of PEDs. Several factors may
explain why the issue of PED use and its adverse health effects has remained neglected
[14426].
- First, public attention is focused almost entirely on PED use among elite athletes, with
an emphasis on how these drugs enable athletes to illicitly gain a competitive
advantage. Hence, there appears to be a widespread misconception that PED use is
primarily a phenomenon among a small group of highly competitive elite athletes.
This misperception has distracted attention from the health risks associated with PED
use and the fact that PED use is not limited to elite athletes but involves a much
larger group of nonathlete weightlifters. And although testing is a major preoccupation
in athletics, it is virtually nonexistent elsewhere, in part because of the high cost of
PED testing.
- Second, researchers cannot ethically conduct controlled studies of the long-term

adverse effects of PEDs in normal volunteers, especially when using
supraphysiologic doses. Therefore, most of our knowledge comes from studies of
PED users in the field (supplemented with studies in animals). These uncontrolled
human studies are subject to inherent methodologic limitations including selection
bias (e.g. individuals experiencing adverse effects may be more likely or less likely to
present for study than those without such effects), information bias (eg, individuals
are retrospectively reporting use of illicit drugs of uncertain potency and authenticity,
often used years before the time of index evaluation), and confounding variables (eg,
PED users frequently consume a wide range of other PEDs, frequently use classical
drugs of abuse, and may also display additional risk factors for diseases that are
associated with weightlifting (diet, use of needles, and other aspects of their lifestyle).
- Third, because widespread illicit PED use did not appear in the general population
until the 1980s and 1990s, the great majority of the world's PED users are still under
the age of 50 today. As such, this relatively young population has not reached the
age of risk for a range of diseases, such as cardiovascular problems, that typically
arise later in life. This likely explains why, to date, only occasional case reports have
highlighted acute medical events and deaths associated with PEDs. And it's likely that
some of the long-term effects of PEDs will only now start to become visible as the
older members of the PED-using population reach the age of risk for these
phenomena. Therefore, current observations likely underestimate the full magnitude
of medical consequences of PEDs that will become evident over the next 2 or 3
decades.
- Fourth, PED use in the general population is usually covert. PED use typically begins
after the teenage years and therefore evades scrutiny of parents or high school
teachers. Consequently, national surveys focusing on teenagers, such as high school
594
students, will underestimate the total number of individuals who ultimately use PEDs,
because the great majority of such individuals initiate use after their teenage years.
Also, it has been our observation that people are less apt to disclose PED use than
other forms of drug use, perhaps because doing so would acknowledge that their
physical prowess is largely due to chemical enhancement.
- Fifth, PED users often do not trust physicians; in one study, 56 percent of AAS users
reported that they had never disclosed their AAS use to any physician. Thus,
physicians are often unaware of the prevalence of PED use.
- Sixth, PED use rarely brings individuals to emergency rooms, because the most
widely used class of PEDs, AASs, rarely precipitate a medical emergency
comparable to an overdose of alcohol or heroin. Thus, surveillance techniques such
as the Drug Abuse Warning Network do not capture AAS users. Collectively, these
many factors may conspire to keep nonathletic AAS use out of view, and thus
obscure the magnitude of this public health problem.
Harm reduction
The illicit use of anabolic steroids among the gym population continues to rise, along with the
number of steroid using clients attending harm reduction services in the UK. This presents
serious challenges to public health. Study objectives were to account for the experiences of
anabolic steroid users and investigate how “risk environments” produce harm. It was made
qualitative face-to-face interviews with 24 users of anabolic steroids engaged with harm
reduction services in the UK. Body satisfaction was an important factor when deciding to
start the use of anabolic steroids. Many users were unaware of the potential dangers of using
drugs from the illicit market, whereas some had adopted a range of strategies to negotiate
the hazards relating to the use of adulterated products, including self-experimentation to
gauge the perceived efficacy and unwanted effects of these drugs. Viewpoints, first-hand
anecdotes, norms and practices among groups of steroid users created boundaries of
“sensibl”' drug use, but also promoted practices that may increase the chance of harms
occurring. Established users encouraged young users to go to harm reduction services but,
at the same time, promoted risky injecting practices in the belief that this would enhance the
efficacy of anabolic steroids. The current steroid-related viewpoints and practices contribute
to the risk environment surrounding the use of these drugs and may undermine the goal of
current public health strategies including harm reduction interventions. The level of harms
among anabolic steroid users are determined by multiple and intertwining factors, in addition
to the harms caused by the pharmacological action or injury and illness associated with
incorrect injecting techniques [14437].
Empirical qualitative studies show that users exchange anabolic steroid-related information
on methods to reduce or avoid adverse effects. As a consequence, many users ignore or
perceive the risk of steroid usage as relatively minor. Steroid users who seek the support of
peer users but avoid social censure by keeping their use of drugs a secret from others may
also serve as a mechanism that promotes harm. By contrast, shared norms and practices in
local groups of steroid users may also establish lines between what is seen as sensible drug
usage and what is not. One study found that the use of ephedrine (a stimulant) and
nalbuphrine (an opioid) for enhancement purposes were seen by some steroid users as
irresponsible risk taking because these drugs were compared to amphetamines and heroin,
respectively [14437].
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An integrated approach
Doping use is an important issue in both competitive and non-competitive sports, and poses
potentially irreversible health consequences to users. Scholars increasingly call for theory-
driven studies on the psychosocial processes underlying doping use that will inform
subsequent policy-making and prevention interventions. The aim of one study was to
implement an integrative theoretical model to assess the direct and indirect effects of
motivational variables, moral orientations, and social cognitions on doping intentions. A
randomly selected and representative sample of 750 elite athletes anonymously completed a
battery of questionnaires on motivational and moral constructs, and social cognitions related
to doping. Hierarchical linear regression analysis and multiple mediation modeling were
used. The effects of achievement goals and moral orientations were significantly mediated by
attitudinal, normative, and self-efficacy beliefs, in both lifetime ever and never doping users.
Moral orientations indirectly predicted the doping intentions of never users, but did not predict
ever users' doping intentions. Achievement goals and sportspersonship orientations
influence doping intentions indirectly, through the effects of attitudes and self-efficacy beliefs.
Sportspersonship (moral) orientations were relevant to doping intentions among athletes with
no prior experiences with doping, while achievement goals and situational temptation were
relevant to both lifetime never and ever dopers [13043].
Difficulty accessing health services for dopers?
To understand health service access and needs of people who use performance and image
enhancing drugs (PIED) in regional Queensland a semi-structured interviews were
conducted with 21 people (n=19 men) who reported the use of a range of PIEDs, including
anabolic-androgenic steroids, human chorionic gonadotropin, growth hormone, clenbuterol,
tamoxifen, insulin and peptides. Participants reported accessing a range of services,
including needle and syringe programs and pharmacies, for sterile injecting equipment. While
PIEDs users attributed some stigma to needle and syringe programs, they were seen as an
important service for injecting equipment. Participants reported receiving either positive care
from health-care providers, such as general practitioners (GP), or having negative
experiences due to the stigma attached with PIED use. Few participants reported disclosing
their PIED use to their GP not only because of the concerns that their GP would no longer
see them but also because they felt their GP was not knowledgeable about these
substances. Participants in the study reported no difficulty in accessing health services
based on living in a regional area, with their concern focused more upon how they were
viewed and treated by service staff [150035].
Doping: proposing a functional analysis based on the color yellow
The authors demonstrate in three experiments (n=241) that yellow impacts on social
perceptions when associated with competitive cycling. In Experiment 1, the image of a
syringe evocated competitive cycling and doping more strongly when presented on yellow as
compared with gray. In Experiment 2, a performance improvement scenario yielded more
discredit of a depicted racer and higher suspicions of doping when ending on a yellow frame,
as opposed to a gray one. In Experiment 3, the image of a racer wearing a yellow jersey
(instead of a gray or a white one) yielded the lowest scores on measures of suitability as a
role model and attractiveness of sport participation. Moreover, no significant differences
emerged for gender, thereby suggesting equivalent effects for female and male participants.
Finally, the authors discuss conceptual and practical implications as well as limitations before
proposing a number of avenues for future research [150036].
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US Army: stimulants, anabolic hormones, and blood doping
The level playing field of competitive sports is an irrelevant concern in asymmetrical warfare.
However, there is a common theme of pressure to use performance-enhancing drugs
because athletic or military opponents may be using them to advantage. This interest is
fueled by personal anecdotes, misconceptions, and myths, and decisions to use or not to use
pharmacological interventions may ignore available scientific data. The U.S. Army has led
research in this area, with an abundance of published data extending back to World War II.
Behavioral effects have been a consistent concern. A key conclusion to be drawn from this
research is that although there may be specialized applications for some of these
interventions, the majority of soldiers will gain the greatest performance benefits from
effective physical and mental training programs combined with good principles of rest and
nutrition. Furthermore, the perceived need to improve human biology with drugs may be
solving the wrong problem, trying to fit the human to the demands of poorly conceived
tactics, tasks, and equipments instead of capitalizing on human capabilities [150037].
Protesting against things which have not yet happened
In the field of science and technology studies, recent works have analyzed the multiplication
of promises and predictions as a major evolution of science management. The authors
involved in this "sociology of technical expectations" have documented the role played by
promises in the elaboration of scientific projects and their impact on the social reception of
scientific issues. Yet, little attention has been paid to the predictions regarding undesirable
technological futures. One article proposes therefore to analyze the discursive and
argumentative practices through which journalists, scientists, and politicians denounce and
propose to counter a public issue "which does not exist yet": gene doping (no case of gene
doping has been recorded to date). After a literature review of the field of the sociology of
technological expectations and a presentation of the corpus, the article describes the
structure of predictions and analyzes the discursive strategies according to which social
actors predict a disaster in the making. The analysis is based on the study of media
discourses about gene doping, in a corpus of 163 French language articles from European
newspapers, published between 1998 and 2012 [150046].
Different countries
Spanish football
The aim of one study was to understand the attitudes, beliefs and knowledge among
technical staff members of Spanish football teams regarding doping. The sample was drawn
from 88 football teams that ranged from elite to under-18 categories. The 237 stakeholders
(34.45 ± 8.59 years) were categorised as follows: coaches (COA) (n=101), physical trainers
(PT) (n=68) and rest of technical staff (RTS) (n = 68). The descriptive exploratory design
used an instrument that combined a validated questionnaire (Performance Enhancement
Attitude Scale; PEAS) with specific, qualitative open-ended questions. The overall mean
score from the PEAS (range, 17-102, with higher scores representing more permissive
attitudes towards doping) was 31.64 ± 10.77; for COA, 31.91 ± 11.42; for PT, 31.28 ± 9.44;
and for RTS, 31.58 ± 11.18. Regarding participants' knowledge and beliefs, most
respondents (57.6 %) did not know the meaning of WADA (World Anti-Doping Agency); 84.9
percent did not know the prohibited list; and 39.2 percent had used/recommended
supplements. In addition, 87.2 percent recognised "differential treatment of doping among
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sports," with cycling considered most affected (62.6 %) and team sports least (27.2 %, with
football at 15 %). The dangerous lack of knowledge highlights the necessity for anti-doping
education and prevention programs for all football stakeholders, not just athletes [150047].
Elite Scottish athletes' attitudes towards doping
Understanding athletes' attitudes to doping continues to be of interest for its potential to

contribute to an international anti-doping system. However, little is known about the
relationship between elite athletes' attitudes to drug use and potential explanatory factors,
including achievement goals and the motivational climate. In addition, despite specific World
Anti-Doping Agency Code relating to team sport athletes, little is known about whether sport
type (team or individual) is a risk or protective factor in relation to doping. Elite athletes from
Scotland (n=177) completed a survey examining attitudes to performance-enhancing drug
(PED) use, achievement goal orientations and perceived motivational climate. Athletes were
generally against doping for performance enhancement. Hierarchical regression analysis
revealed that task and ego goals and mastery motivational climate were predictors of
attitudes to PED use. Compared with individual athletes, team athletes were significantly
lower in attitude to PED use and ego orientation scores and significantly higher in
perceptions of a mastery motivational climate. The study provides insight into how individual
and situational factors may act as protective and risk factors in doping in sport [150048].
Street market in Senegal
A pilot study of market surveillance in Senegal has been performed analyzing best selling
drugs from an official pharmacy and a street market in two principal cities of Senegal and
some traditional preparations from herbal medicine from the same market. A simple and
rapid gas chromatography method with mass spectrometry detection has been applied after
a liquid-liquid extraction of pharmaceutical products and traditional preparations at acidic,
neutral and basic pH with chloroform-isopropanol (9:1, v/v). The assay was validated in the
range from 10 mg to 250 mg/g powder preparations with good determination coefficients for
the calibration curves. At three concentrations spanning the linear dynamic ranges of the
calibration curves, mean recoveries of substances under investigation were always higher
than 90 percent and intra-assay and inter-assay precision and accuracy were always better
than 15 percent. The four best selling drugs purchased from a Dakar local pharmacy exactly
contained the amount of active principles reported in the respective labels while the best
selling drugs freely purchased from Kaolack market contained an amount of active
ingredients lower than that declared on the label. No pharmacological active compound, but
salicylic acid was found in one of the traditional herbal preparations. This pilot study showed
that whereas official drugs sold in pharmacies at prices accessible for a very few portion of
the population contained the amount of active principles as reported in the labels, those from
street market bought by the majority of population contained an amount of active ingredients
lower than that declared on the label and finally traditional herbal preparations seldom
contain pharmacological active principles [150049].
Uganda
Despite the development of advanced drug testing systems, both deliberate and inadvertent
doping in sports is increasing in elite, amateur and school sports. As a result, alternative
approaches that seek to influence an athlete's attitudes are needed to address the growing
doping concerns that threaten both the health and well being of the athlete as well as the
legitimacy of the sport. Therefore, one study set out to establish the doping attitudes,
knowledge and practices of professional Ugandan athletes, gathering information that may
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guide the design of more efficient doping prevention programs. It was a cross-sectional study
of 384 professional Ugandan athletes from four contact team sports (basketball, football,
handball and rugby) and two individual sports (athletics and cycling). An Interviewer
administered questionnaire used contained; questions about the doping behavior, the
performance enhancement attitude scale (PEAS), and doping use belief (DUB) statements.
Approximately 60 percent of the athletes reported familiarity with information on doping and
that most of this information came from fellow colleagues (42 %), individual or team coaches
(30 %) or the media (16 %). However, nearly 80 percent of these athletes could not correctly
define doping. The overall mean PEAS score, a measure of doping attitudes, for all study
participants was 40 ± 15. Female athletes (PEAS: 41 ± 15), athletes with a prior doping
history (PEAS: 44 ± 16) and athletes from the sport of athletics (PEAS: 57 ± 17) had higher
mean PEAS scores than their respective counterparts. Regarding doping behaviors
/practices, 9 percent of the study participants had been offered a doping agent at some point,
although only 4 percent of the athletes acknowledged recent use. Thus, the confessed use of
doping agents in this study was low, which may suggest that fewer athletes use doping
agents in Uganda. However, there is still an urgent need for educational anti-doping
programs to address the knowledge gaps observed amongst athletes in this study. Modifying
the existing Physical education curriculum for inclusion of more content about doping in sport
could provide the basis for doping prevention programs amongst amateur athletes in
Ugandan primary and secondary schools [150050].
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UNINTENTIONAL DOPING
Doping refers to the use of prohibited performance-enhancing substances or methods in
sport. It is considered a serious offence in sport that has many negative consequences,
including titles being stripped, bans from participating, damage to reputation and ill health. As
doping is assumed to be a pre-meditated action, engaging in this behaviour has been
predominantly attributed to athletes’ decision-making processes and moral values or
obligations. An increasing volume of literature has focused on the psychological factors
associated with doping or doping intention, such as motivation, sportsmanship, moral
disengagement and social-cognitive factors. These studies make a central assumption that
doping is a consciously controlled and goal-directed behaviour. However athletes may dope
unintentionally because they are not aware that food, drinks, supplements, or medications
may contain doping substances. Therefore, one of the key antidoping strategies of WADA,
apart from doping control, is to enhance athletes’ antidoping awareness and their capacity to
avoid unintentional doping [150065].
In the past years, an increasing number of dietary supplements containing undeclared

doping substances has been identified. The consumption of these supplements can lead to
inadvertent doping cases. Although warnings about the risk of inadvertent doping have been
communicated, recent studies show that athletes' knowledge of the problem is inadequate.
Furthermore, it seems that the risk has been growing due to the increased availability of
pharmaceutical substances via the internet, which are admixed by criminal manufacturers to
their, arguably, non-effective supplement products. The main candidates from the dietary
supplement market for inadvertent doping with stimulants are products containing ephedrine
and analogues, sibutramine and methylhexaneamine. Such products are mainly advertised
as fat burners or mood enhancers, and their use may lead to positive doping results in
competition. The risk of inadvertent doping with such supplements is based on different
reasons. In the case of supplements containing ephedrines, the natural sources of ephedrine
such as Ma Huang or ephedra sinica are frequently mentioned on the label rather than the
names of the active ingredients (ephedrine, pseudoephedrine, methylephedrine, etc).
Despite extensive education of athletes regarding unclear labelling, or the variety of names
by which banned substances may be referred to, many athletes still fall into this doping trap
[11010].
According to the World Anti-Doping Agency (WADA) Prohibited List, anabolic agents consist
of exogenous anabolic androgenic steroids (AAS), endogenous AAS and other anabolic
agents such as clenbuterol and selective androgen receptor modulators (SARMs). Currently
employed strategies for their improved detection include the prolongation of the detection
windows for exogenous AAS, non-targeted and indirect analytical approaches for the
detection of modified steroids (designer steroids), the athlete's biological passport and
isotope ratio mass spectrometry for the detection of the misuse of endogenous AAS, as well
as preventive doping research for the detection of SARMs. The recent use of these
strategies led to 4-80-fold increases of adverse analytical findings for exogenous AAS, to the
detection of the misuse of new designer steroids, to adverse analytical findings of different
endogenous AAS and to the first adverse analytical findings of SARMs. The strategies of the
antidoping research are not only focused on the development of methods to catch the
cheating athlete but also to protect the clean athlete from inadvertent doping. Within the past
few years several sources of inadvertent doping with anabolic agents have been identified.
Among these are nutritional supplements adulterated with AAS, meat products contaminated
with clenbuterol, mycotoxin (zearalenone) contamination leading to zeranol findings, and
natural products containing endogenous AAS. The protection strategy consists of further
investigations in case of reasonable suspicion of inadvertent doping, publication of the
600
results, education of athletes and development of methods to differentiate between
intentional and unintentional doping [14452].
About 20 percent of legally sold nutritional supplements have been found to be contaminated
with AAS. With global sales of nutritional supplements exceeding USD 32 billion in 2012 and
rapidly rising, this ubiquitous impurity poses significant public health problems [14427].
In the past years, an increasing number of dietary supplements containing undeclared

doping substances has been identified. The consumption of these supplements can lead to
inadvertent doping cases. Although warnings about the risk of inadvertent doping have been
communicated, recent studies show that athletes' knowledge of the problem is inadequate.2
Furthermore, it seems that the risk has been growing due to the increased availability of
pharmaceutical substances via the internet, which are admixed by criminal manufacturers to
their, arguably, non-effective supplement products. The main candidates from the dietary
supplement market for inadvertent doping with stimulants are products containing ephedrine
and analogues, sibutramine and methylhexaneamine. Such products are mainly advertised
as fat burners or mood enhancers, and their use may lead to positive doping results in
competition. The risk of inadvertent doping with such supplements is based on different
reasons. In the case of supplements containing ephedrines, the natural sources of ephedrine
such as Ma Huang or ephedra sinica are frequently mentioned on the label rather than the
names of the active ingredients (ephedrine, pseudoephedrine, methylephedrine, etc).
Despite extensive education of athletes regarding unclear labelling, or the variety of names
by which banned substances may be referred to, many athletes still fall into this doping trap.
In case of supplements enriched with sibutramine, the ingredient is not declared on the label
and the consumer is only provided with the information that the product contains ‘pure herbal
ingredients’ that are advertised to have considerable weight loss capabilities. Sibutramine
can be found in therapeutic or even supratherapeutic doses in slimming capsules, powders
and even slimming teas. Sibutramine is a synthetic anorectic drug, only approved as a
pharmaceutical preparation and available only on prescription. Because of its enormous side
effects (stroke and heart attack risk for patients with a history of cardiovascular disease), the
European Medicines Agency recommended in January 2010 that this drug be withdrawn
from the market. Sibutramine has been on the list of prohibited substances from the World
Anti-Doping Agency (WADA) since 2006. Since 2008–2009, there has been a high risk for
inadvertent doping with the stimulant methylhexaneamine, which was added to the WADA
prohibited list in 2009. The issue of inadvertent doping arises from the fact that
methylhexaneamine can be found on the labels in numerous different names such as
dimethylamylamine, dimethylpentylamine, pentylamine, geranamine, forthane and 2-amino-
4-methylhexane. On WADA's 2011 prohibited list, only the names methylhexaneamine and
dimethylpentylamine are mentioned in the group of stimulants, which complicates the
identification of the substance as a prohibited compound. In some supplements, geranium
root extract or geranium oil is mentioned as an alleged natural source of
methylhexaneamine. However, recent investigations have shown that methylhexaneamine is
not a natural ingredient of geranium oil, which means that synthesised methylhexaneamine
must have been added. Despite warnings by different national antidoping agencies in 2009
and 2010, numerous elite athletes in competition have been found to have a positive test for
methylhexaneamine [11425].
It is clear that there is a real risk that athletes who use dietary supplements may unknowingly
ingest a banned substance that will cause them to record a positive doping outcome. There
are cases in which a doping infringement can be traced back to supplement use and for
which the athlete has undertaken some strategies to reduce this risk. For example, the
athlete has received written advice from a supplement manufacturer that their produce does
not contain banned substances, but following a positive doping test, a sealed container of the
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dietary supplement has been examined and found to contain the banned ingredient.
Unfortunately, strict liability applies to these situations and even if athletes have been
successful in having the terms of their ban from sport reduced, a doping infringement will still
be recorded against their name. The loss of a career, livelihood and reputation are stakes
that an athlete must take into account when using dietary supplements [11425].
Until now only two cases have been detected in which dietary supplements contained
therapeutic (30 µg per tablet) and supratherapeutic (2 mg/capsule) doses of the beta 2-
agonist clenbuterol. In the supratherapeutically dosed product, clenbuterol was not declared
on the label. Both supplements were advertised as weight loss products. Because of the
extremely high concentration of clenbuterol in the second product (100-fold more than the
therapeutic dose), severe side effects could be expected; in addition, a high risk of cross-
contaminations of other products with clenbuterol is likely. Because WADA has classified
clenbuterol as an anabolic agent, its detection in doping control may lead to severe
sanctions. In 2009 and 2010, dietary supplements containing the prohibited growth hormone-
releasing peptide-2 (GHRP-2) were detected. The products were advertised to produce
anabolic, fat-reducing and anticatabolic effects and to improve regeneration. One product, in
an ampoule of a drinking solution, contained an orally active concentration of GHPR-2. Such
a product may lead to inadvertent doping cases because the name GHPR-2 is not
specifically listed on the WADA prohibited list and is unknown to the majority of the sports
community. However, GHRP-2 belongs as a releasing factor to the prohibited substance
group S2 on the WADA list [11425].
According to the World Anti-Doping Agency (WADA) Prohibited List, anabolic agents consist
of exogenous anabolic androgenic steroids (AAS), endogenous AAS and other anabolic
agents such as clenbuterol and selective androgen receptor modulators (SARMs). Currently
employed strategies for their improved detection include the prolongation of the detection
windows for exogenous AAS, non-targeted and indirect analytical approaches for the
detection of modified steroids (designer steroids), the athlete's biological passport and
isotope ratio mass spectrometry for the detection of the misuse of endogenous AAS, as well
as preventive doping research for the detection of SARMs. The recent use of these
strategies led to 4-80-fold increases of adverse analytical findings for exogenous AAS, to the
detection of the misuse of new designer steroids, to adverse analytical findings of different
endogenous AAS and to the first adverse analytical findings of SARMs. The strategies of the
antidoping research are not only focused on the development of methods to catch the
cheating athlete but also to protect the clean athlete from inadvertent doping. Within the past
few years several sources of inadvertent doping with anabolic agents have been identified.
Among these are nutritional supplements adulterated with AAS, meat products contaminated
with clenbuterol, mycotoxin (zearalenone) contamination leading to zeranol findings, and
natural products containing endogenous AAS. The protection strategy consists of further
investigations in case of reasonable suspicion of inadvertent doping, publication of the
results, education of athletes and development of methods to differentiate between
intentional and unintentional doping [14029].
According to the WADA Prohibited List, anabolic agents consist of exogenous anabolic
androgenic steroids (AAS) including for instance stanozolol, metandienone, oxandrolone,
etc, endogenous AAS such as testosterone, dehydroepiandrosterone, androstenedione, etc
and other anabolic agents such as clenbuterol and selective androgen receptor modulators
(SARMs). Analytical challenges for the detection of the misuse of anabolic agents result from
various different facts, among which a few are of major concern. These include the growing
problem of the administration of unapproved and/or new designer substances, the evidently
increasing use of endogenous substances, the constantly decreasing concentrations of the
analytes detected in positive doping control samples (most probably due to the use of
602
microdoses or to an earlier cessation of the drug regimen before doping controls are
expected), and genetic polymorphisms that lead to different metabolic patterns in the tested
individuals. Besides, major problems in connection with anabolic agents arise from “doping
traps”, which can lead to inadvertent doping cases. Candidates for such traps are, for
example, nutritional supplements adulterated with endogenous or exogenous AAS, food
contaminated with clenbuterol and animal tissues used in traditional medicine therapeutics
that contain endogenous AAS, etc. Here, the challenge obviously is to identify such sources
of inadvertent doping to be able to warn and protect athletes [14029].
Compulsory drug testing was introduced in 1968 by the International Olympic Committee.
Since then, several doping cases have been reported in sports competition world wide.
Positive results are based on the detection of prohibited substances, their metabolites and
markers in biological (mainly urine) samples supplied by athletes. In some cases, the
evidences were not contested and athletes admitted the use of banned substances.
However, in other cases, athletes denied the use of doping to enhance performance and
claimed to have inadvertently or passively absorbed the drug. Unfortunately, no current
accepted analytical method is capable of distinguishing between a sample from a cheater
and one from an athlete who was passively exposed to a doping agent. Athletes' allegations
have included the passive inhalation of drug smoke (e.g. marijuana) or the ingestion of food
or products sold as nutritional supplements that contained prohibited substances. In the
scientific literature, several studies have been performed to investigate the possibility of an
accidental exposure being the reason for the appearance of detectable quantities of banned
substances in urine samples. Based on these studies, this article discusses those cases
where the athlete's claims could be possible in generating a positive result in doping control
and in which circumstances it would be improbable to happen. Despite all efforts in fighting
against doping in sports, the misuse of forbidden substances has become common practice
among athletes who want to artificially increase their performances. Doping control, including
the systematic accomplishment of toxicological analyses, has been the most consistent form
of minimising the problem of doping in sports. The International Olympic Committee (IOC)/
World Anti-Doping Agency (WADA) has provided a list of prohibited substances in sports that
is constantly changing because of the introduction of new substances used by athletes. The
IOC list, updated in 2003, included the following classes of substances: stimulants, narcotics,
anabolic agents, diuretics, peptide hormones, agents with anti-oestrogenic activity and
masking agents. For each one of these groups, there are some representative examples.
The evidence of doping use is based on the identification of prohibited substances, their
metabolites and/or markers in biological samples supplied by athletes. Urine is the
mandatory specimen in doping control due to the ease and non-invasive nature of collection.
With the exception of some substances, in which a specific threshold is reported in the IOC
list, the detection of any quantity of a prohibited substance in the athlete’s sample is
considered an anti-doping rule violation [14039].
The market for products sold as “nutritional supplements” grew during the mid 1990s. A
strong indication of this trend comes from the increased sales of these products in the US,
from USD 8.3 billion in 1994 to USD 14 billion in 1999. The belief that this kind of supplement
is “natural” and, therefore, will not cause harmful effects, contributes to the popularity of
these products [14039].
Every year a new ergogenic product presents which is enthusiastically recommended by a

sales person with minimal sports medicine knowledge. On enquiry, the sports medicine
practitioner is assured that there has been undeniable proof and confirmation of the value of
this preparation. However, all papers and product information provided merely present
anecdotal experiences. Interestingly, clinical trials never seem to be forthcoming and the lack
of effectiveness of these products is demonstrated by their use rarely being sustained for
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more than one season. The dangers associated with the use of supplements have been
evidenced in the trial undertaken by the IOC-accredited drug testing laboratory in Cologne . It
analysed 630 nutritional supplements. Ninety four of these supplements or almost 15
percent, contained substances that would have led to a positive drug test. Of these 94
supplements, 23 contained precursors of both nandrolone and testosterone, 64 contained
precursors of testosterone alone and 7 contained precursors of nandrolone alone. None of
the illegal substances was listed on the labels. In addition to these 94 samples, 66 others or
10 percent returned borderline results for various unlabelled substances. As the World Anti-
Doping code of strict liability makes athletes totally responsible for whatever substances are
found in their bodies, the challenge is to educate them as to the danger of taking
supplements which could well contain contaminants that lead to a positive drug test. The
purity of many supplements are suspect as the manufacturers are not subject to the same
stringent controls as is the pharmaceutical industry, thereby exposing athletes to danger
[03027].
Why preventing unintentional doping is important?
Unintentional doping could lead to adverse analytical findings (AAFs) in doping controls (e.g.
testing positive for a banned substance after providing a urine or blood sample). A
substantial number of medications, nutritional supplements, beverages and herbal products
containing doping substances can be obtained from the internet, drug store or supermarket,
without prescription. These products present a serious risk for athletes. More than 10 percent
of nutritional supplements (e.g. multivitamins, minerals and amino acids) in the market
contain doping substances such as stimulants and anabolic steroids. Unintentional doping is
also possible when athletes are offered unfamiliar food, drinks, supplements or medication,
with unknown ingredients, from their trusted social agents, such as coaches, parents or
friends. These substances present athletes with a high risk of an AAF in antidoping
procedures, which could lead to WADA investigation and media attention. At worst, it may
result in a lengthy ban if the athlete cannot provide proof of the contaminated product.
Axiomatically, athletes who are blind to the potential of unintentional doping have a
heightened risk of consuming doping substances. This is also true for athletes who use drugs
that are on the banned list to treat their medical conditions. They may breach the antidoping
code if there is no prior approval (i.e. via a therapeutic use exemption). One might argue that
the presence of doping substances in food or medical products can depend on governmental
policy and legislation. A clear labelling system for product ingredients may make it easier for
athletes to identify doping substances in food or drug products, but it is practically impossible
to ensure all products’ ingredient tables would be updated according to changes in the
WADA's list of doping substances. Furthermore, such ingredient lists would need to be
enforced by law all over the world, which would be very challenging and costly [150065].
Protection of athletes from inadvertent doping with anabolic agents
Within the past few years, several sources of inadvertent doping with anabolic agents have
been identified. Among these are nutritional supplements adulterated with AAS, meat
products contaminated with clenbuterol and natural products containing endogenous AAS.
Since about 2003 a great number of nutritional supplements have become available,
advertised with claims of enormous muscle growth and increase in strength. According to the
advertisements and the labels these biological effects are attributed to new ingredients and
formulae with fantasy-derived and unapproved names. The analyses of many of these
products has shown that they contain exogenous AAS such as metandienone, stanozolol,
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oxandrolone and dehydrochloromethyltestosterone in therapeutic or even supratherapeutic
doses, not declared on the label. Also nutritional supplements adulterated with clenbuterol
have been detected, which were additionally advertised with their fat burning effects. The
consumption of such supplements inevitably leads to AAFs and is connected with
considerable health risks. Criminal nutritional supplement producers use this strategy to
establish their ineffective products in the sports market. To prevent inadvertent doping cases
athletes should avoid the consumption of nutritional supplements, which are advertised with
extreme claims of muscle growth, increase of strength and fat loss. The best strategy to
overcome this problem seems to be the appropriate education of athletes [14029].
How to help responsible athletes prevent unintentional doping?
Team physicians and coaches who see athletes on a regular basis are well-placed to take
the leading role in preventing unintentional use of doping substances. But they are not
always present to monitor athletes outside of training. Athletes must therefore be diligent in
self-monitoring and regulating their own behaviour for the avoidance of unintentional doping.
Developing self-monitoring and self-regulation skills is likely to be paramount in combatting
unintentional doping. To avoid unintentional forms of doping, athletes are advised to update
their knowledge of doping substances and to be aware of their presence in food, drinks,
supplements and medications, and, more importantly, to be ready to manage or avoid
situations where they are likely to be offered unknown food, drinks, supplements or
medications that could contain doping substances. These suggested behaviours for the
avoidance of unintentional doping require conscious effort. It has been reported that the
awareness of, and intention or adherence to avoiding unintentional doping is related to a
number of psychological variables such as motivation, social-cognitive variables and beliefs,
and self-control. Extending this research would help sport governing bodies, antidoping
agencies and sport professionals, to establish essential training and social environmental
conditions that empower athletes to self-monitor and act appropriately to help prevent
unintentional doping. Indeed, the research on this topic is still in its infancy because the
primary concern in the field has been the psychological antecedents of goal-directed doping
behaviours, rather than the factors relating to avoiding unintentional doping [150065].
Black market products and potential doping agents
Most people who use anabolic steroids obtain these drugs from the illicit market. Here, many
products are manufactured in “underground laboratories” operating outside the formal
parameters of the production of licensed medicinal products. In the UK, investigations by the
Medicines and Healthcare products Regulatory Agency (MHRA) have highlighted the issue
of adulterated drugs on the UK illicit market, including anabolic agents, leading to drug
seizures, arrests and illicit online retailers being shut down. In a number of cases, analysis
has identified that drugs claiming to contain anabolic steroids often include a different
amount of pharmacologically active substance than declared on the label, a different active
substance or no active substance, whatsoever. Further, drug analysis has shown that illicit
manufacturers prepare their own mixtures of anabolic steroids containing a blend of active
substances unlike that of any authorized medicinal products. Due to poor manufacturing,
products may also be accidentally contaminated with toxic chemicals and preparations for
injection may be unsterile, resulting in local or systemic infections. In addition, many people
who use anabolic steroids take a variety of off-the-shelf products marketed as dietary or
nutritional supplements that are increasingly being found to contain pharmacologically active
substances, such as appetite suppressants, sildenafil (used for erectile dysfunction) and
605
stimulants, that have not been listed on the package. The fact that in most cases, anabolic
steroid users remain unaware of their intake of specific active substances, means that many
case reports suffer from an over-reliance on self-reported drug use. Healthcare professionals
who come into contact with patients using anabolic steroids should be aware of these issues.
Where possible, and when resources are available, drug vials, tablets and/or biological
samples should be collected and submitted for analysis to identify the pharmacologically
active substance, along with any contamination. These data will add considerably to
information retrieved from the patients’ drug histories, improving both diagnosis and
prognosis. Furthermore, collecting data on the contents of these products will contribute to
our understanding of the causal relationship between various kinds of anabolic steroids and
specific health harms in case reports pertaining to patients’ usage of these drugs [14627].
In Germany
The desire to increase the athletic performance, to “optimize” an individual's appearance,

and to complement but also to arguably substitute exercise by means of drugs and drug
candidates has generated a considerable (illicit) market for compounds such as anabolic-
androgenic steroids, stimulants, growth promoting peptide hormones, and so on. Genuinely
developed for therapeutic use, their abuse/misuse generates enormous health risks, which
has necessitated comprehensive controls of compound trafficking by customs and anti-
doping authorities. From 2012 to 2013, the Bureau of Customs Investigation in Germany
confiscated products containing anabolic-androgenic steroids (AAS; 259 kg), stimulants
(13 kg), selective estrogen receptor modulators (SERMs; 24 kg), and human growth
hormone (hGH; 3500 ampules). In cooperation with the Bureau and under the umbrella of
the European Monitoring Center for Emerging Doping Agents (EuMoCEDA), the Cologne
Anti-Doping Laboratory analyzed an additional 337 (black market) products between 2010
and 2013, allowing to monitor developments in drug use and, hence, the anticipation of new
challenges in sports drug testing. Main tools utilized in characterizing confiscated materials
were liquid chromatography-high resolution mass spectrometry (LC-HRMS), gas
chromatography-high resolution mass spectrometry (GC-HRMS), and polyacrylamide gel
electrophoresis (PAGE) with subsequent bottom-up identification of peptidic compounds
using nano liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS). Among the
337 substances analyzed in the doping control laboratory in Cologne, 67 active ingredients
were found, 49 of which being categorized as doping agents by the World Anti-Doping
Agency (WADA). A total of 84 percent accounted for steroidal substances (predominantly
testosterone, trenbolone, and nandrolone and corresponding esters), 13 percent accounted
for peptide hormones and growth factors (predominantly hGH and growth hormone releasing
peptides (GHRPs)), 3 percent of the products contained hormones and metabolic
modulators, and 0.3 percent accounted for diuretic agents. Outstanding findings were the
detection of the selective androgen receptor modulator (SARM) LGD-4033, the thymic
hormone thymosin beta4, and a fusion protein of unknown biological activity. It was
concluded that trafficking of considerable amounts of arguably performance and/or body-
enhancing compounds has been observed during the past 4 years, the majority of which is
categorized as relevant to sports drug testing. Several substances are of fake/non-approved
nature and represent enormous health risks to the customer [14628].
Black market anabolic steroids
Anabolic steroids have become increasingly popular among athletes even at subcompetitive
or recreational level instead of extensive doping tests, educational campaigns and lethal
incidents. Nowadays, the fitness boom has also produced a population of steroid users at
high school level and also under non-sports practicing children. After opening the borders to
East Europe an explosion of the black-market for anabolic steroids occurred. Beside the well-
606
known side effects of anabolic steroids new problems and risks occurred due to fake drugs
from the black market. One review was subdivided into two parts: It was provided a detailed
review of the literature an anabolic steroids to the reader the information needed to make an
informed decision an the relative risks and benefits of anabolic steroids. Secondly, we
evaluated 40 "anabolic steroids" obtained from the black market using mass spectrometry or
gas chromatography analysis to evaluate the real pharmacological compounds. As the
results of this analysis, it was found that 15 (38 %) these drugs contained different or any
pharmacological compounds as labeled. From the external packing, a differentiation between
original and the fake drugs was impossible. Therefore, a large information and credibility gap
concerning anabolic steroids particular those from the black market exists between the
athletes and the medical and scientific communities [00021].
Estradiol
Estradiol and its metabolites in meat
Most studies related to research on steroids in main edible tissues (muscle, liver or kidney)
have focused on measurement of parent or major metabolite residues. In order to evaluate
the estradiol content in bovine edible tissues, a multi-step extraction procedure was
developed in conjunction with parallel metabolism studies of [14C]-17beta-estradiol in cattle.
Various classes of free estradiol and conjugates were separated: estradiol -17beta and -
17alpha, estradiol-17-fatty acid esters, estradiol 17-glycoside, estradiol 3-glucuronide,
estradiol-17-glycoside and 3-glucuronide (diconjugates) were separated. No sulphates
conjugated forms have been found at the detection level of the method. The quantification
was realized by calibration with deuterated 17beta-estradiol-d3 standard and was validated
at the ng/kg (ppt) level. Muscle, liver, kidney and fat samples from control or Revalor S single
(licensed implantation) or multi-implanted steers have been assayed. The results show a
wide variation between animals, but both the highest value and the mean of total estradiol
content in each group proportionally increase from untreated to multi-implanted animals. In
accordance with international rules, a calculation of the daily food supply of estradiol by such
edible tissues in comparison with the acceptable daily intake was performed [01024].
Clenbuterol
In pork liver
A surface plasmon resonance (SPR) immunoassay with an immobilization of self-assembled

molecular identification membrane for the detection of residual Clenbuterol Hydrochloride
(CLB) in pork liver was systematically investigated and experimentally validated for its high
performance. SPR immunoassay with a regular competitive inhibition assay cannot be
directly verified to detect CLB residuals. In this study, the binding of Au film with
mercaptopropionic acid was investigated using the known form of the strong S-Au covalent
bonds formed by the chemical radical of the mercaptopropionic acid and the Au film. After
that, the immunoglobulin IgG of swine (SwIgG-CLB) was bonded with the mercaptopropionic
acid by covalent -CO-NH- amide bonding. The modified comprehensive analysis of how the
membrane structure works was introduced together with the customized SPR bioanalyzer. In
order to evaluate the performance of this biomembrane structure, the concentrations of CLB-
contained solutions of 0 ng/mL, 10 ng/mL, 20 ng/mL, 33.3 ng/mL, and 40 ng/mL were
prepared by adding CLB reagents into the solutions of CLB antibody (Clenbuterol
Hydrochloride Antibody, CLB-Ab), successively and then the response unit (RU) was
607
measured individually. Using the data collected from the linear CCD array, the fitting curve
was established with the R-Square value of 0.9929. Correspondingly, the recovery rate
ranged from 88.48 percent to 103.21 percent was experimented and the limit of detection of
CLB in 1.26 ng/mL was obtained efficiently. It was concluded that the detection method
associated with biomembrane properties is expected to contribute much to the determination
of residual CLB in pork liver quantitatively by using the customized SPR bioanalyzer
[150076].
In calf hair
In agriforensics, time of administration is often debated when illegal drug residues, such as
clenbuterol, are found in frequently traded cattle. In this proof-of-concept work, the feasibility
of obtaining retrospective timeline information from segmented calf tail hair analyses has
been studied. First, an ultraperformance liquid chromatography-tandem mass spectrometry
(UPLC-MS/MS) hair analysis method was adapted to accommodate smaller sample sizes
and in-house validated. Then, longitudinal 1 cm segments of calf tail hair were analyzed to
obtain clenbuterol concentration profiles. The profiles found were in good agreement with
calculated, theoretical positions of the clenbuterol residues along the hair. Following
assessment of the average growth rate of calf tail hair, time of clenbuterol administration
could be retrospectively determined from segmented hair analysis data. The data from the
initial animal treatment study (n=2) suggest that time of treatment can be retrospectively
estimated with an error of 3-17 days [150077].
Various living tissues for monitoring clenbuterol abuse in food-producing cattle
It was aimed to evaluate whether living tissues such as urine, plasma and hair were suitable
for monitoring clenbuterol (CL) abuse after its subchronic administration of a growth-
promoting dose to the Chinese Simmental beef cattle. Eight male, white and red pied
Chinese Simmental beef cattle were involved in the experiment, and the CL dose was 16
microg/kg BW/day. Liquid chromatography tandem mass spectrometry (LC-MS-MS) was
used to determine CL residues in different tissues, and the addition of D9-clenbuterol internal
standard was applied to increase determination accuracy. The recovery of plasma, urine,
hair and in vivo tissues was 88.5-114.2, 83.9-114.3, 88.6-116.9 and 85.3-121.7 percent,
respectively. The results showed that CL residue concentrations in the plasma, on Days 14
after withdrawal and later, were lower than the limit of detection (LOD) (0.06 ng/mL) and CL
residue in urine was lower than LOD (0.16 ng/mL) 42 days after treatment. CL significantly
accumulated in the white and red hair and maintained more than 7.19 ± 2.19 pg/mg within
the early withdrawal period of 70 days. A large number of CL were determined in all tested
biological tissues, in which residues were higher than the maximum residue limits (MRLs)
after dietary administration of CL for 21 days and pre-slaughter withdrawal period of ∼6 h. A
particular concern is the slow depletion of residues of CL in some tissues like gluteus and
liver still exceeding theirs MRLs, respectively, on Days 14 or 28 days after withdrawal. Our
study indicated that plasma and urine could be available for monitoring CL abuse only within
a short period of time. However, hair (including light-pigmented) as a target matrix can be
selected to perform the long-period monitor of CL [150078].
Dexamethasone
Growth promoters (GPs) such as the glucocorticoid dexamethasone (DEX) and the beta-
adrenergic agonist clenbuterol (CLEN) are still used abusively in beef cattle production.
Transcriptomic markers for indirect detection of such GPs have been discussed in either
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experimentally treated animals or commercial samples separately. In the present study we
examine the transcriptomic signature of DEX alone or in combination with CLEN in skeletal
muscle of experimentally treated beef cattle, and, furthermore, compare them with previously
screened commercial samples from a field-monitoring study, as well as with proteomics data
representing the same set of samples. Using DNA microarray technology, transcriptomic
profiling was performed on 12 samples representing three groups of animals: DEX (0.75
mg/animal/day, n=4), a combination of DEX (0.66 mg/animal/day) and CLEN (from 2 to 6
mg/animal/day, n=4) and a control group (n=4). Analyses showed the differential expression
of 198 and 39 transcripts in DEX and DEX-CLEN groups, respectively. Both groups had no
common modulated genes in between, neither with the proteomics data. Sixteen candidate
genes were validated via qPCR. They showed high correlation with the corresponding
microarray data. Principal component analysis (PCA) on both the qPCR and normalised
microarray data resulted in the separation of treated animals from the untreated ones.
Interestingly, all the PCA plots grouped the DEX-positive samples (experimental or
commercial) apart from each other. In brief, this study provides some interesting
glucocorticoid-responsive biomarkers whose expression was contradictory to what is
reported in human studies. Additionally, this study points out the transcriptomic signature
dissimilarity between commercial and experimentally treated animals [150079].
Sibutramine
In case of supplements enriched with sibutramine, the ingredient is not declared on the label
and the consumer is only provided with the information that the product contains “pure herbal
ingredients” that are advertised to have considerable weight loss capabilities. Sibutramine
can be found in therapeutic or even supratherapeutic doses in slimming capsules, powders
and even slimming teas. Sibutramine is a synthetic anorectic drug, only approved as a
pharmaceutical preparation and available only on prescription. Because of its enormous side
effects (stroke and heart attack risk for patients with a history of cardiovascular disease), the
European Medicines Agency recommended in January 2010 that this drug be withdrawn
from the market. Sibutramine has been on the list of prohibited substances from the World
Anti-Doping Agency (WADA) since 2006 [11010].
Melamine
Nutritional supplements are used or experimented with by consumers, notably these are;
competitive and recreational athletes of all ages, and “weekend warriors”. As a consequence
the supplement industry has grown to meet the increasing demand. A Global Industry
Analysts Inc. report indicates that the herbal supplement market has not declined during the
worldwide recession, but in fact exhibited steady growth over the period 2008 to 2009. It is
anticipated that the market will reach USD 93.15 billion by the year 2015. These
supplements may contain adulterated substances that may potentially have harmful short -
and long-term health consequences to the consumer. "Scrap Melamine" is such an example,
which has been implicated in the kidney failure and death of several cats, dogs and pigs. In
China in 2008, reports described very severe health effects in infants and young children. At
the time over 294,000 infants were screened and diagnosed with urinary tract stones and
sand-like calculi associated with melamine in milk products, of which 50,000 infants were
hospitalised, and at least six associated deaths, recorded. The extent that melamine
contamination occurs in nutritional supplements is not known. Therefore, the aim of this
study was to determine whether commercially available nutritional and traditional supplement
products contain melamine, even though they are not declared by the manufacturer on the
609
product label. A total of 138 nutritional supplements products were obtained from (i) direct
purchases from shops, pharmacies and outlets, (ii) directly from consumers, and (iii) from
suppliers, manufacturers and distributors. The products were laboratory analysed for
melamine, using Tandem Liquid Chromatography Mass Spectrometry. Forty-seven percent
of all the products (n=138) tested positive for melamine. Eight-two percent of the South
African produced products (n = 27) tested positive and 58 percent of the products imported
into South Africa (n=50) tested positive. The median concentration estimate for melamine in
the products tested were, 6.0 microg/g for the 138 supplements tested, 8.9 microg/g for
South African produced products, and 6.9 microg/g for products imported into South Africa. It
was concluded that the melamine (undeclared on product label) levels detected in the
nutritional supplements products investigated were within the tolerable daily intake (TDI) limit
guidelines of 200 μg/g as set by WHO and others. Melamine over exposure within the
context of the nutritional supplements consumption in the products investigated should not
be of concern to the consumer provided the recommended guidelines of daily product use
are adhered to. Further investigation is warranted to determine, (i) the link of melamine as
(part) substitute for the perceived total declared protein content on the product label, (ii)
cyanuric and uric acid presence in the supplement products that could form chemical-
complex formation with melamine and/or analogues that could cause adverse health effects
[150080].
Methylhexaneamine
Since 2008-2009, there has been a high risk for inadvertent doping with the stimulant
methylhexaneamine, which was added to the WADA prohibited list in 2009. The issue of
inadvertent doping arises from the fact that methylhexaneamine can be found on the labels
in numerous different names such as dimethylamylamine, dimethylpentylamine, pentylamine,
geranamine, forthane and 2-amino-4-methylhexane. On WADA's 2011 prohibited list, only
the names methylhexaneamine and dimethylpentylamine are mentioned in the group of
stimulants, which complicates the identification of the substance as a prohibited compound.
In some supplements, geranium root extract or geranium oil is mentioned as an alleged
natural source of methylhexaneamine. However, recent investigations have shown that
methylhexaneamine is not a natural ingredient of geranium oil, which means that synthesised
methylhexaneamine must have been added. Despite warnings by different national
antidoping agencies in 2009 and 2010, numerous elite athletes in competition have been
found to have a positive test for methylhexaneamine [11010]
Illicit blue tablets containing anabolic androgen steroids
The necessity of specific, confirmatory tests in the identification of seized illicit products was
highlighted by the analysis of eighteen heart shaped, blue tablets confiscated by Police at a
street control in the North East of Italy. The tablets responded as amphetamines to a
preliminary color test (Marquis); a subsequent, confirmatory assay by gas chromatography-
mass spectrometry revealed the presence of two anabolic androgen steroids (AAS),
methandienone and methyltestosterone, in concentration of 1.7 and 1.5 mg respectively per
tablet; no trace of amphetamine-like or nitrogen containing compounds was found. The
observed orange coloration was due to the reaction of concentrated sulphuric acid, contained
in the Marquis reagent, with keto group of steroids. The two AAS, banned under the world
antidoping code, are not considered as psychoactive drugs of abuse in most countries,
although their trafficking may entangle severe public health concerns [13076].
610
Counterfeit products
As the patients will generally be using counterfeit products one is unable to advise on the
relative safety of each product. For example, growth hormone being used by bodybuilders,
purchased on the black market, was found to contain no more than glucose, which had not
been prepared in a sterile manner (personal communication from medical information
department, Pharmacia–Upjohn). Often, counterfeit products will contain anabolic steroids of
indeterminate dosage but with no quality control the label on the product may bare little
resemblance to the contents. This in itself constitutes a health risk but substitute prescribing
would be fraught with logistical and monitoring problems [01015].
Doping through supplement use, general aspects
The potential for supplement use to result in doping infringements is likely to be of concern
for anyone involved in sports nutrition. The available data indicates that 40-70 percent of
athletes use supplements, and that between 10 and 15 percent of supplements may contain
prohibited substances. Such data indicates that there is a considerable risk of accidental or
inadvertent doping through using supplements. Accordingly, one paper sets out to provide an
overview of the currently available empirical evidence of accidental doping by supplement
use. In carrying out this task, the authors refer to press releases and proxy measures
associated with nutritional supplement use, as well as statistical data on supplement
contamination rates and doping infractions. A number of different indications as to the
percentage of doping cases that might be attributed to supplement use are presented,
ranging from 6.4 to 8.8 percent. Such percentages are not comparable; instead they are
provided as indications as to how difficult it is to ascertain or estimate the scale of this
problem. Although some forms of estimation can be made, it is suggested that it is currently
not possible to quantify the scale of the problem. By way of conclusion, it is argued that
antidoping regulators may wish to review current data gathering and information provision
systems so that the problem of inadvertent doping can be more directly assessed as a factor
in sports doping overall [150066].
Nutritional supplements are part of the diet of many athletes. With the exception of caffeine
and ephedrine alkaloids, most of these products do not contain substances that are
prohibited to competing sportsmen. In recent years, androgens, pro-hormones such as
DHEA, androstenedione, androstenediol and 19-norsteroids became available for oral self-
administration in many countries and on the Internet. Their claimed actions, efficiency or
potency, and the possible adverse effects have not been thoroughly investigated by
controlled clinical studies. Some products were shown to contain prohibited substances such
as ephedrine, caffeine, or steroids, that were not listed on the label. Urine samples collected
after the administration of these supplements can test positive. The administration of natural
steroids such as testosterone and its precursors cannot be proven by the sole identification
of the substances in the urine. The approach to detection is based upon the deviation of
selected parameters of the metabolic profiles from the range of values normally found in
humans. The individual's norm is also studied to exclude the few cases of systematic and
natural excretion of extreme values. The combination of the GC/MS and the GC/C/IRMS
offers a powerful tool to discriminate between the natural and synthetic origin of the urinary
steroids [01017].
To determine if steroids containing over-the-counter (OTC) dietary supplements conform to

the labeling requirements of the 1994 Dietary Supplement Health and Education Act
611
(DSHEA) 12 brands of OTC supplements containing 8 different steroids were randomly
selected for purchase in stores that cater to athletes. There are two androstenediones (4-
and 5-androstene-3,17-dione), two androstenediols (4- and 5-androstene-3beta, 17beta-
diol), and 4 more are 19-nor cogeners (19-nor-4- and 5-androstene-3,17-dione and 19-nor-4-
and 5-androstene-3beta, 17beta-diol). The 12 brands of OTC anabolic-androgenic
supplements were analyzed by high-pressure liquid chromatography. It was found that 11 of
12 brands tested did not meet the labeling requirements set out in the 1994 Dietary
Supplement Health and Education Act. One brand contained 10 mg of testosterone, a
controlled steroid, another contained 77 percent more than the label stated, and 11 of 12
contained less than the amount stated on the label. These mislabeling problems show that
the labels of the dietary steroid supplements studied herein cannot be trusted for content and
purity information. In addition, many sport organizations prohibit OTC steroids; thus, athletes
who use them are at risk for positive urine test results. In this article we provide the details of
the analyses, a summary of the steroids by name and structure, and information on the
nature of the positive test results. Athletes and their physicians need this information
because of the potential medical consequences and positive urine test results [01018].
Many athletes report using a wide range of special sports foods and supplements. In the
present study of 77 elite Australian swimmers, 99 percent of those surveyed reported the use
of these special preparations, with 94 percent of swimmers reporting the use of non-food
supplements. The most popular dietary supplements were vitamin or mineral supplements
(used by 94 % of the group), herbal preparations (61 %), and creatine (31%). Eighty-seven
percent of swimmers reported using a sports drink or other energy-providing sports food. In
total, 207 different products were reported in this survey. Sports supplements, particularly
supplements presented as pills or other non-food form, are poorly regulated in most
countries, with little assurance of quality control. The risk of an inadvertent "positive doping
test" through the use of sports supplements or sports foods is a small but real problem facing
athletes who compete in events governed by anti-doping rules. The elite swimmers in this
survey reported that information about the "doping safety" of supplements was important and
should be funded by supplement manufacturers. Although it is challenging to provide such
information, we suggest a model to provide an accredited testing program suitable for the
Australian situation, with targeted athlete education about the "sports safety" of sports
supplements and foods [01019].
It was reported the findings of the analysis of 75 different nutritional supplements bought
through the internet. Seven products (all from the class of prohormones) contained other
hormone substances than indicated on the labels, and two further products contained
ephedrine and caffeine without a clear indication on the labels [01020].
Non reported contents in dietary supplements
Several recently published reports have shown that the content and dose of active
ingredients in some dietary supplements deviates significantly from the labeled amounts.
Discrepancies between actual product composition and labeled content have been
demonstrated with ginseng, echinacea, St. John's Wort, and androstenedione. In a few
instances, botanical species misidentification has resulted in contamination with harmful
herbs and produced significant morbidity. Adulteration of herbal products with
pharmaceuticals and contamination with heavy metals have also been described. A 2000
analysis showed that many commercial dietary supplements contain markedly different
amounts of ephedra alkaloids than declared on the product labels. Significant lot-to-lot
variation was discovered. They also found that 7 of 20 products analyzed did not list the
quantity of total ephedra alkaloids on the label, and 16 of 20 products did not list the
612
ephedrine content on the label. The caffeine content of the supplements was not investigated
[14041].
Assessment and management of the risk in Australia
Many athletes report using a wide range of special sports foods and supplements. In the
present study of 77 elite Australian swimmers, 99 percent of those surveyed reported the use
of these special preparations, with 94 percent of swimmers reporting the use of non-food
supplements. The most popular dietary supplements were vitamin or mineral supplements
(used by 94 % of the group), herbal preparations (61 %), and creatine (31 %). Eighty-seven
percent of swimmers reported using a sports drink or other energy-providing sports food. In
total, 207 different products were reported in this survey. Sports supplements, particularly
supplements presented as pills or other non-food form, are poorly regulated in most
countries, with little assurance of quality control. The risk of an inadvertent "positive doping
test" through the use of sports supplements or sports foods is a small but real problem facing
athletes who compete in events governed by anti-doping rules. The elite swimmers in this
survey reported that information about the "doping safety" of supplements was important and
should be funded by supplement manufacturers. Although it is challenging to provide such
information, we suggest a model to provide an accredited testing program suitable for the
Australian situation, with targeted athlete education about the "sports safety" of sports
supplements and foods [01021].
Anabolic steroids in nutritional supplements
A method is described for the determination of anabolic steroids including testosterone, 19-
nor-4-androstene-3,17-dione, 4-androstene-3,17-dione and nandrolone in food supplements.
Initial clean-up is done by HPLC followed by determination with GC/MS. A 'contaminated'
food supplement was analysed and appeared to contain 19-nor-4-androstene-3,17-dione and
4-androstene-3,17-dione. One capsule of this nutritional supplement was ingested by five
male volunteers. Urine samples were collected and analysed by GC/MS and GC/MS-MS.
Neither the ratio testosterone/epitestosterone, nor the ratio androstenedione/epitestosterone
increased significantly. Concentrations above 2 ng/ml for norandrosterone, the major
metabolite of nandrolone, were detected until 48-144 h after ingestion of the food supplement
[01022].
Dietary supplements containing prohibited anabolic agents
The extent of the contamination of dietary supplements with anabolic agents was evaluated
in 2001 and 2002. A well-publicised study showed that about 15 percent of non-hormonal
supplements such as vitamins, minerals, proteins and creatine contained anabolic
androgenic steroids (mainly prohormones) that were not declared on the label. The reason
for the contamination was most probably the fact that manufacturers of prohormones also
manufactured other supplements on the production line without sufficient cleaning. Another
source of cross-contamination could have been the unclean transport containers from raw
material suppliers of prohormones. The amount of detected prohormones, especially
prohormones of nandrolone, could produce positive doping cases. Since 2002, dietary
supplements have appeared on the market, which are probably intentionally spiked with high
amounts (more than 1 mg/g) of “classic” anabolic steroids, not declared or declared with non-
approved or fancy names on the label. Among these, steroids including stanozolol,
metandienone, dehydrochloromethyltestosterone and oxandrolone have been identified. All
these steroids are orally effective drugs based on their 17-alkyl group. These dietary
613
supplements are advertised as leading to enormous enhancement of strength and lean body
mass. The concentrations of the anabolic androgenic steroids are in the therapeutic or
supratherapeutic range per serving leading to positive doping cases detectable for several
days and weeks, respectively, depending on the type of steroid administered. Because the
manufacturers of these faked products also prepare other nutritional supplements on the
same production line, the risk of cross-contaminations with such “classic” anabolic
androgenic steroids is very high. Such contaminations have been found in fizzy tablets of
vitamin C, magnesium and multivitamins produced for Spanish and German supermarkets
containing, for example, small amounts of stanozolol and metandienone with the potential to
produce a positive doping response [11425].
Since 2002, dietary supplements have appeared on the market, which are probably
intentionally spiked with high amounts (more than 1 mg/g) of “classic” anabolic steroids, not
declared or declared with non-approved or fancy names on the label. Among these, steroids
including stanozolol, metandienone, dehydrochloromethyltestosterone and oxandrolone have
been identified. All these steroids are orally effective drugs based on their 17-alkyl group.
These dietary supplements are advertised as leading to enormous enhancement of strength
and lean body mass. The concentrations of the anabolic androgenic steroids are in the
therapeutic or supratherapeutic range per serving leading to positive doping cases
detectable for several days and weeks, respectively, depending on the type of steroid
administered. Because the manufacturers of these faked products also prepare other
nutritional supplements on the same production line, the risk of cross-contaminations with
such classic anabolic androgenic steroids is very high. Such contaminations have been
found in fizzy tablets of vitamin C, magnesium and multivitamins produced for Spanish and
German supermarkets containing, for example, small amounts of stanozolol and
metandienone with the potential to produce a positive doping response. Since 2002, the so-
called designer steroids can also be found on the dietary supplement market. These steroids
are neither listed as ingredients in any currently available medication, nor do their names
appear in the WADA list of prohibited substances. Most of these designer steroids have been
synthesised in the 1960s and were tested only in animal studies for their anabolic and
androgenic effects. Nowadays, these steroidal agents are produced exclusively for the
nutritional supplement market and are advertised for their anabolic- or aromatase-inhibiting
capacities. With regard to the effects and side effects of these steroids for humans, there is
limited or no knowledge. In most cases, the labelling of these products contains non-
approved or fancy names of the steroids. More than 40 such designer steroids have been
detected. The detection of metabolites of such a steroid in an athlete's urine sample is likely
to lead to a positive doping case [11010].
Nutritional supplements can be a source of positive doping cases as some supplements

contain prohibited substances without showing this on their label. This problem has existed
for some time and has been extensively studied in the past 8 years. The sport of tennis has
played a particular role in this problem because of some peculiar doping cases within its
community. For more than a decade, it has been known that nutritional supplements can be
“contaminated” with doping substances, which means that the contents of the supplements
are not identical to the list of ingredients on the label. Tennis has played a particular role in
this debate because of the complexity of the cases of Bohdan Ulihrach and Greg Rusedski,
who tested positive for nandrolone or nandrolone prohormones in 2002 and 2003,
respectively. In June 2007, Guillermo Coria sued an American nutritional company for the
financial damages he suffered during his 2 year suspension after also testing positive for
nandrolone in 2001. This problem is of major concern to elite athletes, who can test positive
in a doping test without knowingly taking banned substances. This so called “inadvertent
doping use” has resulted in an unknown number of positive cases because doping tests
often rely on the presence of metabolites of banned substances in urine, and cannot discern
614
between intentional and inadvertent use [07025].
Twenty-four products suspected of containing anabolic steroids and sold in fitness equipment
shops in the United Kingdom (UK) were analyzed for their qualitative and semi-quantitative
content using full scan gas chromatography-mass spectrometry (GC-MS), accurate mass
liquid chromatography-mass spectrometry (LC-MS), high pressure liquid chromatography
with diode array detection (HPLC-DAD), UV-Vis, and nuclear magnetic resonance (NMR)
spectroscopy. In addition, X-ray crystallography enabled the identification of one of the
compounds, where reference standard was not available. Of the 24 products tested, 23
contained steroids including known anabolic agents; 16 of these contained steroids that were
different to those indicated on the packaging and one product contained no steroid at all.
Overall, 13 different steroids were identified; 12 of these are controlled in the UK under the
Misuse of Drugs Act 1971. Several of the products contained steroids that may be
considered to have considerable pharmacological activity, based on their chemical structures
and the amounts present. This could unwittingly expose users to a significant risk to their
health, which is of particular concern for naïve users [14706].
Colloquially referred to by various misleading monikers (“pro-hormones”, “natural steroids”,

“testosterone boosters”, etc.) designer anabolic steroids have been popular now for over a
decade as a way to achieve classic anabolic steroid-like results from products sold in the
legal marketplace. Recent evidence suggests that anabolic steroid use may be the most
common cause of hypogonadism in men of reproductive age. Despite recent regulatory
efforts that have banned specific compounds, many anabolic-androgenic steroids (AAS)
remain available in over-the-counter dietary supplements that are legally sold in the United
States. Severe side effects including hepatotoxicity, cholestasis, renal failure, hypogonadism,
gynecomastia, and infertility have been reported secondary to the use of these products.
While some of these side effects may be reversible, more aggressive use may result in more
permanent end-organ damage as has been previously described for the case of aggressive
AAS users [Rahnema et al., Fertil Steril, 2014]. Designer AAS remain easily available for
purchase in over-the-counter bodybuilding supplements and these products appear to be
increasingly popular, despite the known health risks associated with their use. It was
conducted a systematic search to identify the designer steroids that are most commonly sold
in dietary supplements as of April 2014 and review what is known regarding their potency
and toxicity. It was proposed that the impact of AAS use on the reproductive and hormonal
health of men is underestimated in the literature owing to previous studies' failure to account
for designer steroid use. Lastly, it was made clinical recommendations to help physicians
steer patients away from potentially harmful supplements, and summarize key regulatory
obstacles that have allowed potent androgens to remain unregulated in the legal marketplace
[150067].
Anabolic steroids detected in 23/24 bodybuilding dietary supplements
Twenty-four products suspected of containing anabolic steroids and sold in fitness equipment
shops in the United Kingdom (UK) were analyzed for their qualitative and semi-quantitative
content using full scan gas chromatography-mass spectrometry (GC-MS), accurate mass
liquid chromatography-mass spectrometry (LC-MS), high pressure liquid chromatography
with diode array detection (HPLC-DAD), UV-Vis, and nuclear magnetic resonance (NMR)
spectroscopy. In addition, X-ray crystallography enabled the identification of one of the
compounds, where reference standard was not available. Of the 24 products tested, 23
contained steroids including known anabolic agents; 16 of these contained steroids that were
different to those indicated on the packaging and one product contained no steroid at all.
Overall, 13 different steroids were identified; 12 of these are controlled in the UK under the
615
Misuse of Drugs Act 1971. Several of the products contained steroids that may be
considered to have considerable pharmacological activity, based on their chemical structures
and the amounts present. This could unwittingly expose users to a significant risk to their
health, which is of particular concern for naïve users [150068].

Gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) is now
established as a robust and mature analytical technique for the doping control of
endogenous anabolic androgenic steroids in human sport. It relies on the assumption that
the carbon isotope ratios of naturally produced steroids are significantly different to
synthetically manufactured testosterone or testosterone prohormones used in commercial
medical or dietary supplement products. Recent publications in journals have highlighted the
existence of black market testosterone preparations with carbon isotope ratios within the
range reported for endogenous steroids. In one study, it was set out to profile domestic and
international law enforcement seizures of illicit testosterone products to monitor the
prevalence of “enriche”' substrates – which if administered to human subjects would be
considered problematic for the use of current GC-C-IRMS methodologies for the doping
control of testosterone in sport. The distribution of delta13C values for this illicit testosterone
sample population (n=283) ranged from -23.4 ‰ to -32.9 ‰ with mean and median of -28.6
‰ – comparable to previous work. However, only 13 out of 283 testosterone samples (4.6 %)
were found to display delta13C values ≥ -25.8 ‰, confirming that in the vast majority of cases
of illicit testosterone administration, current GC-C-IRMS doping control procedures would be
capable of confirming misuse [14629].
Other laboratory techniques
A sensitive method for the identification and quantification of anabolic steroids and
clenbuterol at trace levels in dietary supplements by liquid chromatography-high-resolution
mass spectrometry (LC-HRMS) in atmospheric pressure ionisation (APCI) mode using a
single-stage Orbitrap analyser operating at a resolution power of 100 000 full width at half
maximum (FWHM) was developed and validated. A total of 1 g of dietary supplement was
added with testosterone-d3 as internal standard, dissolved in methanol, evaporated to
dryness, diluted in sodium hydroxide solution and extracted with a mixture of pentane/ethyl
ether 9:1. The extract was directly injected into the LC-HRMS system. The method was fully
validated. Limits of detection (LODs) obtained for anabolic androgenic steroids (AASs) varied
from 1 to 25 ng/g and the limit of quantitation (LOQ) was 50 ng/g for all analytes. The
calibration was linear for all compounds in the range from the LOQ to 2000 ng/g, with
correlation coefficients always higher than 0.99. Accuracy (intended as %E) and repeatability
(%CV) were always lower than 15 percent. Good values of matrix effect and recovery were
achieved. The ease of the sample preparation together with a fast run time of only 16 min
permitted rapid identification of the analytes. The method was applied to the analysis of 30
dietary supplements in order to check for the presence of anabolic agents not labelled as
being present in these supplements. Many AASs were often detected in the same sample:
indeed, androstenedione was detected in nine supplements, 5-androsten-3beta-ol-17-one
(DHEA) in 12, methandienone in three, stanozolol in one, testosterone in seven and
testosterone esters in four of them. A retrospective analysis of suspected compounds not
included at the beginning of the method development was also possible by means of the full
acquisition spectra obtained with the HRMS technique [150069].
Dietary supplements contaminated with prohormones
616
The extent of the contamination of dietary supplements with anabolic agents was evaluated
in 2001 and 2002. A well-publicised study showed that about 15 percent of non-hormonal
supplements such as vitamins, minerals, proteins and creatine contained anabolic
androgenic steroids (mainly prohormones) that were not declared on the label. The reason
for the contamination was most probably the fact that manufacturers of prohormones (legally
marketed as dietary supplements in the USA until 2004) also manufactured other
supplements on the production line without sufficient cleaning. Another source of cross-
contamination could have been the unclean transport containers from raw material suppliers
of prohormones. The amount of detected prohormones, especially prohormones of
nandrolone, could produce positive doping cases [11010].
Several recent studies have shown evidence of some nutritional supplements containing
prohibited anabolic androgenic steroids, so-called prohormones, which were not declared on
the label. Therefore, a broad-based investigation of the international nutritional supplement
market was initiated to clarify the extent of this problem. From October 2000 until November
2001, 634 non-hormonal nutritional supplements were purchased in 13 countries from 215
different suppliers. Most supplements were bought in shops in the respective countries (578
samples = 91 %) and on the internet (52 samples = 8 %). 289 supplements were from
prohormone-selling companies and 345 supplements came from companies which do not
offer prohormones. After isolation from the supplement matrix 11 different anabolic
androgenic steroids, mainly prohormones of testosterone and nandrolone, were analysed by
gas-chromatography/mass spectrometry. Out of the 634 samples analysed 94 (15 %)
contained anabolic androgenic steroids not declared on the label ("positive supplements"). It
was not obtain reliable data for 66 samples (10 %) due to matrix effects. In relation to the
total number of products purchased per country, most of the positive supplements were
bought in the Netherlands (26 %), in Austria (23 %), in the UK (19 %) and the USA (19 %).
According to the label, all positive supplements were from companies located in only five
countries: the USA, the Netherlands, the UK, Italy and Germany. Twenty-one percent of the
nutritional supplements from prohormone-selling companies contained anabolic androgenic
steroids, whereas 10 percent of the supplements from companies not selling prohormones
were positive. The positive supplements showed anabolic androgenic steroid concentrations
of 0.01 micro g/g up to 190 micro g/g. The administration of supplements containing
nandrolone prohormones adding up to a total uptake of more than 1 micro g resulted in
positive doping results for norandrosterone for several hours [14042].
Dietary supplements containing beta₂-Agonists
One paper presents an application of ultrahigh-performance liquid-chromatography-

quadrupole-orbitrap high resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) for the
ultra-trace analysis of 12 beta2-agonists in pork, beef, mutton and chicken meat. The mass
spectrometer was operated in Full MS/dd-MS(2) mode, under which a Full MS scan was
followed by a dd-MS(2) scan with a fragmentation energy. The quantification was achieved
using matrix-matched standard calibration curves with salbutamol-d3 and clenbuterol-d9 as
the internal standards. The method validation included assessment of selectivity, sensitivity,
calibration curve, accuracy, precision, recovery, matrix effect and stability. The results show
an exceptional linear relationship with the concentrations of the analytes over wide
concentration ranges. The detection limits (LODs) were in the range of 0.0033-0.01
microg/kg, which was much lower than the current reported methods. The recoveries were
able to reach 73.0-88.7 percent, while the matrix effects ranged from 83.7 to 92.8 percent.
Analysis of 400 pork, beef, mutton and chicken samples reveal that only 4.25 percent
samples were positive for beta2-agonists. The detected beta2-agonists involved salbutamol,
clenbuterol, ractopamine and clorprenaline. Overall, the novel Q-Orbitrap technique was
617
demonstrated to have great performance for the screening, identification and quantification of
ultra-trace beta2-agonists used in food animal muscles, which helps to ensure food safety
and public health [150072].
Clenbuterol
A further source of inadvertent doping, which attracted attention within the past 3 years was
meat contaminated with clenbuterol. The anabolic agent clenbuterol has been evidently
misused as growth promoter in cattle feeding in selected countries and meat originating from
these farms can contain traces of clenbuterol leading to AAFs or even poisonings. In 2010
and 2011 unexplainable doping cases with clenbuterol of groups of athletes in table tennis
and soccer led to investigations of the origin of these findings. It could be shown that the
travel and stay in China and Mexico was connected with a high risk of inadvertent doping
with clenbuterol. The most probable source is contaminated meat but other animal products
such as milk or offal cannot be excluded. Also other risk countries might exist. The
antidoping research, which also includes investigations to protect athletes from inadvertent
doping, is now focused on the development of methods to differentiate between clenbuterol
originating from medication and from contaminated meat. Promising results are provided by
studies of pharmacokinetics and metabolism of clenbuterol. A further tool for the
differentiation of inadvertent and intentional ingestion of doping agents maybe hair analysis
[14029].
Untill summer 2011 only two cases have been detected in which dietary supplements
contained therapeutic (30 microg per tablet) and supratherapeutic (2 mg/capsule) doses of
the beta2-agonist clenbuterol. In the supratherapeutically dosed product, clenbuterol was not
declared on the label. Both supplements were advertised as weight loss products. Because
of the extremely high concentration of clenbuterol in the second product (100-fold more than
the therapeutic dose), severe side effects could be expected; in addition, a high risk of cross-
contaminations of other products with clenbuterol is likely. Because WADA has classified
clenbuterol as an anabolic agent, its detection in doping control may lead to severe sanctions
[11010].
Dietary supplements containing prohibited peptide hormones
In 2009 and 2010, dietary supplements containing the prohibited growth hormone–releasing
peptide-2 (GHRP-2) were detected. The products were advertised to produce anabolic, fat-
reducing and anticatabolic effects and to improve regeneration. One product, in an ampoule
of a drinking solution, contained an orally active concentration of GHPR-2. Such a product
may lead to inadvertent doping cases because the name GHPR-2 is not specifically listed on
the WADA prohibited list and is unknown to the majority of the sports community. However,
GHRP-2 belongs as a releasing factor to the prohibited substance group S2 on the WADA
list [11010].
Dietary supplements containing caffeine (in unkown dosage)
With ephedrine
Methyl-xanthines and adrenergic stimulants, such as caffeine and synephrine, are commonly
added to food supplements due to their stimulating and thermogenic effects. In addition, the
abusive consumption of food supplements with ergogenic and aesthetic purposes has been
618
observed worldwide. One work describes the study of caffeine, p-synephrine, hordenine,
octopamine, tyramine, ephedrine and salicin as stimulants in dietary supplements marketed
in Brazil for weight loss and physical fitness claims. A total of 94 different products were
acquired from 30 Brazilian websites. Thus, the sampling of marketed supplements was
performed in virtual commerce (e-commerce) with claims of weight loss, appetite reduction,
fat burning and metabolism acceleration. The developed analytical method involved the
separation of the stimulants by HPLC with diode array detection (HPLC-DAD) by using a
gradient elution of flow rate (0.7-2.5 mL/min) and mobile phase composition (0.1 %
H3PO4/methanol). The validated method was applied to the study of 46 dietary supplements.
Caffeine, p-synephrine and ephedrine were found to be present as stimulants in 52 percent
of the studied samples marketed as encapsulated or bulk forms. Caffeine was found to be
present in concentrations that represent doses from 25.0 to 1476.7 mg/day. Synephrine was
found in concentrations that represent doses from 59.1 to 127.0 mg/day. Ephedrine was
found to be associated with caffeine in one formulation at a concentration representing a
26.1 mg/day dosage [150074].
With creatine
Caffeine and creatine are ingredients in the most popular dietary supplements consumed by
soccer players. However, some products may not contain the disclosed amounts of the
ingredients listed on the label, compromising the safe usage and the effectiveness of these
supplements. Therefore, the aim of this study was to evaluate the content of caffeine and
creatine in dietary supplements consumed by Brazilian soccer players. The results obtained
were compared with the caffeine content listed on the product label. Two batches of the
supplement brands consumed by ≥ 50 percent of the players were considered for analysis.
The quantification of caffeine and creatine in the supplements was determined by a high-
performance liquid chromatography system with UV detector. Nine supplements of caffeine
and 7 supplements of creatine met the inclusion criteria for analysis. Eight brands of caffeine
and five brands of creatine showed significantly different values as compared with the values
stated on the label. There were no significant differences between the two batches of
supplements analyzed, except for one caffeine supplement. It can be concluded that caffeine
and creatine dietary supplements consumed by Brazilian soccer players present inaccurate
values listed on the label, although most presented no difference among batches. To ensure
consumer safety and product efficacy, accurate information on caffeine and creatine content
should be provided on all dietary supplement labels [150073].
Dietary supplements containing 2-ethylamino-1-phenylbutane
The quantitative analysis of a new designer doping agent, 2-ethylamino-1-phenylbutane

(EAPB) and its metabolite, 2-amino-1-phenylbutane (APB) in urine samples, and the
determination of EAPB in dietary supplement samples, have been presented. The main
purpose of the present study was to develop simple and reliable gas chromatography-mass
spectrometry method (GC-MS) for excretion study following a single oral administration of
dietary supplements containing EAPB. Three analytical methods for the determination of
EAPB in urine and supplement samples, and APB in urine samples using the GC-MS
system, have been validated. The method of the determination of EAPB in supplement
samples was applied to analyze seventeen dietary supplements, CRAZE and DETONATE.
Two other methods were used to determine the urinary excretion profile of EAPB and APB in
the case of three healthy volunteers and, on further investigation, it was applied to the anti-
doping control in sport. Quantification was obtained on the basis of the ions at m/z 86, 58
and 169, monitored for EAPB, APB and diphenylamine (used as an internal standard),
619
respectively. The limits of detection and quantification were 2.4 and 7.3 microg/g for EAPB in
the case of supplement analysis, 2.9 and 8.8 ng/mL for EAPB in the case of urine analysis,
and 3.2 and 9.7 ng/mL for APB. The other validation parameters as linearity, precision and
trueness have been also investigated with the acceptable results. The extraction yield of all
presented methods was above 69 percent. EAPB was detected in fourteen analyzed
supplements (not included EAPB in their labels) and its content varied between 1.8 and 16.1
mg/g. Following oral administration of three supplements with EAPB to one male and two
female volunteers, the parent compound of EAPB and its metabolite were monitored and the
excretion parameters as the maximum concentration of the analyte in urine (2.2-4.2
microg/mL for EAPB; 1.1-5.1 microg/mL for APB) and the time for the maximum height of the
excretion peak (2-8 h and 22 h in one case for EAPB; 20-22 h and 4 h in one case for APB)
have been indicated. EAPB and APB were detected at the level above 50 ng/mL (50 % of the
minimum required performance level for stimulants in the anti-doping control in-competition
in sport) in the urine up to 46-106 h and 58-120 h, respectively. Additionally, the result of the
anti-doping control during swimming competition of one athlete, whose urine sample was
analyzed for stimulants and narcotics, has been presented. The qualitative and quantitative
analyses of new designer agents in urine samples and the excretion studies of these
substances are of a great importance in the anti-doping control in sport. Moreover, the
presentation of detection examples of these agents in supplements that haven't got included
an information about them in the labeling, make athletes (and other supplement customers)
more and more aware of the risk of the supplement use and possible health and doping
consequences [150075].
Zeranol
Similar to the clenbuterol contamination issue, the unintentional intake of the mycotoxin
zearalenone can result in AAFs concerning one of its human urinary metabolites referred to
as zeranol. Zeranol has been prohibited according to the regulations of WADA as an
anabolic agent and its formation from zearalenone has been observed in humans,
representing an analytical challenge for doping controls as the drug's deliberate intake needs
to be differentiated from the consumption of mycotoxin-contaminated produce. A viable
strategy was established employing metabolite profiling of zearalenone and zeranol in case
of suspicious test results, which confirmed zearalenone as the origin of zeranol findings in
2011 [14029].
Boar meat
In previous work (Le Bizec et al. Rapid Commun Mass Spectrom. 2000; 14: 1058), it was
demonstrated that a boar meal intake could lead to possible false accusations of abuse of
17beta-nortestosterone in antidoping control. The aim of the present study was to identify
and quantify endogenous 19-norsteroids in boar edible tissue at concentrations that can alter
the steroid urinary profile in humans, and lead to excretion of 19-norandrosterone (19-NA)
and 19-noretiocholanolone (19-NE). The samples were analysed in two laboratories. The
methodologies used for extraction and detection (GC/MS(EI) and LC/MS/MS(APCI+)) are
compared and discussed. 19-Norandrostenedione (NAED), 17beta- and 17alpha-
nortestosterone (bNT, aNT), and 17beta- and 17alpha-testosterone (bT, aT) were quantified.
The largest concentrations of NAED and bNT were observed in testicles (83 and 172
microg/kg), liver (17 and 63 microg/kg) and kidney (45 and 38 microg/kg). A correlation
between the bNT and NAED content of a typical meal prepared with boar parts and the
excreted concentrations of 19-NA and 19-NE in human urine was demonstrated [01023].
620
Musk pod
A very unusual source of arguably inadvertent doping with anabolic agents was detected
during the FIFA women’s World Cup 2011 in soccer in Germany. Five players of a team were
tested positive for endogenous AAS. The AAFs were detected by means of atypical steroid
profiles and confirmed by positive IRMS results. Extracts and grains of deer musk pods were
identified as source of the positive results. These animal products contained huge amounts
of 16 different endogenous AAS of which 9 were listed on the WADA Prohibited List 2011.
These musk deer products were claimed to be used by the team to increase mental strength
without knowing that the consumption leads to AAFs [14029].
Designer drugs
In recent years, new doping substances have been continuously introduced to the market in
the form of nutritional supplements. They are often produced in clandestine drug laboratories
by modification or positional rearrangement of well-established doping agents such as
stimulants. The main aim of these activities is to deliver specifically designed, biologically
active substances which are not controlled by the public law. This allows for making a profit
on selling a product yielding “unprecedented results” that, in the case of stimulating agents,
would correspond to such effects as rapid weight loss or the ability to perform extensive
training for extended periods of time. The presence of designer substances in these products
usually remains unknown until anti-doping laboratories identify them. This, in turn, may lead
to a large number of sanctioned athletes that fail anti-doping tests once testing laboratories
have implemented methods for their detection. This is due to the fact that the list of
substances prohibited in sport is open and defines novel doping agents based on their
similarity in action and/or structure to those already listed [14439].
Since 2002, the so-called designer steroids can also be found on the dietary supplement
market. These steroids are neither listed as ingredients in any currently available medication,
nor do their names appear in the WADA list of prohibited substances. Most of these designer
steroids have been synthesised in the 1960s and were tested only in animal studies for their
anabolic and androgenic effects. Nowadays, these steroidal agents are produced exclusively
for the nutritional supplement market and are advertised for their anabolic- or aromatase-
inhibiting capacities. With regard to the effects and side effects of these steroids for humans,
there is limited or no knowledge. In most cases, the labelling of these products contains non-
approved or fancy names of the steroids. More than 40 such designer steroids have been
detected. The detection of metabolites of such a steroid in an athlete's urine sample is likely
to lead to a positive doping case [11425].
Emerging drugs
In 2009 and 2010, the first prohibited selective androgen receptor modulators (SARMs) and
the gene doping substances AICAR and GW1516 were detected on the black market. All
these substances are still in clinical trials and have not yet been approved as medications.
From the experience, it can be expected that these substances will appear very soon on the
dietary supplement market, with advertising that the SARM products will achieve anabolic
effects whereas the gene doping substances will enhance endurance. If these substances
are added to other supplement products without being declared on the label, new sources of
risk for inadvertent doping will be created [11010].
621
Counterfeit drugs
Anabolic-androgenic steroids (AASs) have been illegally used in counterfeit drugs to improve
the performance of athletes. In addition, AASs have been used for cosmetic purpose by non-
athletes. To determine the presence of 26 AASs, an analysis method using ultra-liquid
chromatography-tandem mass spectrometry (UHPLC-MS/MS) was developed and validated.
The validated method was applied to 19 counterfeit drugs collected from the Internet and off-
line markets during 2014. Nearly 50 percent (9/19) of the samples contained one of these 26
AASs. In addition, the concentration ranges of the AASs ranged from 0.09 to 119,228.57
mg/kg in the suspected samples. The determined AASs primarily consisted of testosterone
and testosterone 17-propionate (26 %) followed by boldenone (21 %). These results indicate
the adulteration of over-the-counter counterfeit drugs, and the continuous monitoring of
counterfeit drugs or dubious dietary supplements containing anabolic steroids is warranted
[150070].
Doping substances contaminating food
Meat
Despite the ban by the European Union, anabolic steroids might still be illicitly employed in
bovine meat production. The surveillance of misuse of such potentially harmful molecules is
necessary to guarantee consumers' health. Analytical methods for drug residue control are
based on LC-MS/MS, but their efficacy can be hindered due to undetectable residual
concentrations as a result of low-dosage treatments. Screening methods based on the
recognition of indirect biological effects of growth promoters' administration, such as the
alteration of protein expression, can improve the efficacy of surveillance. The present study
was aimed at identifying modifications in the muscle protein expression pattern between bulls
treated with an ear implant (Revalor-XS®) containing trenbolone acetate (200 mg) and
estradiol (40 mg), and untreated animals. The analysis of skeletal muscle was carried out
using a tandem mass tags shotgun proteomics approach. We defined 28 candidate protein
markers with a significantly altered expression induced by steroids administration. A subset
of 18 candidate markers was validated by SRM and allowed to build a predictive model
based on partial least square discriminant analysis. Our findings confirm the effectiveness of
the proteomics approach as potential tool to overcome analytical limitations of drug residue
monitoring [150082].
Proteomics for the detection of steroids treatment in bovine muscle

using a tandem mass tags shotgun proteomics approach. It was defined 28 candidate protein
622
based on partial least square discriminant analysis. Our findings confirm the effectiveness of
Anabolic steroids in bovine liver
A rapid liquid chromatography tandem mass spectrometry method has been developed and
validated for the determination of alpha-trenbolone, beta-trenbolone, alpha-nortestosterone,
beta-nortestosterone, zeranol, and taleranol in bovine liver. The impact of liquid-liquid
extraction with methyl tert-butyl ether and optimized solid phase extraction on silica
cartridges significantly reduced effort and time of sample preparation. Electrospray ionization
gives a significant signal increase compared with atmospheric pressure chemical ionization
and atmospheric pressure photoionization. The HPLC gradient was optimized to separate
isobaric analytes and matrix constituents from the hormone molecules. The optimized time
and temperature of enzymatic hydrolysis of conjugated trenbolone was 4 h at 52 °C. The
method validated in the range of 0.5-30 microg/kg for alpha-trenbolone, beta-trenbolone,
zeranol, taleranol, and 2-30 microg/kg for alpha-nortestosterone, beta-nortestosterone.
Combined uncertainty of measurements was in the range of 4-23 percent. The matrix effect
was negligible (1 %-5 %) for all analytes except of alpha-nortestosterone (19 %). The
developed method with changes concerning sample size and hydrolysis was also applied for
the analysis of meat, serum, and urine samples [150084].
Anabolic steroids in bovine bile
The administration of boldenone and androstadienedione to cattle is forbidden in the

European Union, while prednisolone is permitted for therapeutic purposes. They are
pseudoendogenous substances (endogenously produced under certain circumstances). The
commonly used matrices in control analyses are urine or liver. With the aim of improving the
residue controls, we previously validated a method for steroid analysis in bile. We now
compare urine (a “classic” matrix) to bile, both collected at the slaughterhouse, to understand
whether the detection of steroids in the latter is easier. With the aim of having clearer results,
we tested the presence of the synthetic corticosteroid dexamethasone. The results show that
bile does not substantially improve the detection of boldenone, or its conjugates,
prednisolone and prednisone. Dexamethasone, instead, was found in 10 out of 53 bovine
bile samples, but only in one urine sample from the same animals. Bile could constitute a
novel matrix for the analysis of residues in food-producing animals, and possibly not only of
synthetic corticosteroids [150085].
Doping substances in urine
Synthetic glucocorticoids in bovine urine

Challenges to testing for the illicit use of anabolic substances in meat-producing animals
stem from the production of new synthetic compounds and the administration of low-dose
cocktails to circumvent detection by the surveillance schemes of European Union member
states. This work evaluated for the first time GR-CALUX, a highly sensitive reporter gene
assay, as a screening tool for the detection of synthetic glucocorticoids in bovine urine. In
order to verify the effect of natural corticosteroids on the method, the bioassay was tested
first using blank urine samples collected at the farm and the slaughterhouse. Next, the dose-
response curves were measured for the most commonly used synthetic glucocorticoids. The
bioassay's ability to detect them in spiked and incurred samples of bovine urine was then
evaluated. Finally, its performance was compared against a commercially available ELISA kit
ordinarily used in screening activities. GR-CALUX performance did not appear to be
623
influenced by physiological levels of endogenous corticosteroids in the farm samples,
whereas an increase in these hormones might invalidate the analysis in samples obtained at
the slaughterhouse. Using pure compounds, GR-CALUX showed a high sensitivity toward
the synthetic glucocorticosteroids tested in order of relative potencies: flumethasone ≫
dexamethasone > betamethasone > methylprednisolone > prednisolone. As expected, the
bioassay failed to detect the prohormone prednisone. The results obtained from analysis of
the spiked and incurred specimens reproduced those of the blank samples and the pure
compounds. GR-CALUX is a promising screening tool for the detection of illicit treatments in
meat-producing bovines. Its ability to detect the most commonly used synthetic
glucocorticoids was comparable with the ELISA test. Importantly, it appeared to be less
susceptible to matrix effects than ELISA [150086].
Urine boldenone in young bulls from the food chain

The administration of boldenone (bold) to bovines, either for growth promotion or therapeutic
purposes, has been banned in the EU since 1981. It is, however, a pseudoendogenous
hormone, thus its detection in bovine urine, in the form of α-boldenone conjugates, is
considered fully compliant up to 2 ng/mL. Greater attention has been placed on beta-
boldenone, the anabolic active epimer, whose conjugated form must be absent in urine.
Recently, the identification of a biomarker representing unquestionable evidence of illicit
treatment with bold or its precursor androstadienedione has been a major topic in the
literature regarding the detection of residues in bovine urine, and beta-boldenone sulphate is
a candidate molecule. In one study, it was used a method previously validated according to
the European Commission Decision 2002/657/EC for the determination of sulphate and
glucuronide conjugates of beta-boldenone. It was assessed the occurrence of these
molecules in young bull urine, with the aim of understanding whether they could be of
endogenous origin, and to check for a possible relationship with particular environmental and
stress conditions. Urine samples from 56 young bulls were collected after transport stress,
under non-stressful conditions and after transport and slaughter stress. Histopathological
investigation of the hormone target organs, i.e. the bulbourethral and prostate glands, was
also performed. The results indicate an inverse relationship between the presence and
concentration of beta-boldenone sulpho- and gluco-conjugates in urine, and stress
conditions, expressed by the absence of detection at the slaughterhouse. No significant
macroscopic and histologic lesions were detected. The study indicates that beta-boldenone
sulphate could be a biomarker of treatment only at the slaughterhouse, while at the farm, in
untreated animals (i.e. after a five-month period under the control of Official Veterinarians),
sulphate and glucuronide metabolites were found with a frequency of 78 and 46 percent,
respectively, showing the endogenous origin of boldenone [150087].
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DETECTION OF DOPING AGENTS IN ENVIRONMENT, FOOD AND
FOOD SUPPLEMENTS
Hormones in international meat production
Beef and its products are an important source of nutrition in many human societies. Methods
of production vary and include the use of hormonal compounds (“hormones”) to increase
growth and lean tissue with reduced fat deposition in cattle. The hormonal compounds are
naturally occurring in animals or are synthetically produced xenobiotics and have oestrogenic
(oestradiol-17beta and its esters; zeranol), androgenic (testosterone and esters; trenbolone
acetate) or progestogenic (progesterone; melengestrol acetate) activity. The use of
hormones as production aids is permitted in North American countries but is no longer
allowed in the European Union (EU), which also prohibits the importation of beef and its
products derived from hormone-treated cattle. These actions have resulted in a trade dispute
between the two trading blocs. The major concern for EU authorities is the possibility of
adverse effects on human consumers of residues of hormones and metabolites. Methods
used to assess possible adverse effects are typical of those used by international agencies
to assess acceptability of chemicals in human food. These include analysis of quantities
present in the context of known biological activity and digestive, absorptive, post-absorptive
and excretory processes. Particular considerations include the low quantities of hormonal
compounds consumed in meat products and their relationships to endogenous production
particularly in prepubertal children, enterohepatic inactivation, cellular receptor- and non-
receptor-mediated effects and potential for interference with growth, development and
physiological function in consumers. There is particular concern about the role of oestradiol-
17beta as a carcinogen in certain tissues. Now subject to a “permanent” EU ban, current
evidence suggests that certain catechol metabolites may induce free-radical damage of DNA
in cell and laboratory animal test systems. Classical oestrogen-receptor mediation is
considered to stimulate proliferation in cells maintaining receptivity. Mathematical models
describing quantitative relationships between consumption of small amounts of oestrogens in
meat in addition to greater concentrations from endogenous production, chemical
stoichiometry at cellular level and human pathology have not been developed. Such an
approach will be necessary to establish 'molecular materiality' of the additional hormone
intake as a component of relative risk assessment. The other hormones, although generally
less well researched, are similarly subject to a range of tests to determine potentially adverse
effects. The resulting limited international consensus relates to the application of the
'precautionary principle' and non-acceptance by the European Commission of the
recommendations of the Codex Alimentarius Commission, which determined that meat from
cattle, hormone-treated according to good practice, was safe for human consumers. The
present review considers the hormone issue in the context of current international social
methodology and regulation, recent advances in knowledge of biological activity of hormones
and current status of science-based evaluation of food safety and risk for human consumers
[02019].
Steroid growth promoters from beef cattle feedyards
Studies of steroid growth promoters from beef cattle feedyards have previously focused on
effluent or surface runoff as the primary route of transport from animal feeding operations.
There is potential for steroid transport via fugitive airborne particulate matter (PM) from cattle
feedyards; therefore, the objective of this study was to characterize the occurrence and
concentration of steroid growth promoters in PM from feedyards. Air sampling was
conducted at commercial feedyards (n=5) across the Southern Great Plains from 2010 to
625
2012. Total suspended particulates (TSP), PM10, and PM2.5 were collected for particle size
analysis and steroid growth promoter analysis. Particle size distributions were generated
from TSP samples only, while steroid analysis was conducted on extracts of PM samples
using liquid chromatography mass spectrometry. Of seven targeted steroids, 17alpha-
estradiol and estrone were the most commonly detected, identified in over 94 percent of
samples at median concentrations of 20.6 and 10.8 ng/g, respectively. Melengestrol acetate
and 17alpha-trenbolone were detected in 31 and 39 percent of all PM samples at median
concentrations of 1.3 and 1.9 ng/g, respectively. Results demonstrate PM is a viable route of
steroid transportation and may be a significant contributor to environmental steroid hormone
loading from cattle feedyards [150081].
Influence on reproduction
Although the hormone-mediated effects of the synthetic androgenic hormone

methyltestosterone (MT) are well characterized in mammals, little is known about endocrine
and other toxic effects on reproduction in birds. In a one-generation study, MT was
administered to adult Japanese quail (12 pairs per group) at dietary dose levels of 0, 10, 50,
and 110 ppm for a period of 3 weeks. Reproductive performance was severely affected in the
groups receiving 50 and 110 ppm MT. In females, the egg-laying rate was reduced not only
related to the dose administered but also to the duration of treatment. The administration of
110 ppm, and to a lesser extent, of 50 ppm MT resulted in an immediate and dramatic
decrease in the total number of eggs laid, which complicated reliable assessment of other
reproduction-related parameters. In males, the findings suggested inhibition of
spermatogenesis at dose levels of 50 ppm and above, resulting in a subsequent reduction in
male fertility [05018].
Influence on resistance to antibiotics
In a longitudinal study (165 days), it was investigated the effect of growth-promoting agents
(monensin and trenbolone acetate-estradiol) and an antibiotic (oxytetracycline) on the
incidence in feedlot steers of Escherichia coli O157, including antibiotic-resistant and
hypermutable isolates. Eighty steers in 16 pens were treated with eight combinations of
promoters, and each treatment was duplicated. Fecal samples were collected at nine
different sampling times for detection of E. coli O157. Overall, 50 E. coli O157 isolates were
detected in treated animals, and none were found in untreated animals. Compared with
untreated controls, there was a significant association between the utilization of growth-
promoting agents or antibiotics and the shedding of E. coli O157 at day 137 (P = 0.03), when
a prevalence peak was observed and 50% of the isolates were detected. Multiplex PCR
assays were conducted for some virulence genes. PCR results indicated that all except one
isolate possessed at least the Shiga toxin gene stx2. MICs for 12 antibiotics were
determined, and eight oxytetracycline-resistant E. coli O157 strains were identified.
Antibiotic-resistant strains were considered a distinct subpopulation of E. coli O157 by
pulsed-field gel electrophoresis typing. Seven of these antibiotic-resistant strains were
isolated early in the study (on or before day 25), and among them two were also
hypermutable as determined by rifampin mutation frequencies. The proportion of
hypermutable strains among E. coli O157 isolates remained relatively constant throughout
the study period. These results indicate that the use of growth-promoting agents and
antibiotics in beef production may increase the risk of environmental contamination by E. coli
O157 [05019].
626
Analytical strategies
Detection of the abuse of synthetic steroids in food production is nowadays relatively

straightforward using modern techniques such as gas or liquid chromatography coupled to
mass spectrometry (GC-MS/MS or LC-MS/MS, respectively). However, proving the abuse of
“endogenous” (or naturally occurring) steroids is more difficult. Despite these difficulties,
significant progress in this area has recently been made and a number of methods are now
available. The aim of the current review was to systematically review the available analytical
approaches, which include threshold concentrations, qualitative marker metabolites, intact
steroid esters, gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-
IRMS), longitudinal testing and omics biomarker profiling. The advantages/disadvantages of
these methods are considered in detail, but the choice of which to adopt is dictated by a
number of practical, political, and economic factors, which vary in different parts of the world.
These include the steroid/species combination requiring analysis, the matrix tested, whether
samples are collected from live or slaughtered animals, available analytical instrumentation,
sample throughput/cost, and the relevant legal/regulatory frameworks. Furthermore, these
approaches could be combined in a range of different parallel and/or sequential
screening/confirmatory testing streams, with the final choice being determined by the
aforementioned considerations. Despite these advances, more work is required to refine the
different techniques and to respond to the ever increasing list of compounds classified as
endogenous. At this advanced stage, however, it is now more important than ever for
scientists and regulators from across the world to communicate and collaborate in order to
harmonize and streamline research efforts [12355].
Potential gene expression biomarker signature
The misuse of anabolic agents in animal husbandry is a ubiquitous problem. The ban of
growth promoters in food producing animals in the European Union is well controlled, but
there are still application regimes, such as new designed drugs or hormone cocktails, that
are difficult to detect. Therefore, the idea of identifying molecular biomarkers that are based
on the physiological effect of treatment has come into focus. In a previous study it was
identified mRNA biomarker candidates in liver samples that enable the separation of
untreated animals from animals treated with a combination of androgens plus estrogens. In
the present study those candidates were validated in calves treated with a combination of
progesterone plus estradiol or clenbuterol, respectively. Therefore, the candidate genes were
quantified in liver samples of those calves via RT-qPCR. Using dynamic principal component
analysis (PCA), a signature of 11 genes could be selected. This set of genes enabled the
separation of treated and control animals independent of the applied drug. Additional
quantification of these genes in a set of control samples from another animal trial resulted in
a PCA that also showed a separation of those samples from treated animals. The study
showed that gene expression biomarkers have a high potential to enable the detection of
physiological changes caused by the application of growth-promoting substances
independent of the given drug, but further studies are necessary to broaden the spectrum of
anabolic substance groups for which those biomarker candidates can be used [14244].
The effect of administration of cortisol, corticosterone, testosterone, progesterone and a

synthetic estrogen, diethylstilbestrol on plasma proteins of tilapia (Oreochromis
mossambicus) was investigated. SDS-PAGE clearly revealed the appearance of several new
bands of protein, which were not present in the control plasma and were comparable to the
known bands of the molecular markers. Of the different bands appeared in the steroids
treated plasma, the most important ones were the presumed vitellogenin and corticotrophin
627
binding globulin with a molecular weight of 180 and 17 kDa, respectively. Increase in protein
bands in the steroid treated plasma of O. mossambicus confirmed the anabolic role of
steroids in teleost [02018].
Revised EU criteria for the confirmation of anabolic steroids in meat
The EU criteria for the confirmation of the presence of illegal compounds in biological
matrices were recently revised. The old and the revised criteria were applied to relative ion
intensities obtained for five anabolic steroids (methylboldenone, methyltestosterone,
ethynylestradiol, beta-boldenone and beta-nortestosterone) in meat (cow, pig, turkey) and
fish at concentrations ranging from 0.5 to 5.0 microg/kg. Confirmatory analysis was done by
GC-MS; therefore four diagnostic ions had to be monitored and three ion ratios had to be
calculated and tested against the criteria. Application of the old and revised criteria, with
either standards or fortified samples as reference, showed mutually rather divergent results.
Confirmation according to the revised EU criteria and using fortified samples as a reference
gave the best results; in other words the highest percentage of diagnostic ion ratios within
the tolerance intervals. A correlation was found between the percentage of these ion ratios
and the signal/noise (S/N) ratio of the least intense ion of interest in the recorded MS
spectrum. Although there were distinct differences in the results obtained for different
analytes and sample types, it is safe to conclude that at S/N=3 the percentage of ratios within
the tolerance intervals generally will be at or below 50 percent, while for S/N >10, the
percentage increases to over 90 percent. In the present study, fully satisfactory results were
obtained down to about 2 microg/kg, but not for lower analyte concentrations [14032].
Metabolomics
Metabolomics is a science of interest in food analysis to describe and predict properties of

food products and processes. It includes the development of analytical methods with the
ultimate goal being the identification of so-called'quality markers, (i.e. sets of metabolites that
correlate with, for example, quality, safety, taste, or fragrance of foodstuffs). In turn, these
metabolites are influenced by factors as genetic differences of the raw food ingredients (such
as animal breed or crop species differences), growth conditions (such as climate, irrigation
strategy, or feeding) or production conditions (such as temperature, acidity, or pressure). In
cases where the routine-based measurement of a food property faces some limitations such
as the lack of knowledge regarding the target compounds to monitor, monitoring based on a
limited set of crucial biomarkers is a good alternative, which is of great interest for food safety
purposes regarding growth promoting practices. Such an approach may be more efficient
than using a classic approach based on a limited set of known metabolites of anabolic
compounds. In this context, screening strategies allowing detection of the physiological
response resulting from anabolic compound administration are promising approaches to
detect their misuse. The global metabolomics workflow implemented for such studies is
presented and illustrated through various examples of biological matrices profiling (tissue,
blood, urine) and for different classes of anabolic compounds (steroids, beta-agonists and
somatotropin) [12356].
Screening for hormone residues in drug residues
An emerging trend is recognised in hormone and veterinary drug residue analysis from liquid
chromatography tandem mass spectrometry (LC/MS/MS) based screening and confirmation
628
towards accurate mass alternatives such as LC coupled with time-of-flight (TOF), Fourier
transform ion cyclotron resonance (FTICR) or Fourier transform orbitrap (FT Orbitrap) MS. In
this study, mass resolution and accuracy are discussed for LC/MS screening and
confirmation of targeted analytes and for the identification of unknowns using the anabolic
steroid stanozolol and the designer beta-agonist "Clenbuterol-R" as model substances. It is
shown theoretically and experimentally that mass accuracy criteria without proper mass
resolution criteria yield false compliant (false negative) results, both in MS screening and
MS/MS confirmation of stanozolol. On the other hand, previous medium resolution accurate
mass TOFMS/MS data of the designer beta-agonist were fully confirmed by high resolution
FT Orbitrap MS(n) experiments. A discussion is initiated through a proposal for additional
criteria for the use of accurate mass LC/MS technologies, to be implemented in Commission
Decision 2002/657/EC [06041].
The Netherlands
Nutritional supplements are riddled with contaminants such as steroids and other banned
substances, despite that supplements contain ingredients such as vitamins, minerals and
amino acids. But for several years contaminants have been suspected of causing some
athletes to test positive for banned substances, leading authorities to recommend that
sportsmen and women stay off them altogether. Although their labels claimed they were
hormone-free, nearly 15 percent of the supplements tested positive for anabolic steroids. In
2002, a study by the Netherlands Centre for Doping Affairs found that nearly a quarter of
samples from supplements that Dutch athletes intended to use while training for the Utah
Olympic Games were likewise contaminated [14040].
Anabolic steroids
A method is described for screening and confirmation of synthetic and endogenous steroids
in muscle tissue. The method is sensitive, selective, and rapid and the consumption of
organic solvents is low, compared to previously published methods. The procedure involves
hydrolysis, defattening with heptane and final clean up with SPE using C18 cartridge. After
filtration, the analytes are analysed by LC/MS/MS and quantification is performed using
deuterated internal standards. Decision limits (CCalpha) varied from 0.02 to 0.33 µg/kg and
the detection capabilities (CCbeta) were <0.50 µg/kg. The mean within-laboratory
reproducibility ranged 5-22 percent (%RSDIR). Endogenous steroids (e.g. testosterone,
epitestosterone and androstenedione) have been included in the method, to provide an
insight into their levels, as the presence of these steroids was detected several times during
analysis of imported meat [11046].
For many years anabolic-androgenic steroids (AAS) are by far the most frequently detected
pharmacological substances in doping control. In order to improve their performances,
professional sportsmen are often tempted to take dietary supplements. However, due to the
frequent and widespread occurrence of contaminated supplements, the use of such products
is not without risk for the athletes involved. In order to minimize the chances of an
unattended positive doping test or serious health problems, fast and reliable screening
methods for the detection of anabolic steroids in dietary supplements are needed. A general
screening procedure requires the fast and unambiguous detection of a large range of
steroids. Gas chromatography-mass spectrometry (GC-MS) has been used intensively in the
detection of doping substances for the past 40 years. Over time, many laboratories have
delivered spectra to be included in standard reference databases, one of which is maintained
629
by the National Institute of Standards and Technology (NIST) (Gaithersburg, MD, USA). In
recent years, however, liquid chromatography coupled to mass spectrometry (LC-MS) has
gained popularity. Unfortunately, existing GC-MS libraries are not applicable to LC-MS
analysis. In one study, a new mass spectral library of 88 steroids was developed, along with
a fast UPLC-MS method. For the construction of this mass spectral library, three different
mass spectra were measured for each steroid, with a sample cone voltage of 30, 60 and 100
V, respectively. This method was then successfully tested on contaminated dietary
supplements which had previously been tested by means of a targeted LC-MS/MS method.
Overall, the library search was shown to identify the same compounds as the MRM method
[11047].
The use of steroids as growth-promoting agents in food production is banned under

European Union legislation. Detecting the abuse of testosterone, nandrolone, boldenone,
oestradiol and progesterone is complicated by the fact that these steroids are known to be
endogenous in certain situations. In one study, the concentrations of characteristic
metabolites of each of these steroids were quantified in populations of untreated steers and
heifers. Steroid concentration population data were then used by a statistical to produce
threshold concentrations for screening and confirming the abuse of these steroids in steer
and non-pregnant heifer urine. In addition to thresholds based on testing one animal, new
methods based on testing multiple animals from a herd allowed threshold concentrations to
be significantly reduced and hence false compliances to be minimised. In the majority of
cases, the suggested thresholds were found to be capable of confirming the abuse of
endogenous steroids in steers and heifers. In the case of estradiol abuse in the female,
however, confirmation based on a threshold is not possible and alternative methods such as
gas chromatography-combustion-isotope ratio mass spectrometry are required. In addition to
the steer and heifer populations, a small number of pregnant animals were also tested,
yielding insights into the biosynthetic pathways of some of the steroids [11048].
Recently, the effect of illicit growth promoters (GPs) upon the cattle transcriptome has drawn
the increasing attention of the scientific community. In the present study, the pre-
transcriptional effects of three different illicit protocols on a set of target genes, including
steroidogenic enzymes and three related transcription factors, were estimated in cattle testis.
Beef cattle were administered with dexamethasone (DEX) orally (group D1) or
intramuscularly in experiment 1 (group DIM). In experiment 2, DEX was orally administered
alone (group D2) or with 17beta-estradiol (group DE), and in experiment 3,
dehydroepiandrosterone and boldione were orally administered alone (group DHEA and
group ADD) or in combination (group DHAD). The GP effects were measured by quantitative
real time RT-PCR. The results of our study were significant but not univocal. A GP-
dependent effect on target gene mRNA levels was noticed for 3beta-hydroxysteroid
dehydrogenase type 1, the cytochrome P450 side chain cleavage, the cytochrome P450
17A1, HSD17beta3, aromatase, the androgen receptor and the mineralocorticoid receptor-
like. The results suggest that different GP schedules are likely to affect genes involved in
steroid synthesis and regulation in cattle testis. Thus, this tissue might be considered a
potential surrogate tissue that warrants further study into its usefulness in the screening of
GP abuse [11049].
A sensitive rapid resolution liquid chromatography-tandem mass spectrometry (RRLC-

MS/MS) method, combined with solid-phase extraction, ultrasonic extraction and silica gel
cartridge cleanup, was developed for 28 steroids including 4 estrogens (estrone (E1),
17beta-estradiol (E2), 1alphaα-ethynyl estradiol (EE2), diethylstilbestrol (DES)), 14
androgens (androsta-1,4-diene-3,17-dione (ADD), 17alpha-trenbolone, 17beta-trenbolone, 4-
androstene-3,17-dione, 19-nortestoserone, 17beta-boldenone, 17alpha-boldenone,
testosterone (T), epi-androsterone (EADR), methyltestosterone (MT), 4-hydroxy-androst-4-
630
ene-17-dione (4-OHA), 5alpha-dihydrotestosterone (5alpha-DHT), androsterone (ADR),
stanozolol (S)), 5 progestagens (progesterone (P), ethynyl testosterone (ET), 19-
norethindrone, norgestrel, medroxyprogesterone (MP)), and 5 glucocorticoids (cortisol,
cortisone, prednisone, prednisolone, dexamethasone) in surface water, wastewater and
sludge samples. The recoveries of surface water, influents, effluents and sludge samples
were 91-119 percent (except 5alpha-DHT was 143 %), 44-200 percent, 61-123 percent and
63-138 percent, respectively. The method detection limits for the 28 analytes in surface
water, influents, effluents and freeze-dried sludge samples were 0.01-0.24 ng/L, 0.02-1.44
ng/L, 0.01-0.49 ng/L and 0.08-2.06 ng/g, respectively. This method was applied in the
determination of the residual steroidal hormones in two surface water of one river, 12 waste-
water and 8 sludge samples from two wastewater treatment plants in Guangdong (China).
Ten analytes were detected in surface water samples with concentrations ranging between
0.4 ng/L (17beta-boldenone) and 55 ng/L (5alpha-DHT); twenty analytes in the wastewater
samples with concentrations ranging between 0.3 ng/L (progesterone) and 621 ng/L (5alpha-
DHT); and 12 analytes in the sludge samples with concentrations ranging between 1.6 ng/g
(E1) and 372 ng/g (EADR) [11050].
In the present paper it was reported the LC-MS/MS determination of residues of 12 anabolic
steroids in bovine serum, as an expansion of the work protocols for steroids determination in
biological matrices. Steroids analyzed included alpha-zearalanol, beta-zearalanol, alpha-
trenbolone, beta-trenbolone, methyltestosterone, alpha-estradiol, beta-estradiol,
ethynylestradiol, alpha-boldenone, beta-boldenone, alpha-nortestosterone, and beta-
nortestosterone. Following protein precipitation, serum samples were cleaned up by solid-
phase extraction using Oasis HLB and Amino cartridges. Atmospheric pressure chemical
ionization (APCI) in both positive and negative ionization modes was used and mass
spectrometry detection was carried out in multiple reaction monitoring mode following two or
(in most cases) three product ions per precursor ion. The method was validated in
accordance with the Commission Decision 2002/657/EC. The decision limit (CCalpga) values
obtained, ranged from 0.01 to 0.07 ng/mL and the detection capability (CCbeta) values
obtained ranged from 0.02 to 0.12 ng/mL. The recoveries ranged from 70 to 118 percent.
The developed method is suitable for routine and confirmatory purposes such as control of
illegal use in livestock production [11051].
Anabolic steroids are banned in food producing livestock in Europe. Efficient methods based
on mass spectrometry detection have been developed to ensure the control of such
veterinary drug residues. Nevertheless, the use of "cocktails" composed of mixtures of low
amounts of several substances as well as the synthesis of new compounds of unknown
structure prevent efficient prevention. New analytical tools able to detect such abuse are
today mandatory. In this context, metabolomics may represent new emerging strategies for
investigating the global physiological effects associated to a family of substances and
therefore, to suspect the administration of steroids. The purpose of one study was to set up,
assess and compare two complementary mass spectrometry-based metabolomic strategies
as new tools to screen for steroid abuse in cattle and demonstrate the feasibility of such
approaches. The protocols were developed in two European laboratories in charge of
residues analysis in the field of food safety. Apart from sample preparation, the global
process was different in both laboratories from LC-HRMS fingerprinting to multivariate data
analysis through data processing and involved both LC-Orbitrap-XCMS and UPLC-ToF-MS-
MetAlign strategies. The reproducibility of both sample preparation and MS measurements
were assessed in order to guarantee that any differences in the acquired fingerprints were
not caused by analytical variability but reflect metabolome modifications upon steroids
administration. The protocols were then applied to urine samples collected on a large group
of animals consisting of 12 control calves and 12 calves administrated with a mixture of
17beta-estradiol 3-benzoate and 17beta-nandrolone laureate esters according to a protocol
631
reflecting likely illegal practices. The modifications in urine profiles as indicators of steroid
administration have been evaluated in this context and proved the suitability of the approach
for discriminating anabolic treated animals from control ones. Such an approach may
therefore open a new way for the screening of anabolic steroid administration through
targeted monitoring of relevant biomarkers highlighted as a result of the metabolomics study
[11052].
632
The presence and metabolism of endogenous steroid hormones in meat-producing animals
has been the subject of much research over the past 40 years. While significant data are
available, no comprehensive review has yet been performed. Species considered in this
review are bovine, porcine, ovine, equine, caprine and cervine, while steroid hormones
include the androgenic-anabolic steroids testosterone, nandrolone and boldenone, as well as
their precursors and metabolites. Information on endogenous steroid hormone
concentrations is primarily useful in two ways: (1) in relation to pathological versus “normal”
physiology and (2) in relation to the detection of the illegal abuse of these hormones in
residue surveillance programmes. Since the major focus of one review was on the detection
of steroids abuse in animal production, the information gathered to date is used to guide
future research. A major deficiency in much of the existing published literature is the lack of
standardization and formal validation of experimental approach. Key articles are cited that
highlight the huge variation in reported steroid concentrations that can result when samples
are analysed by different laboratories under different conditions. These deficiencies are in
most cases so fundamental that it is difficult to make reliable comparisons between data sets
and hence it is currently impossible to recommend definitive detection strategies.
Standardization of the experimental approach would need to involve common experimental
protocols and collaboratively validated analytical methods. In particular, standardization
would need to cover everything from the demographic of the animal population studied, the
method of sample collection and storage (especially the need to sample live versus slaughter
sampling since the two methods of surveillance have very different requirements, particularly
temporally), sample preparation technique (including mode of extraction, hydrolysis and
derivatization), the end-point analytical detection technique, validation protocols, and the
statistical methods applied to the resulting data. Although efforts are already underway to
produce more definitive data and promote communication among the scientific community on
this issue, the convening of a formal European Union working party is recommended
[09078].
In livestock production, illegal use of natural steroids is hard to prove because metabolites
are either unknown or not significantly above highly fluctuating endogenous levels. In one
work it was outlined for the first time a metabolomics based strategy for anabolic steroid
urine profiling. Urine profiles of controls and bovines treated with the prohormones
dehydroepiandrosterone (DHEA) and pregnenolone were analyzed with ultraperformance
liquid chromatography in combination with time-of-flight accurate mass spectrometry (UPLC-
TOFMS). The obtained full scan urinary profiles were compared using sophisticated
preprocessing and alignment software (MetAlign) and multivariate statistics, revealing
hundreds of mass signals which were differential between untreated control and
prohormone-treated animals. Moreover, statistical testing of the individual accurate mass
signals showed that several mass peak loadings could be used as biomarkers for DHEA and
pregnenolone abuse. In addition, accurate mass derived elemental composition analysis and
verification by standards or Orbitrap mass spectrometry demonstrated that the observed
differential masses are most likely steroid phase I and glucuronide metabolites excreted as a
direct result from the DHEA and pregnenolone administration, thus underlining the relevance
of the findings from this untargeted metabolomics approach. It is envisaged that this
approach can be used as a holistic screening tool for anabolic steroid abuse in bovines and
possibly in sports doping as well [09079].
An existing gas chromatography-mass spectrometry-based quantitative screening method for

the regulatory analysis of the resorcylic acid lactones zeranol, taleranol, and zearalanone
and the stilbene anabolic steroids diethylstilbestrol and dienestrol was extended to include
natural precursors of zeranol (zearalenone, alpha-zearalenol, and beta-zearalenol) in veal
liver. No changes in sample preparation were required; the instrumental conditions were
633
selected to effect a suitable chromatographic separation and detection of the analytes.
Validation experiments were performed to verify the performance and applicability of the
extended method for the quantitative screening of the original and additional analytes in veal
liver in the concentration range from 0.5 to 2.0 microg/kg. The limits of detection were 0.08-
0.19 microg/kg. The limits of quantitation were 0.27-0.64 microg/kg. Recoveries were 29-67
percent. Combined relative measurement uncertainty estimates were 6-21 percent [09080].
The administration of anabolic steroids, for the purposes of growth promotion, to food-
producing animals is banned in the EU. Among the compounds covered by this prohibition is
ss-nortestosterone (beta-NT). This hormone is known to occur naturally in stallions and
boars, and its main bovine metabolite, alpha-nortestosterone (alpha-NT), occurs naturally in
pregnant cows and neonatal calves. However, neither compound is believed to occur
naturally in male cattle. During 2006, the presence of alpha-NT and, on occasion, beta-NT
was confirmed in male cattle (bulls and steers) slaughtered in Northern Ireland on welfare
grounds, as a result of acute injury. Subsequent investigations revealed no evidence of
abuse at any of the farms involved and revealed that the phenomenon also occurred in three
other regions of the EU, in similarly injured animals. A hypothetical link to release of the
adrenal steroid, dehydroepiandrosterone (DHEA), in response to the stress of the injury was
tested. Following the intravenous administration of DHEA to two normal steers, beta-NT (but
not alpha-NT) was confirmed in the urine of one steer. Thus, it may be concluded that both
beta-NT and, by implication, alpha-NT can occur naturally in male cattle (or a specific cohort
thereof) in contrast to previously accepted scientific knowledge [09081].
A new LC-MS/MS method was developed for the analysis of 29 veterinary drug residues,
spanning three different drug classes, in animal tissues. The procedures used to measure
the characteristic performance parameters of the method and the results obtained using
fortified blank bovine muscle and kidney tissue are described. For a quantitative and
confirmatory method, the characteristic performance parameters to be determined are the
limits of quantification, trueness, recovery, precision, selectivity, ruggedness, and stability.
The characteristic performance parameters defined for the method will be verified during a
validation study by an independent experienced analyst to determine whether the method is
suitable for use in a regulatory monitoring and control program for residues of the 29
analytes [09082].
The abuse of synthetic esters of natural steroids such as testosterone and estradiol in cattle
fattening and sports is hard to detect via routine urine testing. The esters are rapidly
hydrolysed in vivo into substances which are also endogenously present in urine. An
interesting alternative can be provided by the analysis of the administered synthetic steroids
themselves, i.e. the analysis of intact steroid esters in hair by liquid chromatography tandem
mass spectrometry (LC/MS/MS). However, retrospective estimation of the application date
following a non-compliant finding is hindered by the complexity of the kinetics of the
incorporation of steroid esters in hair. In this study, the incorporation of intact steroid esters in
hair following pour-on treatment has been studied and critically compared with results from
intramuscular treatment. To this end animals were pour-on treated with a hormone cocktail
containing testosterone cypionate, testosterone decanoate and estradiol benzoate in
different carriers. The animals were either treated using injection and pour-on application
once or three times having 1 week between treatments using injection and pour-on
application. Animals were slaughtered from 10-12 weeks after the last treatment. Both hair
and blood plasma samples were collected and analysed by LC/MS/MS. From the results, it is
concluded that after single treatment the levels of steroid esters in hair drop to CCbeta levels
(5-20 microg/kg) after 5-7 weeks. When treatment is repeated two times, the CCbeta levels
are reached after 9-11 weeks. Furthermore, in plasma, no steroid esters were detected; not
even at the low microgramme per litre level but – in contrast with the pour-on application –
634
after intramuscular injection, significant increase of 17beta-testosterone and 17beta-estradiol
were observed. These observations suggest that transport of steroid esters after pour-on
application is not only performed by blood but also by alternative fluids in the animal so
probably the steroid esters are already hydrolysed and epimerized before entering the blood
[09083].
The detection of hormone abuse for growth promotion in food animal production is a global
concern. Initial testing for hormones in Canada was directed at the compounds approved for
use in beef cattle, melengestrol acetate, trenbolone acetate and zeranol, and the banned
compound diethylstilbestrol (DES). No hormonal growth promoters are approved for use in
veal production in Canada. However, instances of use of trenbolone and clenbuterol were
detected in Canada in the 1990s. During the development of a new analytical method for
testosterone and progesterone, there were reports of suspicious injection sites being found in
veal calves. Upon implementation of the method, analysis of investigative samples revealed
significant residues of testosterone in some injection sites. To prove that the source of these
residues was exogenous, a fully validated method for hormone esters was developed to
confirm the presence of exogenous hormones in these injection sites. The QUECHERS
model was employed in methods development and resulted in a simple, effective extraction
technique that consisted of sample pre-homogenization, liquid/liquid partitioning, extract
dilution, filtration and use of LC/MS/MS to provide detection selectivity. The result was an
adaptable MS/MS confirmation technique that meets the needs of Canadian regulatory
authorities to confirm the misuse of injectable testosterone, and potentially other hormones,
in food animal production [09084].
Anabolic steroids have been used for the promotion of weight gain and feed efficiency in
livestock since the 1940s. In some countries, such as the US and Brazil, the use of
veterinary growth promoters are restricted to therapeutic uses and they are totally prohibited
in countries such as those within the European Economic Community. Despite the
regulation, there is the possibility of illegal use of anabolic steroids and consequently the
presence of hormone residues in animal products. In general, the growth promoters are
found in very low levels in animal tissues, being more concentrated in liver, kidney and
intestine. The presence of these substances in meat could lead to human ingestion, even in
small quantities. Consequently, athletes who have consumed such meat could have a
positive test for anabolic agents in doping control and the risk is increased with repeated
consumption [14039].
Nandrolone
17beta-Nandrolone (17beta-NT) is one of the most recurrent forbidden anabolic steroid used
in meat producing animals breeding. Because efficient control must both take into account
metabolic patterns and associated kinetics of elimination, the metabolism of 17beta-NT in
bovines has already been investigated and is well documented, but only focussing on its
main metabolites (i.e. 17alpha-nandrolone, 19-noretiocholanolone and 19-norandrostene-
dione). The goal of one study was to enlarge this panel of 17beta-NT metabolites, especially
through the urinary estranediols fraction in order to perform a more global steroid profiling
upon 17beta-nortestosterone laureate ester administration in calves. A GC-MS/MS method
has been developed to monitor and quantify 5 estranediols isomers including 5alpha-
estrane-3beta,17beta-diol (abb), 5beta-estrane-3alpha,17beta-diol (bab), 5alpha-estrane-
3beta,17alpha-diol (aba), 5alpha-estrane-3alpha,17beta-diol (aab) and 5beta-estrane-
3alpha,17alpha-diol (baa). Their urinary elimination kinetics have been established allowing
detection of 4 estranediols up to several days after administration. All animals demonstrated
homogeneous patterns of elimination both from a qualitative (metabolite profile) and
quantitative point of view (elimination kinetics in urine). 5alpha-Estrane-3beta,17alpha-diol
635
(aba) was found as the major metabolite with concentrations up to 100microg/L [10345].
Trenbolone
For several years it has been known that sex hormones excreted via human and/or animal
feces can exhibit endocrine-disrupting activity; for example, estrogens present in chicken
manure caused hyperestrogenism when fed to cattle. Natural and synthetic estrogens such
as estradiol-17 and ethinylestradiol-17 were frequently detected in lower nanogram per liter
ranges in discharges of sewage treatment plants, caused by their incomplete removal during
passage through the sewage treatment plants. Exposure of fish to sewage treatment plant
effluents increased plasma levels of vitellogenin, a protein synthesized by the liver of
oviparous fish in response to estradiol stimulation. Public concern focuses especially on the
synthetic estrogen and progestin components of oral contraceptives, which have high
physiologic activity at low doses. Compared with the natural hormones, they show a
relatively greater stability in aqueous media and a greater resistance to microbial
degradation. These properties pose the potential for accumulation and persistence in the
environment. It can be presumed that other structurally related xenobiotic hormones that are
used for veterinary treatment show a similar behavior. The steroids trenbolone acetate (TbA)
and melengestrol acetate (MGA) are licensed as growth promoters for farm animals in
several meat-exporting countries. Although many studies have explored their safety for both
animals and consumers, little is known about their fate after excretion by the animal. One
study aimed to determine the residues and degradation of trenbolone and MGA in solid
dung, liquid manure, and soil. In animal experiments lasting 8 weeks, cattle were treated with
TbA and MGA. Solid dung and, in case of trenbolone, liquid manure were collected and
spread on maize fields after 4.5 and 5.5 months of storage, respectively. Determination of
the hormone residues in all samples included extraction, clean-up (solid-phase extraction),
separation of metabolites and interfering substances by HPLC (RP-18), and quantification by
sensitive enzyme immunoassay. Procedures were validated by mass spectrometry (MS)
methods. During storage of liquid manure the level of trenbolone decreased from 1,700 to
1,100 pg/g (17alpha-isomer), corresponding to a half-life of 267 days. Before storage, the
concentrations in the dung hill ranged from 5 to 75 ng/g TbOH and from 0.3 to 8 ng/g MGA.
After storage, levels up to 10 ng/g trenbolone, and 6 ng/g MGA were detected. In the soil
samples trenbolone was traceable up to 8 weeks after fertilization, and MGA was detected
even until the end of the cultivation period. The results show that these substances should be
investigated further concerning their potential endocrine-disrupting activity in agricultural
ecosystems [01016].
Over the last years, extensive research has documented endocrine-disrupting activities for a
significant number of substances including, among others, hormones, pharmaceuticals,
pesticides and surfactants. Nonetheless, for most endocrine disruptors, toxicological profiles
are still incomplete or even lacking. A systematic review has shown that a number of
endocrine disruptors with steroid-modulating effects may also exert mutagenic and
carcinogenic activities. For trenbolone, an androgenic compound, there is controversy about
its genotoxic properties in the literature, apparently with a strong dependence on the choice
of the test system. Since fish and other aquatic animals are at risk of exposure to run-offs
from cattle feedlots or sewage-discharge sites containing trenbolone, potential
consequences to aquatic ecosystems need to be assessed. To this end, the potential
genotoxic hazard of trenbolone was tested in vitro in the permanent rainbow trout-liver cell-
line RTL-W1, as well as in primary cell cultures derived from zebrafish (Danio rerio) embryos
after in vivo exposure. In either test system, a potential genotoxic hazard characterized by
biphasic dose-response curves could be documented even at exposure concentrations of 30
microg/L. These results thus confirm the conclusion that the steroid trenbolone may act as a
genotoxic substance [11053].
636
Trenbolone acetate (TbA) is a potent synthetic anabolic steroid that was approved by the
FDA as a growth promoter in beef cattle in 1987. Given the endocrine-modulating activity of
TbA and its metabolites in all vertebrates, a sensitive and reliable analytical method is
needed to detect TbA and related residues in environmental matrices. It was developed a
method that incorporates solid phase extraction and liquid chromatography-tandem mass
spectrometry (LC-MS/MS) for the simultaneous determination of the three major TbA
metabolites (trendione, 17beta-trenbolone, 17alpha-trenbolone) in total suspended
particulate matter (TSP) samples. Sample preparation involved pressurized liquid extraction
followed by cleanup on solid-phase extraction cartridges. The procedure was optimized to
obtain maximum recovery and minimum signal suppression/enhancement from matrix
effects. Analytes were separated with a Phenomenex Gemini-NX C18 analytical column (150
mm×2.0 mm, 3 microm particle size) using an aqueous methanol gradient at a flow rate of
0.2 mL/min. Column effluent underwent positive electrospray ionization (ESI). Two or more
diagnostic product ions were acquired from analyte specific precursor ions for unambiguous
confirmation and quantification. The method detection limit was 3.27-4.87 ng/g of particulate
matter (PM). Method accuracy, determined with analyte recoveries, ranged between 68 and
117 percent, and method precision, expressed as relative standard deviation, was below 15
percent at spiked levels of 6.67, 33.3, and 167 ng/g PM. Analysis of TSP samples
demonstrated the presence of the target species associated with PM in the vicinity of beef
cattle feeding operations [11054].
Molecular mechanisms in skeletal muscle associated with anabolic steroid treatment of cattle
are unclear and we aimed to characterize transcriptional changes. Cattle were chronically
exposed (68 ± 20 days) to a steroid hormone implant containing 200 mg trenbolone acetate
and 20 mg estradiol (Revalor-H). Biopsy samples from 48 cattle (half treated) from
longissimus dorsi (LD) muscle under local anesthesia were collected. Gene expression
levels were profiled by microarray, covering 16,944 unique bovine genes: 121 genes were
differentially expressed (DE) due to the implant (99.99 % posterior probability of not being
false positives). Among DE genes, a decrease in expression of a number of fat metabolism-
associated genes, likely reflecting the lipid storage activity of intramuscular adipocytes, was
observed. The expression of IGF1 and genes related to the extracellular matrix, slow twitch
fibers, and cell cycle (including SOX8, a satellite cell marker) was increased in the treated
muscle. Unexpectedly, a very large 21- (microarray) to 97 (real time quantitative PCR)-fold
higher expression of the mRNA encoding the neuropeptide hormone oxytocin was observed
in treated muscle. We also observed an ∼50-fold higher level of circulating oxytocin in the
plasma of treated animals at the time of biopsy. Using a coexpression network strategy
OXTR was identified as more likely than IGF1R to be a major mediator of the muscle
response to Revalor-H. A re-investigation of in vivo cattle LD muscle samples during early to
mid-fetal development identified a >128-fold increased expression of OXT, coincident with
myofiber differentiation and fusion. It was propose that oxytocin may be involved in mediating
the anabolic effects of Revalor-H treatment [11055].
To assess the relative ecological risks of trenbolone acetate (TBA) use in agro-ecosystems,
we evaluated the spatiotemporal dynamics of TBA metabolite transport during irrigation and
rainfall events. Within a pasture, TBA-implanted heifers (40 mg TBA, 8 mg estradiol) were
briefly penned (24 h) at high stocking densities (500 animal units (AU)/ha), prior to irrigation.
Irrigation runoff concentrations of 17alpha-trenbolone (17alpha-TBOH) 0.3 m downslope
were 11 ng/L in the wetting front, but quickly decreased to about 0.5 ng/L, suggesting mass
transfer limitations to transport. At 3 and 30 m downslope, efficient attenuation of 17alpha-
TBOH concentrations is best explained by infiltration and surface partitioning. At plot scales,
transport through vegetated filter strips resulted in <0.5-7 ng/L 17alpha-TBOH concentrations
in rainfall-induced runoff with partial subsequent attenuation. Thus, even under intense
637
grazing scenarios, TBA-metabolite transport potential is expected to be low in rangelands,
with ecological risks primarily arising from uncontrolled animal access to receiving waters.
However, 17alpha-TBOH concentrations in initial runoff were predicted to exceed threshold
levels (i.e. no observed adverse effect levels, NOAELs) for manure concentrations exceeding
2.0 ng/g-dw, which occurs throughout most of the implant life. For comparison, estrone and
17beta-estradiol were modeled and are likely capable of exceeding NOAELs by a factor of
about 2-5 in irrigation runoff, suggesting that both endogenous and exogenous steroids
contribute to endocrine disruption potential in agro-ecosystems [14730].
Trenbolone acetate and estradiol metabolite excretion profiles in implanted steers

Exogenous growth promoters have been used in US beef cattle production for over 50 years.
The environmental fate and transport of steroid growth promoters suggest potential for
endocrine-disrupting effects among ecological receptors; however, the initial excretion of
steroid metabolites from cattle administered growth promoters has not been well
characterized. To better characterize excretion of trenbolone acetate and estrogen
metabolites, steers were assigned to 1 of the following treatment groups: control, given no
implant, or treatment, administered a combination implant (200 mg trenbolone acetate, 40
mg estradiol). Blood, urine, and fecal samples were collected over the course of 112 d
following implantation. Samples were extracted and analyzed by liquid chromatography
tandem mass spectrometry for trenbolone acetate and estrogen metabolites. In both urine
and feces, 17alpha-trenbolone and 17alpha-estradiol were the predominant metabolites
following implantation. Mean concentrations of 17alpha-trenbolone and 17alpha-estradiol in
feces of implanted steers were 5.9 ± 0.37 ng/g and 2.7 ± 0.22 ng/g, respectively. A best-fit
model is presented to predict 17alpha-trenbolone and 17alpha-estradiol excretion from
steers receiving implants. The study provides the first characterization of both trenbolone
and estrogen metabolites in excreta from implanted cattle and will help provide estimates of
steroid production from feedyards in the United States [14731].
Bioavailability and fate of sediment-associated trenbolone and estradiol in aquatic systems

Endocrine disrupting effects in aquatic organisms have been observed in systems influenced
by steroid hormones. Associating endocrine disruption with aqueous concentrations of
steroids alone may overlook the influence of source-sink dynamics in sediments on steroid
hormone bioavailability. The objective of this study was to determine the fate of 17beta-
estradiol and 17beta-trenbolone in two field sediments and to evaluate the corresponding
bioavailability of the compounds to the fathead minnow (Pimephales promelas). Steroid fate
was evaluated using analytical chemistry and verified by assessing the biological activity
using yeast based in vitro assays. Effective bioavailability of the steroids was inferred from
changes in hepatic vitellogenin expression (increased expression in males exposed to
17beta-estradiol, and reduced expression in females exposed to 17beta-trenbolone). In
experiments conducted with 17beta-estradiol, no induction of hepatic vitellogenin mRNA
expression was observed in male fish exposed to sediment-associated 17beta-estradiol. In
contrast, female minnows exposed to sediment-associated 17beta-trenbolone experienced
significant reductions in hepatic vitellogenin compared to negative controls. In both systems,
the parent compounds were shown to degrade rapidly to the more persistent metabolites,
estrone and trendione, both of which were found predominantly associated with the
sediments. Results from the yeast estrogen screen indicate a reduction in biological activity
as biotransformation of 17beta-estradiol occurs; results from the yeast anti-estrogen screen
were inconclusive and unable to substantiate 17beta-trenbolone fate in aquatic systems.
Collectively, these data support the contention that steroid hormones associated with the
sediment can become bioavailable to fish, and that sediment characteristics influence the
observed bioavailability of these compounds [14732].
638
17beta-estradiol and 17beta-trenbolone
Endocrine disrupting effects in aquatic organisms have been observed in systems influenced
by steroid hormones. Associating endocrine disruption with aqueous concentrations of
steroids alone may overlook the influence of source-sink dynamics in sediments on steroid
hormone bioavailability. The objective of this study was to determine the fate of 17beta-
estradiol and 17beta-trenbolone in two field sediments and to evaluate the corresponding
bioavailability of the compounds to the fathead minnow (Pimephales promelas). Steroid fate
was evaluated using analytical chemistry and verified by assessing the biological activity
using yeast based in vitro assays. Effective bioavailability of the steroids was inferred from
changes in hepatic vitellogenin expression (increased expression in males exposed to
17beta-estradiol, and reduced expression in females exposed to 17beta-trenbolone). In
experiments conducted with 17beta-estradiol, no induction of hepatic vitellogenin mRNA
expression was observed in male fish exposed to sediment-associated 17beta-estradiol. In
contrast, female minnows exposed to sediment-associated 17beta-trenbolone experienced
significant reductions in hepatic vitellogenin compared to negative controls. In both systems,
the parent compounds were shown to degrade rapidly to the more persistent metabolites,
estrone and trendione, both of which were found predominantly associated with the
sediments. Results from the yeast estrogen screen indicate a reduction in biological activity
as biotransformation of 17beta-estradiol occurs; results from the yeast anti-estrogen screen
were inconclusive and unable to substantiate 17beta-trenbolone fate in aquatic systems.
Collectively, these data support the contention that steroid hormones associated with the
sediment can become bioavailable to fish, and that sediment characteristics influence the
observed bioavailability of these compounds [14631].
Mobility of trenbolone and melengestrol acetate in agricultural soil

There is growing concern about environmentally released man-made chemicals suspected to
be responsible for a number of adverse effects on endocrine function in wildlife species and
possibly also in humans. Sex hormones are of particular interest due to their regulatory role
in developmental processes such as sexual differentiation. Endogenous hormones of human
or animal origin as well as exogenous sex steroids used for contraception or as anabolics for
farm animals are excreted and reach the environment. It was investigated the transport of the
synthetic growth promoters trenbolone (TbOH) and melengestrol acetate (MGA) in
agricultural soil by means of column experiments with aggregated soil materials (Ap and Bt
horizons of a Luvisol). Column effluent concentrations and depth profiles of TbOH and MGA
were determined with sensitive enzyme immunoassay systems and HPLC (RP-18),
respectively. All procedures were confirmed by liquid chromatography-mass spectrometry.
Small amounts of TbOH and MGA passed the columns very quickly. However, both
hormones exhibited a high affinity to the organic matter of both horizons leading to a high
retardation within the upper layers of the soil columns. Although it cannot be deduce whether
hormones of animal origin reach the ground water under field conditions, the model
experiments show that their transition can be presumed [14035].
Anabolic steroids in dietary supplements
Several recent studies have shown evidence of some nutritional supplements containing
prohibited anabolic androgenic steroids, so-called prohormones, which were not declared on
the label. Therefore, a broad-based investigation of the international nutritional supplement
market was initiated to clarify the extent of this problem. From October 2000 until November
2001, 634 non-hormonal nutritional supplements were purchased in 13 countries from 215
different suppliers. Most supplements were bought in shops in the respective countries (578
samples = 91 %) and on the internet (52 samples = 8 %). 289 supplements were from
prohormone-selling companies and 345 supplements came from companies which do not
offer prohormones. After isolation from the supplement matrix 11 different anabolic
639
androgenic steroids, mainly prohormones of testosterone and nandrolone, were analysed by
gas-chromatography/mass spectrometry. Out of the 634 samples analysed 94 (15 %)
contained anabolic androgenic steroids not declared on the label ("positive supplements"). It
was not obtained reliable data for 66 samples (10 %) due to matrix effects. In relation to the
total number of products purchased per country, most of the positive supplements were
bought in the Netherlands (26 %), in Austria (23 %), in the UK (19 %) and the USA (19 %).
According to the label, all positive supplements were from companies located in only five
countries: the USA, the Netherlands, the UK, Italy and Germany. Twenty-one of the
nutritional supplements from prohormone-selling companies contained anabolic androgenic
steroids, whereas 10 percent of the supplements from companies not selling prohormones
were positive. The positive supplements showed anabolic androgenic steroid concentrations
of 0.01 micro g/g up to 190 micro g/g. The administration of supplements containing
nandrolone prohormones adding up to a total uptake of more than 1 micro g resulted in
positive doping results for norandrosterone for several hours [14043].
Methenolone
It was investigated the possibility of the ingestion of poultry treated with metenolone in
generating a positive result for anabolic steroids in a doping test. Urine samples were
collected from volunteers who consumed chicken meat that had received metenolone
intramuscularly or orally in their feed. No metenolone or metabolites were detected in urine
samples from subjects who ate meat from orally dosed chickens. However, 50 about of the
samples collected 24 hours after consumption of the intramuscularly dosed chicken were
confirmed positive. In one case, a Norwegian athlete who had a positive doping test for
metenolone claimed in his defence that he had eaten contaminated chicken. However, the
tribunal was not convinced by the claim and he lost his appeal [14039].
Methenolone acetate in a veal calf
The use of anabolic steroids has been banned in the European Union since 1981. In this
study, the metabolism of the anabolic steroid methenolone acetate, was investigated in a
male veal calf. After daily oral administration of methenolone acetate, three main metabolites
were detected in both urine and faeces samples. Among these metabolites, alpha-
methenolone was apparently the main one, but 1-methyl-5alpha-androstan-3,17-diol and
3alpha-hydroxy-1-methyl-5alpha-androstan-17-one were also observed. The parent
compound was still detectable in faeces. As a consequence, abuse of methenolone acetate
as growth promoter can be monitored by analysing urine and faeces samples. A few days
after the last treatment, however, no metabolites were observed. Alpha-methenolone was
detectable in urine until 5 days after the last treatment, but in faeces no metabolites were
detectable after 3 days [06240].
Detection of anabolic residues in implantation sites in cattle
Eight weeks before slaughter, 26 heifers, 2 calves, and 1 steer were implanted with licensed
anabolic preparations at off-label injection sites. After slaughter, 24 of 31 implantation sites
(77 %) were detected. Residual pellets of Revalor H contained a mean of 42.9 mg trenbolone
acetate (range 19.8-57.7 mg) and 4.6 mg (1.96-6.45 mg) estradiol, corresponding to 30
percent (19.8-57.7 %) and 32.7 percent (14.0-46.6 %) of the originally applied dose,
respectively. In the tissue areas containing residual Revalor H pellets, total residues ranged
from 14.8 microg to 12.6 mg trenbolone acetate, 41.7 microg to 1.45 mg trenbolone, and
11.1 microg to 3.39 mg estradiol. The outer tissue areas of the injection sites contained <2
microg hormones. The preparations Synovex H, Finaplix H, Implus S, and Component EC
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behaved similarly to Revalor H. Residues of Synovex Plus were low, whereas the
Compudose silicone rubber contained 58.8% of the implanted dose, but left no significant
tissue residues. If implantation sites are processed in meat manufacturing, international
threshold levels of the respective substances will be exceeded in tons of meat products
[00018].
Plasma steroid variations in bull calves
The aim of this work was to investigate the secretion of dehydroepiandrosterone (DHEA),
testosterone (T), dihydrotestosterone (DHT) and oestradiol (E) as biological markers in
response to illegal administration of testosterone, 19-nortestosterone (N) and oestradiol,
either alone or in combination. Twenty male Friesian calves (age 13-14 months) were
allotted to a control group (n=5), and five experimental groups (n=3) each. Each
experimental animal was repeatedly injected with one of the following hormonal treatments:
E, T, N, T+E and N+E. Circulating DHEA, T, DHT and E were determined by
radioimmunoassay. The administration of T alone did not induce any variation in plasma
DHEA, T, DHT and E, which were similar to those in the control group. In contrast, DHEA, T
and DHT were on average significantly lower in the T+E and N-treated groups, whereas the
administration of N+E resulted in the reduction of plasma T and DHT without any
modification of plasma DHEA. The administration of E alone or in combination increased
circulating levels of E but did not affect androgen plasma profiles. The results indicate that
plasma levels of T do not permit detection of illegal treatments because plasma androgens
always remained within the physiological range. Illegal E treatment could be detected in
blood samples when they were collected at least every 20 days [14033].
Sex steroid levels in urine of cattle of different ages
Levels of several natural urinary steroids have been determined in the urine of a large
number of animals of different cattle categories in the context of steroid abuse in beef
production. Bovine animals of different breeds, sex and age included in the Slovene national
residue detection plan for steroid abuse were studied. Urine from 120 males and 174
females was analysed. Urinary boldenone, boldione, androstenedione, equiline,
medroxyprogesterone, medroxyprogesterone acetate, melengestrol acetate, progesterone,
stanozolol, trenbolone, trenbolone acetate, 17alpha-ethinylestradiol, 17alpha-methyl-
testosterone, epitestosterone, 17beta-estradiol, testosterone, and nandrolone were
determined by LC-MS/MS. Epitestosterone was found in all bulls; while the proportion of
animals containing testosterone and androstenedione increased with age. Testosterone was
not detected in bulls less than 5 months of age. Epitestosterone levels, however, were not
age dependent. The ratio of testosterone to epitestosterone thus increased with age, from
0.13 ± 0.09 at 1-7 months to 0.42 ± 0.10 at 25-38 months. It was significantly higher in bulls
above 13 months than in younger animals. In contrast to males, no urinary testosterone was
found in females, whereas epitestosterone, androstenedione, progesterone and estradiol
were present. The proportion of animals of various age groups in which epitestosterone was
detected ranged from 68% to 100%, but the differences were not significant. The presence of
both estradiol and progesterone in the same sample was not observed in any animal. The
results of this study could be helpful in determining physiological urinary steroid levels in
order to provide a baseline for the control of steroid abuse in beef production [14031].
Characterization of exogenous testosterone in livestock

The detection of exogenous testosterone in bovine urine was investigated by using gas
chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). The carbon
isotopic ratio measurement of epitestosterone, etiocholanolone (testosterone metabolite) and
641
DHEA (testosterone precursor) in female bovine urines after testosterone enanthate
administration was carried out. An important modification in the 13C/12C ratio of testosterone
metabolites was observed, such that significant differences between precursor and
metabolites of testosterone occurred until three weeks after intramuscular administration of
testosterone enanthate. The factors influencing the 13C/12C of endogenous steroids were
studied especially through cattle feeding and age. The DHEA mean delta13C value was found
to vary between -25 and -26/1000 when hay and concentrate diet were used for fattening. On
the other hand the delta13C value observed when maize silage was used increased to -
20/1000. Testosterone metabolites showed the same delta13C increase as their precursor.
Moreover, we observed a clear relationship between age and efficiency of misuse
determination. Indeed, because of the lower concentration of natural hormones in young
animals, the contribution of exogenous molecules increases significantly compared with older
subjects. Consequently, demonstration of administration is easier to achieve in calves than in
mature animals [00019].
Eating non-castrated male pork induce increase of nandrolone in urine
For the first time in the field of steroid residues in humans, demonstration of 19-
norandrosterone (19-NA: 3alpha-hydroxy-5alpha-estran-17-one) and 19-noretiocholanolone
(19-NE: 3alpha-hydroxy-5beta-estran-17-one) excretion in urine subsequent to boar
consumption is reported. Three male volunteers agreed to consume 310 g of tissues from
the edible parts (meat, liver, heart and kidney) of a boar. The three individuals delivered urine
samples before and during 24 h after meal intake. After deconjugation of phase II
metabolites, purification and specific derivatisation of target metabolites, the urinary extracts
were analysed by mass spectrometry. Identification was carried out using measurements
obtained by gas chromatography/high resolution mass spectrometry (GC/HRMS) (R = 7000)
and liquid chromatography/tandem mass spectrometry (LC/MS/MS) (positive electrospray
ionisation (ESI+)). Quantification was realised using a quadrupole mass filter. 19-NA and 19-
NE concentrations in urine reached 3.1 to 7.5 microg/L nearby 10 hours after boar tissue
consumption. Levels returned to endogenous values 24 hours after. These two steroids are
usually exploited to confirm the exogenous administration of 19-nortestosterone (19-NT:
17beta-hydroxyestr-4-en-3-one), especially in the antidoping field. It was thus proved that
eating tissues of non-castrated male pork (in which 17beta-nandrolone is present) might
induce some false accusations of the abuse of nandrolone in antidoping [00020].
Estrogenic endocrine disruptors
Sports supplements are becoming a regular dietary addition for consumers who view such
products as a means of improving their health and performance. Previously estrogenic
endocrine disruptors (EDs) were detected in 80 percent of 116 sports supplements
investigated by biological in vitro reporter gene assays (RGAs). The aim of one study was to
quantify the hormonal activity in 50 of these sports supplement samples using a validated
estrogen RGA and perform an exposure and risk assessment for human health. Results
showed that 17beta-estradiol equivalent levels were higher than those reported as being
present in the typical human omnivore diet in 33 of the sports supplements and higher than
the acceptable daily intake (ADI) in 13 of these products. The highest activity samples
presented a potential to influence the human daily exposure to 17beta-estradiol like activity in
various risk groups with a predicted hormonal impact of greatest concern in young boys and
postmenopausal women. In conclusion, consumers of sports supplements may be exposed
to high levels of estrogenic EDs [14032].
Clostebol through sexual intercourse

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Clostebol is a synthetic androgenic steroid with anabolic effects that is frequently used in
sports to increase physical performance. Because of medical and ethical reasons, the use of
clostebol is prohibited by the International Olympic Committee (IOC), and its misuse would
fall under the strict liability rule of the IOC and the World Antidoping Agency. It is therefore
the responsibility of athletes to submit evidence contrary to any ruling issued against them by
the appropriate sports body. Despite the prohibition against the use of clostebol, abuse of
this steroid is increasing, mainly in Brazilian athletes. In Brazil, clostebol acetate is present in
medicines for dermatologic and gynecologic treatments, whereas in the US, the Food and
Drug Administration does not approve of the use of medicines that contain anabolic agents.
One laboratory, LABDOP, is accredited by the IOC and in the past 3 years has encountered
four urine samples that contained clostebol metabolites. One male athlete whose urine
tested positive for traces of clostebol metabolites claimed that he was contaminated as a
result of sexual intercourse with a woman taking a medication containing clostebol. The IOC
did not exonerate him from the results reported by LABDOP. The remaining athletes
maintained that the presence of clostebol metabolites in their urine was the result of using
clostebol-containing medications. Despite this controversy, the directive from the IOC has
been followed, and positive results are always enforced. A previous publication showed the
presence of clostebol metabolites in human urine after oral intake of contaminated meat, but
did not mention sexual intercourse. The laboratory now undertook the present study to
determine whether the urine of men exposed to intravaginal clostebol acetate during sexual
intercourse contains clostebol metabolites. A gas chromatographic–mass spectrometric
method was used to test for the presence of two metabolites of clostebol, clostebol-M1 (4-
chloroandrost-4-en-3alpha-ol-17-one) and clostebol-M2 (4-chloroandrostan-3alpha-ol-17-
one), and other steroids in urine samples. The procedure involves preextraction with XAD-2
resin, elution with tert-butyl methyl ether, hydrolysis with beta-glucuronidase from
Escherichia coli, extraction with n-pentane, and derivatization at 60 °C for 60 min with a
solution containing 1 mL of N-methyl-N-(trimethylsilyl)trifluoroacetamide, 2 microg of NH4I,
and 6 microL of 2-mercaptoethanol. The analytes were monitored in selected-ion monitoring
mode. In Brazil, clostebol acetate is available for intravaginal administration. One such
preparation (Trofodermin®; Searle) contains 200 mg of clostebol acetate and 200 mg of
neomycin sulfate per 40-g blister. The package insert states that Trofodermin is indicated for
cervicitis, postoperative vaginitis, and ulcerative vaginitis, and the recommended dose is 5 g
once or twice a day. Two healthy couples (group I) and two healthy men (group II) were
involved in the study. A baseline urine was obtained from all volunteers before exposure to
clostebol acetate. Participants were healthy and without a history of drug use or gynecologic
disease. The study was approved by the University ethics committee (protocol 168/02).
Immediately after intravaginal application of 5 g of clostebol acetate, group I had sexual
intercourse lasting about 20 min (experiment I). In experiment II, the men in group II applied
200 mg of clostebol acetate topically to their penis for 20 min. Urine samples were collected
from all participant volunteers for the following 2 days. The urine of the men in experiment I
contained trace amounts of clostebol-M1 (0.9-3.5 microg/L) with a tmax of 16 h. The
concentration of clostebol-M1 in the urine of the females reached a maximum of 35 microg/L
after 23 h. Small amounts of clostebol-M2 were also detected. The urine of the men in
experiment II contained higher amounts of clostebol-M1, with a peak concentration of 22
microg/L after 3.5 h, and was detectable for 15 h. The baseline urines contained no
clostebol, clostebol-M1, or clostebol-M2. The possibility of incidental contamination from
sexual intercourse was confirmed, despite the fact that the amount of clostebol-M1 (long-
term metabolite) was near the limit of detection (microg/L). Because the IOC does not make
a distinction among circumstances or means of administration of anabolic compounds,
athletes should be warned not to use clostebol-containing medications and to be aware of
their partner’s medical treatments [04044].
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Sexual behavior of fish after trenbolone
Endocrine disrupting chemicals (EDCs) are a large group of environmental pollutants that
can interfere with the endocrine system function of organisms at very low levels. One
compound of great concern is trenbolone, which is widely used as a growth promoter in the
cattle industry in many parts of the world. The aim of one study was to test how short-term
(21-day) exposure to an environmentally relevant concentration of 17beta-trenbolone
(measured concentration 6 ng/L) affects reproductive behaviour and fin morphology in the
eastern mosquitofish (Gambusia holbrooki). The mosquitofish is a sexually dimorphic
livebearer with males inseminating females using their modified anal fin, the gonopodium, as
an intromittent organ. Although the species has a coercive mating system, females are able
to exert some control over the success of male mating attempts by selectively associating
with, or avoiding, certain males over others. It was found that females exposed to trenbolone
approached males less and spent more time swimming away from males than non-exposed
(control) females. By contrast, it was found no difference in the behaviour of exposed and
non-exposed males. Furthermore, exposure did not affect the anal fin morphology of males
or females. This is the first study to demonstrate that exposure to an androgenic EDC can
impair female (but not male) behaviour. The study illustrates how anthropogenic
contaminants can have sex-specific effects, and highlights the need to examine the
behavioural responses of environmental contaminants in both sexes [13077].
Milk
A quantitative LC/MS/MS method was developed for the determination of 14 steroidal

compounds and three basic nonsteroidal anti-inflammatory drugs (detected as metabolites)
in bovine milk and animal muscle tissue. The proposed method is sufficiently sensitive and
highly selective for residue applications. The described approach offers the possibility to
detect, quantify, and confirm anti-inflammatory drugs belonging to two widely diverging
chemical categories. The employed single-stage SPE step (mixed mode cation exchange)
retains both steroids and basic metabolites of nonsteroidal anti-inflammatory drugs. The
method is capable of handling widely diverging relevant concentration ranges (0.1 microg/kg
for dexamethasone and 100 microg/kg for metamizol metabolites) for the individual
compounds with a single extraction, cleanup, and LC/MS/MS procedure. It provides good
analyte precision and accuracy data [14240].
Growth hormone
Black market products of a pharmaceutical nature and nutritional supplements have received
substantial and increasing attention because of potential performance enhancement in elite
and non-professional sports. In addition, improved general health is claimed for non-
competing individuals. The risks and foreseeable dangers of the uncontrolled use of highly
potent and non-approved pharmaceutical compounds in healthy individuals are of
considerable concern. In the present case report, the emerging drug candidate GHRP-2 with
verified growth-hormone-releasing properties was identified and quantified in tablets offered
as an over-the-counter nutritional supplement. The impact of this orally active peptide on the
hGH/IGF-axis has been established for several years and its illicit use in elite sports has
been assumed. As a releasing factor for hGH, GHRP-2 belongs to the list of substances
prohibited by the World Anti-Doping Agency (WADA). Unfortunately, to date there is no
routinely performed assay for the determination of these peptides potentially occurring in
biological fluids of competing athletes, but the present data will facilitate the implementation
by providing principle analytical information on liquid chromatographic and mass
644
spectrometric behaviour. Qualitative identification of the target analyte after extraction from
the tablet matrix was performed by high resolution/high accuracy mass spectrometry after
liquid chromatographic separation under consideration of the accurate masses and the ratios
of the protonated molecules and their fragment ions derived from their collisionally induced
dissociation. Quantitative results were obtained by means of liquid chromatography coupled
to a triple quadrupole mass spectrometer and linear regression using an external calibration
curve (with GHRP-2 reference compound) adjusted via internal standard (Hexarelin).
Hereby, the content of GHRP-2 was determined with approximately 50 microg per tablet
[10154].
Growth promoters given to livestock
In vitro cell-based bioassays are also used to test for illegal substances such as growth
promoters that are given to livestock. These androgens or androgen-like molecules enhance
the growth of the animals and are used as a means to increase profit. Naturally occurring
steroids such as testosterone or synthetic androgens such as 19-nortestosterone, trenbolone
acetate, and medroxyprogesterone are used to illicitly augment growth of animal livestock. In
addition, prohormones such as dehydroepiandrosterone (DHEA) are being used, and these
are often hard to detect if they have been exogenously administered. GC-MS-based
screening of material such as meat extracts, feed or urine may fail to detect these
substances because deconjugating steps are required prior to GC-MS analysis and these
processing steps can destroy the structures. GC-MS may also fail to detect novel structures
or those with unknown metabolic profiles. Bioassays can complement the screening of
samples as they are capable of detecting hormonally active compounds in prepared extracts
and if the appropriate host cell is used for the bioassay such as hepatocytes then such
assays may also detect prohormones. For these reasons, bioassays are ideal for the
detection of androgens or proandrogens added to nutritional supplements [13084].
Beta-agonists in pork
A method was developed to determine 20 illegal residual beta-agonists in pork tissues,

including muscle and liver simultaneously. The samples were hydrolyzed by beta-
glucuronidase, purified by PCX SPE cartridges, and detected by HPLC coupled with
electrospray ionization MS/MS operating in the positive ion mode. Matrix-fortified calibration
was performed to compensate for the matrix effect and loss in sample preparation. Decision
limit ranged from 0.05 to 0.23 microg/kg in muscle and 0.05 to 0.57 microg/kg in liver.
Decision capacity ranged from 0.11 to 0.4 microg/kg in muscle and 0.16 to 0.79 microg/kg in
liver. In Food Analysis Performance Assessment Scheme proficiency test 0287, a pig liver
test material containing 13 beta-agonists was analyzed using the method developed, and
clenbuterol and ractopamine were confirmed as being present. Z-scores for clenbuterol and
ractopamine were 0.2 and 0.6, respectively [11056].
Clenbuterol
The misuse of the sympathomimetic and anabolic agent clenbuterol has been frequently
reported in professional sport and in the livestock industry. In 2010, a team of athletes
returned from competition in China and regular doping control samples were taken within the
next two days. All urine samples contained low amounts (pg/ml) of clenbuterol, drawing the
attention to a well-known problem: the possibility of an unintended clenbuterol intake with
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food. A warning that Chinese meat is possibly contaminated with prohibited substances
according to international anti-doping regulations was also given by Chinese officials just
before the Bejing Olympic Games in 2008. To investigate if clenbuterol can be found in
human urine, a study was initiated comprising 28 volunteers collecting urine samples after
their return from China. For the quantification of clenbuterol at a low pg/ml level, a very
sensitive and specific isotope dilution liquid chromatography-tandem mass spectrometry (LC-
MS/MS) assay was developed using liquid/liquid re-extraction for clean-up with a limit of
detection and quantification of 1 and 3 pg/ml, respectively. The method was validated
demonstrating good precision, accuracy, and mean recovery. Clenbuterol was detectable in
22 (79 %) of the analyzed samples, indicating a general food contamination problem despite
an official clenbuterol prohibition in China for livestock [12358].
In one study, poly(sodium 4-styrenesulfonate) (PSS) functionalized graphene (GR) was

synthesised via a simple one-step chemical reduction of exfoliated graphite oxides in the
presence of PSS. Characterisation of as-made nanocomposite using Fourier transform
infrared spectroscopy (FT-IR) and ultraviolet and visible spectroscopy (UV-vis) clearly
demonstrate the successful attachment of PSS to graphene sheets. A novel clenbuterol
(CLB) electrochemical sensor was fabricated based on isopropanol-Nafion-PSS-GR
composite film modified glassy carbon electrode. In the Britton-Robinson buffer (pH 1.2), the
sensor exhibited superior electrocatalytic activity towards the oxidation of CLB. Applying
linear sweep voltammetry, a good linear relationship of the oxidation peak current with
respect to concentrations of CLB cross the range of 7.5×10-8-2.5×10-5 mol/L and a detection
limit of 2.2×10-8 mol/L were achieved. The proposed method was successfully applied for the
determination of CLB in pork [14243].
Clenbuterol in muscle
A new pretreatment method, solid-phase extraction combined with dispersive liquid-liquid

microextration (SPE-DLLME), was proposed in first time for the determination of clenbuterol
(CLB) in porcine tissue samples. The tissue samples were firstly extracted by SPE, then its
eluents were used as dispersant of the followed DLLME for further purification and
enrichment of CLB. Various parameters (such as the type of SPE sorbent, the type and
volume of elution solvent, the type and volume of extractant and dispersant, etc.) that
affected the efficiency of the two steps were optimized. Good linearity of CLB was ranged
from 0.19 microg/kg to 192 μg/kg with correlation coefficient (r²) of 0.9995. The limit of
detection (LOD) was 0.07 μg/kg (S/N=3) and the recoveries at three spiked levels were
ranged from 88 percent to 104 percent with the relative standard deviation (RSD) less than
3.9% (n=3). Under the optimized conditions, the enrichment factor (EF) for CLB could up to
62 folds. The presented method that combined the advantages of SPE and DLLME, had
higher selectivity than SPE method and was successfully applied to the determination of CLB
in tissue samples [11057].
Clenbuterol in pig retina
The aim of one study was to assess the persistence of clenbuterol residues in retinal tissue
of pigs after repeated administration in a growth-promoting dose, using enzyme-linked
immunosorbent assay (ELISA) as a screening method for quantitative determination. A
growth-promoting dose of clenbuterol (20 μg/kg body mass per day) was administered orally
to the experimental group (n=6) for 21 days, whereas control animals (n=3) were left
untreated. Clenbuterol-treated pigs were randomly sacrificed (n=3) on days 0 and 45 of
treatment discontinuation, and clenbuterol residues were determined in retinal tissue
dissected from the eye. ELISA was found to be acceptable for quantitative determination of
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clenbuterol in retinal samples because previous method validation yielded mean recovery
values of 84-97 percent with variation coefficients < 14 percent. The mean (± SD) retinal
clenbuterol concentration was 1874 ± 114 ng/g immediately upon clenbuterol withdrawal
(day 0) and 73 ± 4 ng/g on the last day post-withdrawal (day 45). Study results pointed to a
very high potential of clenbuterol accumulation in retinal tissue and marked persistence of
clenbuterol residues upon anabolic dose administration, suggesting retinal tissue to be a very
useful matrix for effective control of residual clenbuterol in food-producing pigs [11058].
Clenbuterol in pork and potable water

A novel molecularly imprinted polymer (MIP) for efficient separation and concentration of
clenbuterol (CLB) was synthesized by covalent imprinting approach using CLB derivative as
functional monomer. The MIPs synthesized were characterized by scanning electron
microscope, nitrogen adsorption analysis, Fourier transform infrared spectrometer, and
thermo-gravimetric analysis. The binding experimental results showed that the MIPs
synthesized had fast adsorption kinetic (20 min at 25 mg/L), high adsorption capacity and
specific recognition ability for the analyte. In addition, the MIPs synthesized were
successfully used as solid-phase sorbent for CLB sample preparation to be analyzed by high
performance liquid chromatography with ultraviolet detector. Under optimized experimental
conditions, the linear range of the calibration curve was 5-80 microg/L. The proposed method
was also applied to the analysis of CLB in pork and potable water samples [150095].
Clenbuterol in milk
A simple and sensitive analytical method was developed for the simultaneous determination
of clenbuterol, chloramphenicol and diethylstilbestrol in bovine milk by isotope dilution ultra
performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Samples
were directly purified through HLB cartridge. The organic phase was dried under nitrogen
and residues were redissolved in mobile phase. Samples were analyzed by UPLC-MS/MS
on an Acquity UPLC® BEH C(18) column with gradient elution. The samples were quantified
using clenbuterol-D(9), chloramphenicol-D(5) and diethylstilbestrol-D(8) as internal
standards. The proposed method was validated according to the European Union regulation
2002/657/EC determining specificity, decision limit (CCalpha), detection capability (CCbeta),
trueness, precision, linearity and stability. The method is demonstrated to be suitable for the
determination of clenbuterol, chloramphenicol and diethylstilbestrol in bovine milk. The total
time required for the analysis of one sample was about 50 min [11059].
Acute intoxication
Acute intoxication cases by clenbuterol (a beta2-agonist used as an animal growth promoter)

due to the ingestion of contaminated veal liver were reported in the literature. The consumed
liver presented clenbuterol levels that varied from 160-500 microg/kg. These studies support
the hypothesis that the consumption of meat originating from animals treated with anabolic
agents could yield illegal positive results in urine samples. Hence, the ingestion of anabolic
agents present in contaminated meat constitutes a serious liability to the athlete [04039].
Caffeine
Caffeine is naturally present in several foodstuffs such as coffee, some kinds of tea and
chocolate. Caffeine is also an ingredient of some soft drinks and ‘energetic beverages’.
According to the IOC list, a result is considered positive for doping control purposes when
caffeine urinary concentration exceeds 12 mg/L. This result could only be obtained with the
647
ingestion of 500 mg of caffeine in a short time before sample collection, which corresponds
to approximately 5-10 cups of coffee, 6-12 cups of tea or 2-3 chocolate bars. Therefore, it
would be improbable that an athlete inadvertently ingested this quantity of caffeine from food
[04039].
Cannabis
Several research investigations have been carried out to determine if passive inhalation of
cannabinoids is possible. These studies have shown that the amount of cannabinoids
detected in the urine of a passive smoker is dependent on the size and ventilation of the
room in which he or she was present, the concentration of tetrahydrocannabinol (THC) in
marijuana cigarettes that are being smoked and the amount of the smoke he or she
passively inhaled. Although being an illicit drug in most countries, marijuana (Cannabis
sativa) is smoked in social situations in which not all individuals present smoke the drug.
Theoretically, it is possible that nonsmokers could passively inhale enough marijuana smoke
to excrete detectable amounts of cannabinoids in their urine. In cases like this it is very hard
to state whether the individual was engaged in the use of the drug or just passively exposed.
Several research investigations have been carried out to determine if passive inhalation of
cannabinoids is possible. These studies have shown that the amount of cannabinoids
detected in the urine of a passive smoker is dependent on the size and ventilation of the
room in which he or she was present, the concentration of tetrahydrocannabinol (THC) in
marijuana cigarettes that are being smoked and the amount of the smoke he or she
passively inhaled. In a study carried out in 1988, it consistently reported that <10 microg/L of
cannabinoids were in the urine of three subjects who passively inhaled marijuana smoke in a
closed room (3.1 × 3.1 × 2.4m) with no windows. The smoke was produced by eight
individuals who smoked four cigarettes with 27 mg THC per cigarette. It was also reported
another marijuana passive exposure study inside a car where subjects smoked eight
marijuana or hashish (mixed with tobacco) cigarettes equivalent to 90mg of THC in the
presence of volunteer passive inhalers. Analyses of urine samples from passive inhalers
showed that there were no detectable levels of cannabinoids in the urine of subjects involved
in the hashish study. Nevertheless, urine specimens of subjects passively exposed to
marijuana smoke were positive for cannabinoids, ranging from 14 to 30 microg/L, when
analysed by radioimmunoassay. It was also reported a study in which five healthy men were
passively exposed to smoke from either four or 16 marijuana cigarettes (2.8 % of THC) for 1
hour each day for six consecutive days in an unventilated room (2.5 × 2.1 × 2.4m). Urinary
specimens were collected from the subjects during the study and were analysed by enzyme
multiplied immuno technique (EMIT), radioimmunoassay and gas chromatograph/mass
spectrometry (GC-MS). After the first study, with four marijuana cigarettes, just a few urine
samples tested positive for the cannabinoid metabolites at the 20 microg/L cut-off
concentration of the EMIT assay. None of the samples were positive using the 75 µg/L cut-off
level. In higher extreme exposure conditions (16 cigarettes), more urine specimens were
positive for the lower cut-off (20 microg/L) and eventually samples exceeded the 100
microg/L cut-off EMIT calibrator. In summary, studies have shown it is possible that an
individual could produce detectable levels of cannabinoids in urine samples only after
extremely severe conditions of passive exposure to marijuana smoke. However, according to
the IOC list, sports federations might consider urine samples positive for cannabis use if the
concentration of 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid (the main metabolite
of THC) is >15 microg/L. Hence, it would be improbable that this result was obtained under
normal and realistic conditions of passive exposure [04039].¨
Hemp-containing food
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Use of food products advertised as hemp products” has increased considerably. The hemp
contained in these products is from the plant Cannabis sativa, commonly referred to as
marijuana. Brownies, cookies and cakes prepared with hemp are some of these products
that are becoming popular in some countries. Probably the attraction of this kind of food is
not its taste but the belief that they contain THC or just the simple novelty of it. Other
foodstuffs prepared with hemp or part of it include hemp seed oil, hemp tea and hemp beer.
Recent works published in the literature cite a number of studies that correlate the ingestion
of hemp products and urinary levels of THC metabolites. It was reported the results of
urinalyses of five drug-free male subjects who ingested marijuana-laced brownies with a
THC content equivalent to one or two cigarettes (2.8 % THC). Individual peak concentrations
of 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid (THC metabolite) in urine varied
from 108 to 436 microg/L after the brownies ingestion. In another study five volunteers
consumed daily doses of hemp oil (THC 0.17–1.77 mg/day) and their urine specimens were
collected. Peak concentrations of THC metabolite in the participants’ urine ranged from 1.8 to
48.7 microg/L. It was evaluated the impact of extended daily ingestion of THC via hemp oil
on urine levels of THC metabolite for four daily THC doses (THC 0.09–0.6 mg). The highest
THC metabolite level determined by GC-MS in any of the specimens was 5.2 microg/L, well
below the 15 microg/L reference value used in doping control. According to the authors, daily
consumption of these quantities is difficult to attain, even by those avid consumers of hemp
seed product [04039].
Cocaine
Passive exposure to cocaine has become a concern since the advent of “crack” (freebase
cocaine) in the 1980s. The high volatility of freebase cocaine allows this substance to be
smoked in pipes. Therefore, inhalation of smoke, vapour and expired breath is the most likely
way to be passively exposed to cocaine. Other possibilities include inhalation of the drug
dust, percutaneous absorption and accidental oral ingestion. It was conducted two studies to
evaluate the passive inhalation of crack smoke. In the first study, six healthy subjects were
exposed for 1 hour to either 100 or 20 0mg freebase cocaine vapour produced inside a small
unventilated room. Urine samples were collected and analysed for cocaine and metabolites.
None of the specimens collected following passive exposure tested positive by immunoassay
at the 300 microg/L cut-off. Peak concentration of benzoylecgonine (the main metabolite of
cocaine) in urine, determined by GC/MS, ranged from 22 to 123 microg/L. In the second
study, urine specimens were collected and analysed from medical personnel who were
passively exposed to sidestream cocaine smoke while assisting in a research study of crack
smokers. The research staff remained in close vicinity while the crack smokers smoked three
doses of freebase cocaine over a period of 4 hours. Urine specimens from the staff members
contained a maximum of 6 microg/L benzoylecgonine. Overall, these studies demonstrated
that only individuals exposed to cocaine smoke under extremely harsh conditions would
eliminate cocaine metabolites in urine [04039].
Metamphetamine
Metamphetamine is an illicit drug also available in smoked form known as “ice”. Hence,
passive inhalation of the smoke could be used as a defence in positive metamphetamine
results in doping control. However, as far as we know, no studies have been performed to
date in evaluating the conditions under which passive inhalation of ‘ice’ smoke could result in
a positive test for metamphetamine in urine [04039].
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Poppy seed-containing food
Poppy seeds (derived from Papaver somniferum) are frequently used in the baking of bread
and cakes all over the world. Studies have shown the presence of two major opium alkaloids,
codeine and morphine, in these seeds. The isolation of opiates from poppy seeds has lead to
the supposition that their consumption would result in significant concentrations in urine
specimens. Therefore, positive opiate results obtained during analyses for doping control
could be related to the ingestion of food containing poppy seeds, rather than doping use of
narcotics. It was shown the maximum morphine and codeine concentrations determined in
poppy seeds (originating from Australia, the Netherlands and Turkey) to be 33.2 and 13.7
microg/g of seed, respectively. Following the ingestion of poppy seed cake containing an
average of 4.69 g of seed per slice, a maximum concentration of morphine in urine was only
302 microg/L. In 1990, it was reported urinary concentrations that reached almost 1000
microg/L in subjects who ingested one, two or three poppy seed rolls, each containing 2 g of
seed (108 microg morphine/g seed). In the same study, one subject ingested poppy seed
cake containing 15 g seed. The greatest morphine concentration in urine was 2010 microg/L,
9 hours after ingestion and the concentration decreased to <300 microg/L, 28 hours after
ingestion. Considering the fact that the threshold value (urinary concentration) for morphine
in the IOC list is 1000 microg/L, the findings from demonstrate that the poppy-seed defence
is a plausible argument to be considered and until a reliable marker of seed ingestion has
been isolated, each positive case in doping control must be carefully evaluated. In 1997,
during the Brazilian Soccer Cup, morphine was detected in the urine of a player. In his
defence, he claimed to have eaten bread containing poppy seeds 6 hours prior to the match
[04039].
Herbal coca tea
The consumption of herbal coca tea is common in some countries of South America, such as
Peru and Bolivia. The tea consists of pure coca leaves or coca leaves mixed with different
herbs and is often package in individual servings as tea bags that contain approximately 1 g
of plant material. In a study performed by urine samples were analysed for 36 hours after the
consumption of a cup of Health Inca coca tea. Peak benzoylecgonine concentrations ranged
from 1400 to 2800 microg/L and occurred 4-11 hours post-ingestion. Positive immunoassay
results were obtained for 21-26 hours after tea ingestion. In 1996, it was shown that
approximately 5 mg of cocaine was present in tea bags from Peru and Bolivia. Following the
consumption of a cup of Peruvian coca tea, a peak urine benzoylecgonine concentration of
3940 microg/L was obtained 10 hours after ingestion. Consumption of Bolivian coca tea
resulted in a peak benzoylecgonine concentration of 4979 microg/L at 3.5 hours. The studies
show that consumption of coca tea results in detectable concentrations of cocaine
metabolites in the urine for at least 20 hours. Therefore, if an athlete consumed coca tea
within hours before the competition, his or her urine test for doping control would probably
indicate a positive result for cocaine. In 1993, during the qualifying soccer games for the
Soccer World Cup held in La Paz, Bolivia, the doping control revealed the presence of
cocaine metabolites in the urine of a Brazilian player. The athlete claimed to have taken a
cup of traditional tea with coca leaves as a component. After an investigation by the
Féderation Internationale de Football Association (FIFA), the athlete was reinstated [04039].
Ephedra
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Nutritional supplements containing the plant Ephedra sinica (Ma-Huang) are marketed
primarily as adjuvants in weight-loss programmes and for enhancement of athletic
performance. In these circumstances, questions of safety, adverse effects and efficacy have
been raised. These supplements contain Ephedra alkaloids, including the stimulants
ephedrine, pseudoephedrine, methylephedrine, norpseudoephedrine and phenyl-
propanolamine (norephedrine), included in the IOC list of prohibited substances. Since the
IOC list comprises substances that occur naturally in botanicals such as ephedrines and
caffeine, athletes could take them unwittingly in the form of herbal supplements. . In 1997 a
soccer player had a positive test for doping control. Ephedrines were found in an athlete’s
urine who claimed to have used a herbal supplement without knowing that it contained
Ephedra alkaloids. The ingestion of the supplement was authorised by the team physician
who assumed responsibility for the incident [04039].
Prohormone-containing nutritional supplements
One of first prohormone-containing products sold as “nutritional supplements” that became

available on the market was dehydroepiandrosterone (DHEA) in 1996. Nowadays, the list
has expanded and includes 4-androstenedione, 4-androstenediol, 5-androstenediol
(precursors of testosterone), 19-norandrostenedione and 19-norandrostenediol (precursors
of nortestosterone). Although being anabolic agents that are banned in sports, until now they
have been legally sold without a medical prescription in most countries. Therefore, the ease
of availability and the great marketing appeal stimulate the consumption of these products,
many times sold as ‘natural’. Another problem is that, in some cases, supplements contain
prohibited substances that are not indicated on the label. In a study performed in the German
Sport University, Cologne, 634 samples of nutritional supplements deriving from 13 different
countries were analysed and 94 (15 %) contained prohormones not declared on the label.
Out of all positive supplements, 23 samples (25 %) contained prohormones of nandrolone
and testosterone, 64 samples (68 %) only contained prohormones of testosterone and seven
samples (8 %) only contained prohormones of nandrolone. It was also studied the detection
time for doping substances not listed on the product label after the ingestion of contaminated
nutritional supplements. Boldenone, 19-nor-4-androstenediol, testosterone, 5alpha-
androstane-3alpha, 17beta-diol were detected in capsules only identified as 1-androstene-
3beta, 17beta-diol. The presence of 19-nor-4-androstene-3-17-dione and 4-androstenedione
was verified in a supplement where the label indicated only the presence of hydroxycitric
acid, levocarnitine, phenylalanine, vanadyl sulfate and extracts from citrus auranitium and
guarana. In a product only identified as a pyruvate supplement, DHEA, 4-androstenedione,
testosterone and 19-nor-4-androstene-3-17-dione were also present. After the ingestion of
these contaminated supplements by male volunteers, the major boldenone metabolite was
detected for up to 6 hours. Norandrosterone was found in concentrations >2 microg/L (the
IOC doping threshold in males) from 2 to 24 hours after the intake of one capsule. In another
study a contaminated supplement was found to contain 0.7 mg of 4-androstene-3,17-dione
and 4.8mg of 19-nor-4-androstene-3,17-dione. After the ingestion of one capsule, five male
volunteers tested positive for norandrosterone (>2 microg/L) until 48–144 hours post-
ingestion. The manufacturer recommended seven capsules on a daily basis. These studies
alert us to the fact that the possibility exists for inadvertent ingestion of anabolic agents in the
form of nutritional supplements. Therefore, athletes should consider that these kinds of
products are not subjected to the same pre-approval requirements and appropriate quality
tests as registered medications. Hence, despite the many times it is not printed on the label,
there is no guarantee that these supplements do not contain hormones banned by the IOC
[04039].
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Codeine
Codeine, present in some medicines due to its analgesic and antitussive properties, is an
opiate drug that is permitted by the IOC. However, this substance is metabolised to
morphine, an opiate that is prohibited in sports, in the human body. It was reported urinary
concentrations of codeine and morphine after the administration of codeine preparations.
Volunteers who took preparations containing 30 mg of codeine phosphate showed morphine
urinary concentrations >1 mg/L (the threshold limit for morphine according to the IOC list).
Codeine was also detected in the urine of these volunteers until 12-24 hours post-ingestion.
Recently, the IOC added an updated explanatory note to the list: “morphine at a
concentration greater than 1 microg/mL is a doping offence unless it may have been caused
as a result of the administration of a permitted substance such as codeine. Laboratories
should consider the presence of other substances that would provide evidence of the
administration of codeine and related substances” [04039].
Famprofazone
Famprofazone is an analgesic and antipyretic drug and is a component of the drug

Gewodin® available mainly in Germany. Each tablet contains famprofazone 25 mg,
paracetamol (acetaminophen) 250 mg, propyphenazone 75 mg and caffeine 30 mg. It is
recommended for headache, migraine, tooth ache, rheumatism, etc. Nevertheless, when
administered to humans, famprofazone is biotransformed to metamphetamine and
amphetamine, stimulants that are included in the IOC list. Detecting the presence of its other
metabolite 3-hydroxymethylpyrazolone is clear evidence of famprofazone use. Unfortunately,
if famprofazone and amphetamine were used in association, it would not be possible to
differentiate from the use of famprofazone alone [04039].
Screening of residues in food
Egg
A cheap, reliable and practical high-performance liquid chromatography-tandem mass

spectrometric method was developed for the simultaneous determination of seven anabolic
steroids in eggs, including trenbolone, boldenone, nandrolone, stanozolol, methandienone,
testosterone and methyl testosterone. The analytes were extracted from the egg samples
using methanol. The extracts were subjected to the removal of fat by freezing-lipid filtration
and then further purified by liquid-liquid extraction using tert-butyl methyl ether. The analytes
were separated on a Luna C18 column by a gradient elution program with 0.1% formic acid
and acetonitrile. This method was validated over 1.00-100 ng/g for all steroids of interest.
The correlation coefficients (r) for each calibration curve are higher than 0.99 within the
experimental concentration range. The decision limits of the steroids in eggs ranged from
0.20 to 0.44 ng/g, and the detection capabilities were below 1.03 ng/g. The average
recoveries were between 66 and 83 percent in eggs at three spiked levels of 1.00, 1.50 and
2.00 ng/g for each analyte. The between-day and within-day relative standard deviations
were in the range of 2-11 percent. High matrix suppression effects were observed for all
compounds of interest [12357].
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Bovine
One paper presents the generation of monoclonal antibodies (mAbs) with high specificity
against 19-nortestosterone (NT) through cell fusion procedures, and the development of
mAb-based heterologous direct competitive enzyme-linked immunoabsorbent assay
(dcELISA) methods to detect NT residue using one of these hybridomas (clone 3B8-E6).
Under optimal experimental conditions, this assay exhibited a working range of 0.004 to 19
ng/mL with IC₅₀ and limit of detection values of 0.28 and 0.002 ng/mL, respectively, when it
was run in 0.01M phosphate-buffered saline (pH 7.4). Except for minor cross-reactivity with
beta-boldenone (6.9 %) and trenbolone (1.2 %), other interference to the assay was
negligible. No significant differences were found for IC₅₀ values when the pH of the assay
buffer ranged from 6 to 8 and phosphate ion concentration was less than 20 mM. The
dcELISA can tolerate higher concentrations of methanol than other organic solvents tested.
When applied to bovine sample, the correlation coefficients of the dcELISA and GC-MS data
were 0.9918 in muscle, 0.9834 in liver, and 0.9976 in kidney. Therefore, this assay has the
potential to be incorporated into a quantitative monitoring program for the rapid screening of
NT residue in food [12359].
Swine
The occurrence and fate of fourteen androgens, four estrogens, five glucocorticoids and five
progestagens were investigated by rapid resolution liquid chromatography-tandem mass
spectrometry (RRLC-MS/MS) in a typical swine farm with lagoon waste disposal systems, in
south China. Nineteen, 22 and 8 of 28 steroids were detected at concentrations ranging from
2.2 ± 0.1 ng/g (androsta-1,4-diene-3,17-dione) to 14,400 ± 394 ng/g (progesterone) in the
feces samples, from 6.1 ± 2.3 ng/L (17beta-boldenone) to 10,800 ± 3190 ng/L (norgestrel) in
the flush water samples, and from 5.0 ± 0.2 ng/g (progesterone) to 225 ± 79.4 ng/g (5alpha-
dihydrotestosterone) in the suspended particles, respectively. By comparing the types and
concentrations of steroids in different treatment stages of the lagoon systems, it
demonstrated that the lagoon systems used in the farm were not effective method to reduce
various steroids in wastewater. Among the thirteen synthetic steroids detected in the swine
feces and flush water, only seven (methyl testosterone, 17alpha-trenbolone, 17beta-
trenbolone, 17alpha-ethynyl estradiol, dexamethasone, medroxyprogesterone, and
norgestrel) were regarded as the parent/metabolite compounds of animal exogenous usage.
According to the estimated masses of steroids from feces and flush water, the excretion of
steroids for sows were mainly from feces, but for piglets or barrows, most excreted steroids
were through flush water rather than feces. The total daily excreted masses of androgens,
estrogens, glucocortcoids and progestagens in the sow feces were in the range of 91-
6310 microg/d, which were up to a thousand fold of those in the feces of other growth stages
indicating that the proportion of sow number in the swine farm directly influenced the total
excretion mass of steroids. In addition, two natural steroids 4-androstene-3,17-dione and
progesterone were worth notice due to their relatively high concentrations per sow excretion,
277 microg/d and 6380 microg/d, respectively, which are approximately equivalent to the
daily excretion of 100 persons. Some steroids were also detected in the well water,
vegetable field and receiving stream, and may pose potential high risks to some sensitive
organisms in the receiving environment [12360].
Fish
A method was developed for the determination of 11 anabolic hormones (boldenone,

androstenedione, nandrolone, methandrostenolone, methyltestosterone, testosterone,
testosterone acetate, trenbolone, testosterone propionate, stanozolol, fluoxymesterone) in
653
fish by multi-function impurity adsorption solid-phase extraction-ultrafast liquid
chromatography-tandem mass spectrometry. After the sample was extracted by methanol,
the extract was cleaned-up quickly by C18 adsorbent, neutral alumina adsorbent and amino-
functionalized nano-adsorbent. The separation was performed on a Shim-Pack XR-ODS II
column (100 mm x 2.0 mm, 2.2 microm) using the mobile phases of 0.1% (v/v) formic acid in
acetonitrile and 0.1% (v/v) formic acid solution in a gradient elution mode. The identification
and quantification were achieved by using electrospray ionization in positive ion mode (ESI+)
in multiple reaction monitoring (MRM) mode. The matrix-matched external standard
calibration curves were used for quantitative determination. The results showed that the
calibration curves were in good linearity for the eleven analytes with the correlation
coefficients more than 0.999. The limits of detection (LODs, S/N > 3) for the 11 anabolic
hormones were from 0.03 microg/kg to 0.4 microg/kg and the limits of quantification (LOQs,
S/N > 10) were from 0.1 microg/kg to 1.5 microg/kg. The method is simple, rapid, sensitive,
accurate and suitable for the quantitative determination and confirmation of the 11 anabolic
hormones in fish [12361].
Screening of anabolic substances in solid nutritional supplements
A sensitive and selective method for the screening of 28 different compounds including
testosterone and prohormones, nandrolone and prohormones, stanozolol and metandienone
in solid nutritional supplements is described and validated. The different substances are
extracted from the solid nutritional supplements by liquid-liquid extraction with a mixture of
pentane and freshly distilled diethylether (9/1) after dissolving the supplement in NaOH (1 N).
The anabolizing agents are derivatized with a mixture of MSTFA/NH(4)I/ethanethiol
(320/1/2), routinely used for the derivatization of anabolic steroids extracted from urine. The
TMS-derivatives are analysed by GC-MS in the SIM mode. The limits of detection were in the
range from 2 to 40 ng/g. One supplement was analysed with this method and was found to
contain several forbidden substances according to IOC doping regulations. All detected
compounds, except dihydrotestosterone, could be confirmed with GC-MS(2), proving that the
proposed method is suitable for the screening of anabolizing agents in solid nutritional
supplements [14034].
Anabolic steroid in meat by gas chromatography-ion trap-mass spectrometer
The use of natural and synthetic anabolic steroids in animal fattening has been prohibited in
Taiwan and many countries because of their potential toxic effect on public health. This
paper describes a newly developed gas chromatography-ion trap-mass spectrometry (GC-IT-
MS) method for the quantitative determination of various residual anabolic steroids in meat.
Anabolic steroid was derivatized with N-methyl-N-trimethylsilytrifluoroacetamide prior to GC-
IT-MS analysis. MS(2) was employed for quantitative measurement. In addition, 2d-estradiol
was used as an internal standard. Quantitative determination was based on the ratio of peak
area of steroid derivative to peak area of internal standard derivative. Good linearity of each
compound, 0.03-1.0 microg/mL, was determined. Solvent extraction was used to extract
residual anabolic compounds in meat samples and a solid phase extraction (SPE) procedure
was utilized for sample cleanup and pre-concentration. The limits of detection of anabolic
compounds approximately ranged from 0.1 to 0.4 microg/kg. The detection limit was
comparable with or better than reported methods and was below the minimum required
performance limits (MRPLs) established by the European Community (EC). The application
of this newly developed method was demonstrated by analyzing various beef, pork, chicken
and several animal internal organ samples from local markets [14037].
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Non-labelled anabolic androgenic steroids in nutritional supplements
Recent studies showed that non-hormonal supplements such as vitamins, minerals and
amino acids can contain anabolic androgenic steroids not declared on the labels of the
products. These undeclared substances (often prohormones of testosterone or 19-
nortestosterone) can cause health risks to consumers and might lead to positive results in
sports doping control, especially for the nandrolone metabolite norandrosterone. The
analysis of nutritional supplements for anabolic steroids has proven to be rather difficult due
to the different matrices in the various products. To conduct a broad-based analysis, a few
robust methods capable of analysing various matrices are needed. To obtain a sensitive gas
chromatography-mass spectrometry (GC-MS) analysis, a method including extraction and
purification of the analytes followed by GC-MS analysis of the trimethylsilyl (TMS) derivatives
of the steroids was developed. The limit of detection was improved by the addition of a
mixture of 1-N,N-diisopropylamino-n-alkanes (DIPAs) to the final extract. In pure creatine
monohydrate powder the limits of detection were demonstrated to be 0.1 ng microg (-1) for
dehydroepiandrosterone (DHEA) and estr-4-ene-3beta,17beta-diol, 0.7 ng g(-1) for 5alpha-
androstane-3beta,17beta-diol and androsta-1,4-diene-3,17-dione, 1 ng g(-1) for estr-5-ene-
3beta,17beta-diol, estr-4-ene-3,17-dione, 19-nortestosterone, androst-4-ene-3, 17-dione and
testosterone, and 2 ng g(-1) for androst-4-ene-3beta,17beta-diol and androst-5-ene-
3beta,17beta-diol. The recovery (determined at 200 ng/g) ranged from 32 percent for 19-
nortestosterone to 92 percent for androst-5-ene-3beta,17beta-diol. During the investigation of
different nutritional supplements, several analytical difficulties occurred. Aspects of
homogenization, extraction, separation, derivatization and GC-MS measurement as well as
strategies for the solution of problems arising were optimized. For quantitative
measurements of the steroids in nutritional supplements, deuterated internal standards of the
specific steroids or standard addition are necessary to compensate for matrix effects [14038].
Detection of doping agents in drinking- and wastewater
Doping substances in general
A wastewater treatment plant may receive various types of wastewater namely, urban,
industrial, agricultural, washout from the streets, wet or/and dry atmospheric deposition. As
such, scientists have detected in wastewaters all major categories of pollutants like
persistent organic pollutants (POPs), polycyclic aromatic hydrocarbons (PAHs) and
pesticides, but also substances that are widely used as pharmaceuticals and cosmetics,
classified as "PPCPs" (pharmaceuticals and personal care products). Finally, the latest
categories of compounds to be looked upon in these types of matrices are illicit drugs (drugs
of abuse, like cocaine, etc.) and doping substances. This review article summarises major
categories of organic microcontaminants that have been detected in wastewaters and
studies their fate during the wastewater treatment process. Occurrence of these compounds
in the influents and effluents are reported, as well as percents of removal, mass balances
and phase distributions [12089].
Anabolic steroids, including testosterone
As part of a regional screening to evaluate the risk, for the health of populations, to certain
classes of emerging substances, several families of pharmaceuticals and hormones were
looked for in waters intended to drinking. Thus, 52 substances were investigated in 71
surface waters and 70 groundwaters. Results indicate that no water was free of pollutants,
regardless of its origin (surface or groundwater) and the season of collect. The
pharmaceuticals most frequently detected and with the highest concentration levels were
655
salicylic acid, carbamazepine and acetaminophen. Among hormones, testosterone,
androstenedione and progesterone were detected in almost all the samples. Globally the
groundwaters were less contaminated than surface waters in regards pharmaceuticals
frequencies and levels. On the other side, androgens and progestagens were present with
comparable frequencies and levels in both compartments. The risk linked to the presence of
these substances on human health is discussed [11558].
A new method was developed for the analysis of natural and synthetic androgenic steroids
and their selected metabolites in aquatic environmental matrixes using direct large-volume
injection (LVI) high-performance liquid chromatography (HPLC) tandem mass spectrometry
(MS/MS). Method accuracy ranged from 87.6 to 108% for analytes with well-matched internal
standards. Precision, quantified by relative standard deviation (RSD), was less than 12
percent. Detection limits for the method ranged from 1.2 to 360 ng/L. The method was
demonstrated on a series of 1 h composite wastewater influent samples collected over a day
with the purpose of assessing temporal profiles of androgen loads in wastewater.
Testosterone, androstenedione, boldenone, and nandrolone were detected in the sample
series at concentrations up to 290 ng/L and loads up to 535 mg/h. Boldenone, a synthetic
androgen, had a temporal profile that was strongly correlated to testosterone, a natural
human androgen, suggesting its source may be endogenous. An analysis of the sample
particulate fraction revealed detectable amounts of sorbed testosterone and
androstenedione. Androstenedione sorbed to the particulate fraction accounted for an
estimated 5 to 7 percent of the total androstenedione mass [11060].
Municipal wastewater has been examined for steroids, beta2-agonists, stimulants, diuretics,
and phosphodiesterase type V inhibitors (PDE type V inhibitors), which are "dual-use-drugs"
applied either as anabolic, doping, and lifestyle drugs or for treatment of diverse diseases. To
identify their origin, fitness centre discharges under suspicion of being point sources and
sewage-treatment plant feed and effluents were sampled and concentrations determined.
Sensitive and selective methods for determination and quantification based on solid-phase
extraction (SPE) followed by high-performance liquid chromatography-high resolution mass
and tandem mass spectrometry (HPLC-(HR)MS and HPLC-MS-MS) were developed and
established for analysis of these compounds in wastewater and to assess their effect on the
environment. The methods developed enabled quantification at trace concentrations (limit of
quantification (LOQ): 5 ng/L). Of the steroids and stimulants under investigation,
testosterone, methyltestosterone, and boldenone or ephedrine, amphetamine, and MDMA
(3,4-methylendioxy-N-methylamphetamine) were observed at up to 5 microg/L (ephedrine).
Of the beta2-agonists salbutamol only, and of the diuretics furosemide and hydrochloro-
thiazide were confirmed in the extracts. Quite high concentrations of the PDE type V
inhibitors sildenafil, tadalafil, and vardenafil and their metabolites were confirmed in fitness
centre discharges (sildenafil: 1,945 ng/L) whereas their concentrations in municipal
wastewater did not exceed 35 ng/L. The study thus identified anabolic and doping drugs in
wastewater for the first time. Results obtained from wastewater treatment plant effluents
proved that these "dual-use-drugs", with the exception of hydrochlorothiazide, were mostly
eliminated [10342].
China
Fate and occurrence of fourteen androgens, four estrogens, five glucocorticoids and five
progestagens were investigated in three swine farms and three dairy cattle farms with
different farming scales and wastes disposal systems in China. Twenty-one, 22, and 12 of
total 28 steroids were detected in feces samples with concentrations ranging from below
method limit of quantitation (<LOQ for estrone) to 8100 ± 444 ng/g (progesterone), in
wastewater samples with concentrations ranging from <LOQ (estrone) to 20,700 ± 1490 ng/L
(androsterone), in suspended particles with concentrations ranging from <LOQ (17beta-
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trenbolone) to 778 ± 82.1 ng/g (5alpha-dihydrotestosterone) in the six farms, respectively.
The steroids via swine farms and human sources were mainly originated from wastewater
into the receiving environments while those steroids via cattle farms were mainly from cattle
feces. The total contributions of steroids to the environment in China are estimated to be
139, 65.8 and 60.7 t/year from swine, dairy cattle and human sources, respectively [12091].
The occurrence and fate of fourteen androgens, four estrogens, five glucocorticoids and five
progestagens were investigated in two different types of wastewater treatment plants (Plant
A: activated sludge with chlorination, and Plant B: oxidation ditch with UV) of Guangdong
province, China. 14, 14, and 10 of 28 target compounds were detected in the influent,
effluent and dewatered sludge samples with the concentrations ranging from below 1.2 ± 0.0
ng/L (stanozolol) to 1368 ± 283 ng/L) (epi-androsterone), below 1.0 ± 0.0 ng/L
(progesterone) to 23.1 ± 1.0 ng/L (5α-dihydrotestosterone), 1.0 ± 0.1 ng/g (estrone) to 460 ±
4.4 ng/g (5alpha-dihydrotestosterone), respectively. The concentrations of total androgens
(1554-1778 ng/L in influent, 13.3-47.8 ng/L in effluent, 377-923 ng/g in dewatered sludge)
were much higher than those of total estrogens (41.5-60.2 ng/L in influent, 5.6-13.5 ng/L in
effluent, 13.9-57.8 ng/g) in dewatered sludge), glucocorticoids (171-192 ng/L in influent, 2.2-
6.3 ng/L in effluent, N.D.-4.4 ng/g in dewatered sludge), and progestagens (39.6-40.5 ng/L in
influent, 6.9-12.1 ng/L in effluent, N.D. in dewatered sludge) in these two WWTPs. According
to mass balance analysis, the removal rates of most target steroids in Plant A had exceeded
90 percent, while those in Plant B for nearly half of detected target steroids were lower than
80 percent. It is obvious that the treatment capacity of the activated sludge system (Plant A)
is superior to the oxidation ditch (Plant B) in the degradation of steroids in sewage treatment
systems. Androgens, estrogens and progestagens were mainly removed by sorption and
degradation, while the reduction of glucocorticoids was primarily due to degradation [12362].
Amphetamine-like psychoactive substances
Besides the common illicit drugs, such as cocaine, heroin, and marijuana, there is a growing
concern about the use of modern "designer drugs" that have emerged in large numbers over
the past few years. In this work, a sensitive and selective method for simultaneous
determination of 25 synthetic amphetamine-like psychoactive compounds, including
amphetamine, sympathomimetic substituted amphetamines, synthetic cathinones and
ketamine, in raw wastewater (RW), secondary effluent (SE) and river water was developed.
Samples were enriched by solid-phase extraction (SPE) on mixed-mode reversed-
phase/strong cation-exchange sorbent (Oasis MCX) and analysed by reversed-phase liquid
chromatography coupled to electrospray ionisation tandem mass spectrometry (LC-MS/MS).
The target compounds were separated on a Synergi Polar column and detected using
multiple reaction monitoring (MRM) in positive ionisation mode. Accurate quantification was
achieved by using several deuterated analogues as surrogate standards. Careful
optimisation and validation of the procedure resulted in a reliable determination of all target
analytes in low ng/L range for all matrices, which makes the method suitable for the
application in wastewater-based epidemiology. The method was applied for assessment of
selected compounds in municipal wastewater and river water from Croatia. It was shown that
most of the wastewater samples contained detectable levels of the well-known synthetic illicit
drugs, amphetamine and 3,4-methylenedioxy-methamphetamine (MDMA) (concentrations up
to 545 ng/L and 55 ng/L in RW, respectively), as well as ephedrine (up to 108 ng/L) and
pseudoephedrine (up to 698 ng/L), which are used as ingredients of popular over-the counter
cough and cold medications. Other target amphetamine-like psychoactive substances,
recently reported for their potential abuse, were detected only occasionally and in low
concentrations (<10 ng/L) [150098].
657
The concentrations of 17 drugs of abuse, including cocaine, several amphetamines, opioid
drugs, and 2 metabolites-benzoylecgonine, a metabolite of cocaine, and 2-ethylidene-1,5-
dimethyl-3,3-diphenylpyrolidine, a metabolite of methadone-were investigated in an urban
watershed that is heavily impacted by discharges of municipal wastewater. The artificial
sweetener sucralose was also monitored as a persistent tracer of contamination from
municipal wastewater. Monitoring was conducted in a municipal wastewater treatment plant
(WWTP) and at sites upstream and downstream of the WWTP discharge, as well as in a
drinking water treatment plant (DWTP) located 19 km downstream of the WWTP discharge
that withdraws raw water from the river. Drug concentrations were monitored with polar
organic chemical integrative samplers deployed for 2 wk in the river and in the WWTP and
DWTP. Several of the investigated compounds exhibited a decrease in concentration with
distance downstream from the wastewater discharge into the river, but there was little
attenuation of sucralose, cocaine, benzoylecgonine, morphine, acetylmorphine,
acetylcodeine, and oxycodone. Heroin and methadone were not detected at any sample
locations. Amphetamine, methamphetamine, 3,4-methylenedioxy-methamphetamine, and 2-
ethylidene-1,5-dimethyl-3,3-diphenylpyrolidine were not detected in the samples collected at
the drinking water intake. Many of the drugs of abuse were not removed effectively in the
DWTP, including cocaine, benzoylecgonine, methylenedioxyamphetamine, ephedrine, and
several prescription opioids, most probably because the DWTP was operating at or above its
rated treatment capacity. These data indicate that there can be transport of drugs of abuse
from wastewater sources into drinking water in urban watersheds [150088].
Ketamine and mephedrone
Wastewater analysis was applied in a four-year monitoring study to assess temporal and
spatial patterns of ketamine and mephedrone use in the general population in Italy.
Composite raw wastewater samples were collected from sewage treatment plants (STPs) in
17 cities. Target analytes were measured using a validated method based on solid phase
extraction and liquid chromatography coupled to tandem mass spectrometry analysis. Mass
loads were use to assess ketamine and mephedrone use and were normalized to the
population served by the plants. Ketamine was detected in wastewater in all except one
(Palermo) of the cities investigated, while mephedrone was detected only in Bologna and
Florence. Ketamine mass loads progressively increased from 2010 to 2013, and in Milan
rose from 1 to 1.5 g/day in 2008-2010 to 3.4-3.6 g/day in 2013-2014. Mass loads were higher
in north and central Italy than in the south, and in larger rather than small cities. Wastewater
analysis was suitable to provide objective and up-to-date information on the use of ketamine
in Italy, to identify ketamine spatial and temporal changes, and to confirm the low use of
mephedrone. These results can complement information from population surveys which
often provide only scant and incomplete figures for these substances [150089].
Drugs of abuse, cytostatic drugs and iodinated contrast media in tap water
One work analyses the presence of forty-eight emerging pollutants, including twenty-five
drugs of abuse and metabolites, seventeen cytostatic drugs and six iodinated contrast
media, in tap water from the Madrid Region. Analysis of the target compounds in the tap
water was performed by means of (on-line or off-line) solid-phase extraction followed by
analysis by liquid chromatography-tandem mass spectrometry. A preliminary human health
risk characterization was undertaken for each individual compound and for different groups
of compounds with a common mechanism of action found in tap water. The results of the
study showed the presence of eight out of the twenty-five drugs of abuse and metabolites
analysed, namely, the cocainics cocaine and benzoylecgonine, the amphetamine-type
658
stimulants ephedrine, 3,4-methylenedioxymethamphetamine and methamphetamine, the
opioid methadone and its metabolite 2-ethylene-1,5-dimethyl-3,3-diphenylpyrrolidine and,
finally caffeine at concentrations ranging from 0.11 to 502 ng/L. Four out of the six analysed
iodinated contrast media, namely, diatrizoate, iohexol, iomeprol and iopromide, were
detected in at least one sample, with concentration values varying between 0.4 and 5 ng/L.
Cytostatic compounds were not detected in any sample. Caffeine was the substance
showing the highest concentrations, up to 502 ng/L, mainly in the drinking water sampling
point located in Madrid city. Among the other drugs of abuse, the most abundant compounds
were cocaine and benzoylecgonine, detected at concentrations ranging from 0.11 to 86 ng/L
and from 0.11 to 53 ng/L, respectively. Regarding iodinated contrast media, iohexol was the
most ubiquitous and abundant compound, with a frequency of detection of 100 percent and
concentrations from 0.5 to 5.0 ng/L in basically the same range in all sampling points. Taking
into account the results and types of treatment applied, ozonisation plus granular activated
carbon filtration appears to be efficient in the removal of cocaine and benzoylecgonine. For
the amphetamine-type stimulants, opioids and caffeine, ozonisation plus granular activated
carbon filtration and ultrafiltration plus reverse osmosis showed higher removal efficiency
than sand filtration. The human health risk characterization performed indicates that the
lifetime consumption of the tap waters analysed has associated a negligible human health
concern [150100].
France
The occurrence of 20 human pharmaceutical compounds and metabolites from 10

representative therapeutic classes was analysed from resource and drinking water in two
catchment basins located in north-west France. Ninety-eight samples were analysed from 63
stations (surface water and drinking water produced from surface water). Of the 20 human
pharmaceutical compounds selected, 16 were quantified in both the surface water and
drinking water, with 22 percent of the values above the limit of quantification for surface
water and 14 % for drinking water). Psychostimulants, non-steroidal anti-inflammatory drugs,
iodinated contrast media and anxiolytic drugs were the main therapeutic classes of human
pharmaceutical compounds detected in the surface water and drinking water. The results for
surface water were close to results from previous studies in spite of differences in
prescription rates of human pharmaceutical compounds in different countries. The removal
rate of human pharmaceutical compounds at 11 water treatment units was also determined.
Only caffeine proved to be resistant to drinking water treatment processes (with a minimum
rate of 5 %). Other human pharmaceutical compounds seemed to be removed more
efficiently (average elimination rate of over 50 %) by adsorption onto activated carbon and
oxidation/disinfection with ozone or chlorine (not taking account of the disinfection by-
products). These results add to the increasing evidence of the occurrence of human
pharmaceutical compounds in drinking water that may represent a threat to human beings
exposed to a cocktail of human pharmaceutical compounds and related metabolites and by-
products in drinking water [11559].
Italy
Wastewater analysis is a new approach developed to estimate illicit drug (ID) consumption in
large communities, such as a city. It was tested the ability of this approach to detect
differences in consumption in different districts of a city. Consumption of cocaine, heroin,
tetrahydrocannabinol (THC) (cannabis active principle), amphetamine, methamphetamine
and ecstasy was estimated by analysis of selected drug excretion residues in composite 24 h
samples of untreated urban wastewater by liquid chromatography-tandem mass
spectrometry. Samples were collected from the inlet of the three main Milan wastewater
659
treatment plants (WWTPs), each serving a district of the city (west, center and east). In each
WWTP, samples were taken daily for seven consecutive days in November 2010 and March
2011. It was observed significant differences of ID consumption (expressed as mg/day/1000
inhabitants) among districts: consumption of some ID was significantly higher in the eastern
district (THC, cocaine and for heroin by one-way analysis of variance), while consumption of
methamphetamine and amphetamine was higher in the central area. Overall, from 2010 to
2011, ID consumption decreased in all the districts, in line with a recent population survey
showing decreases from 25 to 55 percent in the annual prevalence of ID users in Italy. This
approach may help to detect ID consumption in different districts of a city and may be useful
for planning interventions aimed at specific city areas and substances [14729].
Wastewater analysis can provide estimates of illicit drug consumption in local communities. It
wasused repeated raw wastewater analysis in urban wastewater treatment plants to estimate
loads of cocaine, heroin, methamphetamine, and cannabis consumed daily by the
inhabitants of two cities in Northern Italy, Milan and Como, from 2005 to 2009. Daily cocaine
loads did not change in Milan from 2005 to 2008 but fell 45 percent in 2009 and there was a
similar drop in Como. Heroin also fell from 2008 to 2009 in Milan (66 %) and Como (26 %).
However, methamphetamine, which had risen in Milan from 2005 to 2008, rose further in
2009 and cannabis, which was falling from 2005 to March 2009, rose again in September
2009. The results suggest a trend toward a decrease in consumption of costly illicit drug,
such as cocaine and heroin. This might be due to a reduction in the number of consumers
and/or to a change in their behaviour since there was also an increase in the consumption of
less expensive illicit drug. This itselfe might reflect a drop in consumers' money supply,
caused by the economic crisis. Wastewater analysis was useful to estimate illicit drug
consumption levels in local communities in real time and promptly identify changes in trends
[11560].
Czech Republic
It was reported a monitoring study analysing wastewater and associated suspended

particulate matter (SPM) to determine the concentration of drugs of abuse and metabolites in
wastewater influent. The monitoring of SPM is crucial for target analytes because, depending
on their physico-chemical properties, they may partition to particulates; thus, analysis of
wastewater only will result in under-reporting of the concentration of target analytes in the
sample. A daily one week monitoring study was carried out at a WWTP serving one of the
largest cities in the Czech Republic; representing the first comprehensive application of the
sewage epidemiology approach in the Czech Republic. In total, 60 analytes were targeted in
the monitoring programme including stimulants, opioid and morphine derivatives,
benzodiazepines, antidepressants, dissociative anaesthetics, drug precursors and their
metabolites. Analysis of SPM determined that significant proportions of some compounds
were present on the solids. For example, 21-50 percent of the total concentration of EDDP
(2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine) in the sample was determined on SPM
and 11-20 percent of methadone. The highest proportion on SPM was determined for
fluoxetine in the range 68-80 percent, norfluoxetine 47-62 percent and amitriptyline 22-51
percent. In contrast, some compounds presented very little partitioning to SPM. Less than 5
percent was determined partitioned to SPM over the week period for analytes including
cocaine, benzoylecgonine, cocaethylene, amphetamine, methamphetamine, MDMA (3,4-
methylenedioxymethamphetamine), codeine, dihydrocodeine, tramadol, nortramadol,
oxazepam and ephedrine. Determined concentrations in wastewater influent were
subsequently utilised in the sewage epidemiology approach to estimate drug consumption, in
the community from which the wastewater was derived. This back-calculation was updated
for the first time to include the concentration of analytes present on SPM. The consumption
of methamphetamine and MDMA was determined to be especially high in the studied
660
community in relation to other European countries, while cocaine and methadone
consumption was relatively low. This manuscript shows that in order to apply the sewage
epidemiology approach, SPM analysis is required for some compounds; whereas for others
the partitioning is small and one may regard this as negligible [12090].
Influence on fish
17beta-Trenbolone
Despite the pivotal role sexual selection plays in population dynamics and broader
evolutionary processes, the impact of chemical pollution on female mate choice is poorly
understood. One group of chemical contaminants with the potential to disrupt the
mechanisms of female mate choice is endocrine disrupting chemicals (EDCs); a broad class
of environmental pollutants that can interfere with the endocrinology of organisms at
extremely low concentrations. Recent research has revealed that estrogenic EDCs can affect
female mate choice in fish, but the impact of androgenic EDC exposure is yet to be studied.
To address this, we investigated the effects of an environmentally relevant concentration of
trenbolone - an androgenic steroid used as a growth promoter in the cattle industry - on
female mate choice in wild-caught guppies (Poecilia reticulata). It was exposed male and
female guppies to 17beta-trenbolone for 21 days (measured concentration 4 ng/L) via a flow-
through system, and found that trenbolone-exposed female guppies spent less time
associating with males, and were less choosy, compared to unexposed females. In contrast,
trenbolone had no impact on male reproductive behavior or morphology. This is the first
study to show that androgenic EDC exposure can disrupt female mate choice, highlighting
the need for studies to investigate the behavioral impacts of environmental contaminants on
both sexes [150099].
Chemical pollution is a pervasive and insidious agent of environmental change. One class of
chemical pollutant threatening ecosystems globally is the endocrine disrupting chemicals
(EDCs). The capacity of EDCs to disrupt development and reproduction is well established,
but their effects on behaviour have received far less attention. Here, we investigate the
impact of a widespread androgenic EDC on reproductive behaviour in the guppy, Poecilia
reticulata. It was found that short-term exposure of male guppies to an environmentally
relevant concentration of 17beta-trenbolone-a common environmental pollutant associated
with livestock production-influenced the amount of male courtship and forced copulatory
behaviour (sneaking) performed toward females, as well as the receptivity of females toward
exposed males. Exposure to 17beta-trenbolone was also associated with greater male mass.
However, no effect of female exposure to 17beta-trenbolone was detected on female
reproductive behaviour, indicating sex-specific vulnerability at this dosage. The study is the
first to show altered male reproductive behaviour following exposure to an environmentally
realistic concentration of 17beta-trenbolone, demonstrating the possibility of widespread
disruption of mating systems of aquatic organisms by common agricultural contaminants
[150090].
Other anabolic steroids

Due to potential use in aquacultured fish products, the Canadian Food Inspection Agency
has identified residue testing for steroids as a priority. These compounds are used in
aquaculture primarily to direct sexual differentiation with both androgens and estrogens
applied depending on the desired outcome. Published research is lacking with respect to
steroid residue testing in fish; however, recent studies in other matrixes provided transferable
cleanup techniques. A simple, rapid, and sensitive method was developed and validated for
use in monitoring aquacultured fish products for the presence of methyltestosterone,
nandrolone, epi-nandrolone, boldenone, and epi-boldenone residues. The developed method
consists of solvent extraction followed by cleanup using hexane and dual cartridge SPE with
661
analysis by ultra-HPLC-MS/MS. The method is capable of detecting and confirming steroid
residue levels ranging from 0.05 to 25 ng/g in salmon and tilapia, depending on the analyte.
Recoveries ranged from 88 to 130 percent for the analytes. Instrument repeatability was less
than 13 percent for all compounds, while intermediate precision ranged from 5 to 25 percent
RSD. HorRat values were within acceptable ranges [150091].
Influence on frog
Trenbolone, as a growth promoter in animal agriculture, has become an environmental

androgen in surface water. Here, we aimed to reveal the effects of 17beta-trenbolone on
survival, growth, and gonadal differentiation in the frog Pelophylax nigromaculatus, which is
widespread in East Asia and undergoing population decline. P. nigromaculatus tadpoles
were exposed to 17beta-trenbolone (0.1, 1, 10 microg/L) from Gosner stage 24/25 to
complete metamorphosis. It was found that 17beta-trenbolone resulted in significantly high
mortality in a concentration-dependent manner, with a decrease in body weight in the high
concentration group compared with the solvent control. Based on gross gonadal morphology,
no females were observed, instead of about 15 percent ambiguous sexes and 85 percent
males, in all 17beta-trenbolone treatment groups. Like normal testes, the gonads with sex-
ambiguous morphology exhibited testicular histology, showing that the sex-ambiguous
gonads were incomplete ovary-to-testis reversals (IOTTRs) with certain ovarian
morphological features. In the IOTTRs, the transcriptional levels of ovary-biased genes
decreased drastically relative to normal ovaries, and even declined to the levels in normal
testes. These observations confirmed that all test concentrations of 17beta-trenbolone
resulted in 100 percent sex reversal, although some sex-reversed testes retained some
ovarian characteristics at the morphological level. To our knowledge, this is the first report
strongly demonstrating that trenbolone can cause female-to-male reversal in amphibians.
Given that the lowest concentration tested is environmentally relevant, our study highlights
the risks of trenbolone and other environmental androgens for P. nigromaculatus and other
amphibians, in particular the species with high sensitivity of gonadal differentiation to
androgenic chemicals [150093].
Stability of illicit drugs in sewers and wastewater samples
Wastewater-based epidemiology (WBE) applies advanced analytical methods to quantify

drug residues in wastewater with the aim to estimate illicit drug use at the population level.
Transformation processes during transport in sewers (chemical and biological reactors) and
storage of wastewater samples before analysis are expected to change concentrations of
different drugs to varying degrees. Ignoring transformation for drugs with low to medium
stability will lead to an unknown degree of systematic under- or overestimation of drug use,
which should be avoided. This review aims to summarize the current knowledge related to
the stability of commonly investigated drugs and, furthermore, suggest a more effective
approach to future experiments. From over 100 WBE studies, around 50 mentioned the
importance of stability and 24 included tests in wastewater. Most focused on in-sample
stability (i.e. sample preparation, preservation and storage) and some extrapolated to in-
sewer stability (i.e. during transport in real sewers). While consistent results were reported
for rather stable compounds (e.g. MDMA and methamphetamine), a varying range of stability
under different or similar conditions was observed for other compounds (e.g., cocaine,
amphetamine and morphine). Wastewater composition can vary considerably over time, and
different conditions prevail in different sewer systems. In summary, this indicates that more
systematic studies are needed to: i) cover the range of possible conditions in sewers and ii)
compare results more objectively. To facilitate the latter, we propose a set of parameters that
should be reported for in-sewer stability experiments. Finally, a best practice of sample
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collection, preservation, and preparation before analysis is suggested in order to minimize
transformation during these steps [150094].
Androgenic and estrogenic activity in water bodies receiving cattle feedlot effluent
Studies reveal that surface waters worldwide are contaminated with hormonally active
agents, many released from sewage treatment plants. Another potential source of aquatic
hormonal contamination is livestock feedlot effluent. In this study, we assessed whether
feedlot effluent contaminates watercourses by measuring a) total androgenic
[methyltrienolone (R1881) equivalents] and estrogenic (17beta-estradiol equivalents) activity
using the A-SCREEN and E-SCREEN bioassays and b) concentrations of anabolic agents
via gas chromatography-mass spectroscopy and enzyme-based immunoassays. Water
samples were collected over 3 years from up to six sites [all confluent with the Elkhorn River,
Nebraska, USA: a feedlot retention pond (site 1), a site downstream from site 1 (site 2), a
stream with intermediate livestock impact (site 3), and three sites with no observable
livestock impact (sites 4-6)] and two sources of tap water. In 1999, samples from site 1
contained 9.6 pM R1881 equivalents and 1.7 pM 17beta-estradiol equivalents. Site 2
samples had estrogen levels similar to those in site 1 samples but lower androgen levels (3.8
pM R1881 equivalents). Androgen levels in site 3 samples were similar to those in site 2
samples, whereas estrogen levels decreased to 0.7 pM 17beta-estradiol equivalents. At site
6, androgen levels were approximately half those found at site 3, and estrogen levels were
comparable with those at site 3. Sampling in later years was limited to fewer sites because of
drought and lack of permission to access one site. Instrumental analysis revealed estrone
but no significant levels of resorcylic acid lactones or trenbolone metabolites. Tap water was
devoid of hormonal activity. It was concluded that feedlot effluents contain sufficient levels of
hormonally active agents to warrant further investigation of possible effects on aquatic
ecosystem health [14036].
Rests in other parts of nature
Land application of manure may contribute endocrine disrupting compounds (EDCs) such as
steroid hormones to the environment. Little attention has been paid to the potential for
degradation of steroid hormones by manure-borne bacteria and their degradation kinetics
and pathways. In a laboratory study, the potential for biodegradation of testosterone, 17beta-
estradiol (E2) and progesterone by swine (Sus scrofa) manure-borne bacteria was examined.
In addition, the impact of temperature, pH (6, 7, and 7.5), glucose amendments (0, 3, and 22
mmol L(-1)), and presence of oxygen on testosterone degradation kinetics was determined.
Testosterone, 17beta-estradiol and progesterone were biodegraded within 25 h of reaction
initiation under aerobic conditions. The degradation of testosterone followed pseudo first-
order and zero-order reaction kinetics under aerobic and anaerobic conditions, respectively,
in tryptic soy broth (TSB) pre-enriched systems. The half-life (t½) for the degradation of
testosterone under anaerobic conditions was six times longer than aerobic conditions.
Testosterone degradation was found to significantly increase (-17 %) when incubated at 37
degrees C versus 22 degrees C. The impact of pH (t½ ranged from 4.4-4.9 h) and glucose
amendments (t½ ranged from 4.6-5.1 h) on the testosterone degradation rate were found to
be small. Testosterone was transformed to dehydrotestosterone (DHT) (major degradation
product), androstenedione (AD), and androstadienedione (ADD) under aerobic conditions as
revealed by liquid chromatography-time-of-flight mass spectrometry (LC/TOF-MS). These
results indicate that testosterone is rapidly degraded by manure-borne bacteria under a wide
range of environmentally relevant conditions. However, the formed degradation products are
still of potential concern due to their endocrine disrupting potential [10343].
663
Anonymous pooled urine
Analysis of anonymous pooled urine samples from street urinals has been used to
demonstrate time-trends in the detection of classical recreational drugs and novel
psychoactive substances (NPS). One study aimed to expand this to undertake a
geographical trend analysis of classical recreational drugs/NPS across UK. Samples of
anonymous pooled urine were collected from street urinals that had been in place for one
night in April 2014 in nine cities across the UK. Collected samples were then analysed for the
presence of recreational drugs, NPS anabolic steroids using high-performance liquid
chromatography coupled to high-resolution accurate mass full-scan mass spectrometry and
gas chromatography coupled to electron impact ionization mass spectrometry operating in
selected ion monitoring and full-scan modes. Ten classical recreational drugs, nine NPS and
four anabolic steroids were detected across the nine cities; the range of detection was from 1
in Leeds to 14 in London. The most common classical drugs were cocaine (9 cities) and 3,4-
methylenedioxy-methamphetamine (8 cities); the most common NPS was 4-
methylmethcathinone (5 cities). In addition there was variation in the detection of NPS, with
methylhexaneamine detected only in Bristol and London, piperazines (3-
trifluoromethylphenylpiperazine and 1-benzylpiperazine) and pentedrone only detected in
Birmingham and the cathinone methylone only detected in London. There is variability in the
detection of classical recreational drugs, NPS and anabolic steroids across UK, likely
reflecting variation in their use. This technique can be used to supplement drug use surveys
to determine geographical and time trends in the use of these substances. This is important
to ensure appropriate targeting of drug-related interventions [150097].
Analysis of urine samples collected across a city centre, for the detection of novel
psychoactive substances (NPS) was done in a cross-sectional study of anonymized urine
samples used for the analysis of classical recreational drugs, NPS and metabolites. Pooled
urine samples collected from portable stand-alone four-person urinals across a city centre
were analysed using full-scan accurate-mass high-resolution liquid chromatography coupled
to tandem mass spectrometry. Data were processed against compound databases
containing >1700 drug compounds and metabolites. Seven established recreational drugs
(3,4-methylenedioxyamphetamine, cocaine, cannabis, ketamine, 3,4-methylenedioxy-N-
methylamphetamine, methamphetamine and amphetamine) and six potential NPS
[hordenine (all 12 urinals), cathine (11), methylhexaneamine (9), 4-methylmethcathinone (6),
methiopropamine and metabolites (2) and methoxetamine and metabolites (1)] were
detected. Methylhexaneamine, methiopropamine and hordenine are currently uncontrolled in
the UK, whereas methoxetamine is currently subject to a Temporary Class Drug Order.
Metabolites of the anabolic steroid nandrolone were found in two urinals and trenbolone
metabolites and clenbuterol in one urinal. Thus, analysis of pooled urine samples collected
anonymously from stand-alone urinals in a large inner city can detect the use of recreational
drugs, NPS and anabolic steroids. Metabolite detection indicates actual drug use,
metabolism and elimination rather than simply discarded drugs in the urinals. This technique
by confirming the actual drug(s) used has the potential to be additive to currently used
datasets/key indicators providing more robust information for healthcare authorities,
legislative and law enforcement on the drugs actually being used [12092].
A cross-sectional study of anonymized urine samples collected across a city centre was used
for the analysis of classical recreational drugs, novel psychoactive substances (NPS) and
metabolites. Pooled urine samples collected from portable stand-alone four-person urinals
across a city centre were analysed using full-scan accurate-mass high-resolution liquid
chromatography coupled to tandem mass spectrometry. Data were processed against
664
compound databases containing >1700 drug compounds and metabolites. Seven
established recreational drugs (3,4-methylenedioxyamphetamine, cocaine, cannabis,
ketamine, 3,4-methylenedioxy-N-methylamphetamine, methamphetamine and amphetamine)
and six potential NPS [hordenine (all 12 urinals), cathine (11), methylhexaneamine (9), 4-
methylmethcathinone (6), methiopropamine and metabolites (2) and methoxetamine and
metabolites (1)] were detected. Metabolites of the anabolic steroid nandrolone were found in
two urinals and trenbolone metabolites and clenbuterol in one urinal. It was concluded that
analysis of pooled urine samples collected anonymously from stand-alone urinals in a large
inner city can detect the use of recreational drugs, NPS and anabolic steroids. Metabolite
detection indicates actual drug use, metabolism and elimination rather than simply discarded
drugs in the urinals. This technique by confirming the actual drug(s) used has the potential to
be additive to currently used datasets/key indicators providing more robust information for
healthcare authorities, legislative and law enforcement on the drugs actually being used
[12093].
Beef palatability
The use of anabolic implants has a long-standing place in the cattle feeding industry, due to
their positive impact on growth performance and subsequent profitability. However, implants
can have adverse effects on carcass quality, shear force, and eating quality depending on
the dose and frequency, or what some refer to as the aggressiveness of the implant regimen
administered. Within the past decade, a new class of growth promotants, known as beta-
adrenergic agonists (beta-AA), has emerged in the beef feeding industry in the United
States. Currently, 2 have gained U.S. Food and Drug Administration approval for use in beef
finishing diets to improve performance and carcass yields. Much like anabolic implants, these
repartitioning agents can have negative effects on Warner-Bratzler shear force (WBSF), but
the differences do not necessarily translate directly to consumer responses for palatability
and acceptance in some instances, especially when tenderness is managed through proper
postmortem aging. As researchers continued to investigate the mechanisms responsible for
the impact of beta-AA, inevitably this led to consideration of the interaction between beta-AA
and anabolic implants. Early work combining zilpaterol hydrochloride (ZH) with anabolic
implants improved performance, carcass yield, and meat yield with additive negative effects
on WBSF. Similar results were produced when pairing ZH with anabolic steroids equipped
with various release patterns. As with any tool, the key to success is proper management.
Certain cattle populations may be better suited to receive growth promotants such as
implants and beta-AA, and postmortem management of subprimal cuts becomes vital when
producers take more aggressive approaches to improve performance and yield. The
objective of one review is to overview research findings related to the impact of growth
promotant technologies on beef palatability, focusing specifically on the role of implants and
beta-AA on carcass quality, beef tenderness, and consumer responses for meat palatability
[13115].
In meat
Within the scope of the European Community member states' residue monitoring plan, illicit
administration of anabolic steroids is monitored at slaughterhouse level as well as on living
animals. At farm level, urine is one of the target matrices to detect possible abuse of anabolic
steroid growth promoters. Optimisation of the routinely applied analysis method resulted in a
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procedure for which high performance liquid chromatographic (HPLC) fractionation prior to
GC-MS(n) analysis was no longer required. Analytical results could be obtained within 1 day
and only 5 mL urine was needed to carry out the screening procedure. Using the downscaled
methodology, all validation criteria described in the European Commission document
2002/657/EC could be fulfilled, and the minimum required performance limits (MRPLs)
established for anabolic steroids in urine, could be achieved. A higher GC-MS technique's
specificity was achieved by detecting the steroids using GC-MS3. Nevertheless, it was
decided to screen routinely sampled urine with GC-MS2 whereas GC-MS3 was applied to
confirm the presence of anabolic steroid residues in suspected sample extracts [06329].
A gas chromatography/mass spectrometry (GC/MS) method was developed for the

determination of multi-residues of steroid anabolic hormones epitestosterone (ETS),
testosterone 17-propionate (PTS), nandrolone (17beta-NT), 17alpha-methyltestosterone
(MTS), 17beta-estradiol (17beta-ES), estriol ( EST), 17alpha-ethinylestradiol (EES), estrone
(ESN) and 17beta-estradiol 3-benzoate (BES) in the muscle tissues of various animal
species. Homogenized tissue samples were enzymatically digested in acetate buffer (pH
5.0). Consequently, methanol was added and the mixtures were extracted under
ultrasonication incubation. Clean-up was carried out for at least two times with methyl tert-
butyl ether (MTBE) liquid-liquid partitioning followed by a reversed-phase solid phase
extraction (SPE) cartridge purification. The eluate with methanol was evaporated to dryness
by N2 at 40 degrees C and derivatization was achieved with N-methyl-N-( trimethylsilyl)
trifluoroacetamide/iodotrimethylsilane/dithioerythritol (MSTFA-TMIS-DTE) at 60 degrees C
for 45 min. The reaction mixture was injected into a gas chromatograph with a DB-1 capillary
column coupled with a mass spectrometer. The samples were tested by different selected
ion monitoring modes with electron impact (EI) source for the androgens and estrogens. The
limits of quantitation (LOQ) for the above 9 hormones were in the range of 1.0 - 2.0
microg/kg. At the 2.0 microg/kg LOQ spiked level, the mean recoveries were within 63-81
percent, and the relative standard deviations were within 13-27 percent. The real sample
tests showed this method can be used for the sensitive and accurate determination of multi-
steroid anabolic hormones residues in biological muscle samples [06330].
Estradiol, testosterone, progesterone, zeranol and diethylstilbestrol including estradiol

metabolites were determined simultaneously in meat. Extraction of growth hormones was
carried out by ultasonication using a methanol-water mixture. The growth hormones in the
meat extract can be effectively separated from lipids by freezing-lipid filtration, followed by
C8-solid phase extraction (SPE). During freezing-lipid filtration, about 90 percent of lipids are
removed without any significant loss of growth hormones. For further clean-up, silica- and
aminopropyl-SPE were used. To enhance detection sensitivity, the growth hormones are
derivatized with trimethylsilyl reagents. Quantitation using isotope-labelled internal standards
was performed by gas chromatography-mass spectrometry in the selected ion monitoring
mode. The method detection limits were 0.1-0.4 microg/kg for all growth hormones. Overall
recoveries of synthetic and natural growth hormones were 68-106 percent with coefficients of
variation of 5-16 percent for the complete procedure [05028].
Proteomics
The use of beta-agonists, sexual steroids, and corticosteroids as growth-promoting agents
(GPAs) in veal calves is forbidden in the European Union (EU) and subjected to restrictions
in the US because it may be potentially noxious for both treated animals and the consumer.
Although official controls performed in the EU have revealed a limited number of positive
samples, the analysis of seized preparations indicate that the use of illegal GPAs is far from
being abandoned. The presence of these compounds in matrixes of biological origin often
goes unnoticed because of the use of very low dosages and/or of molecules of unknown
chemical structure. It is therefore necessary to develop screening methods based on the
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biological effects of these substances that allow the simultaneous screening of many
components, as proteome analysis. When hepatic cytosols and microsomes from calves
treated with a combination of GPAs were analyzed by 2-DE, it was found changes in the
expression of two proteins, which we identified as adenosine kinase and reticulocalbin. The
aim was not to speculate about molecular mechanisms, but to show the ability of the
proteomic approach to find biomarkers of illicit treatments and to use it as a basis to develop
large-scale screening methods [06331].
In faeces
Feces are a possible medium to be used for horse doping control. Efficient methods for
detecting drugs in feces collected from various animals are routinely applied in institutes of
food safety in Belgium. It has already tested whether they are applicable to horse feces. In
this report, accelerated solvent extraction (ASE), an efficient method for extracting
compounds from solid material, has been tested. ASE has been used to replace the diethyl
ether liquid-liquid extraction step present in the method initially set up. This technique has
been optimized for detecting several non-steroidal anti-inflammatory drugs (NSAIDs) in horse
feces. Extraction recovery and limit of detection have been determined for several NSAIDs,
such as meclofenamic acid, flunixin, vedaprofen, celecoxib, carprofen, diclofenac, and
ketoprofen. The method has been successfully applied to meclofenamic acid, flunixin, and
phenylbutazone post-administration feces samples, and the main metabolites identified in
urine were also detected in feces. In the case of meclofenamic acid, the detection profile in
feces presented in this report is in accordance with our previous finding in feces obtained
with the original method. The use of ASE decreases the time necessary for sample
preparation. This method is applicable on a large scale, which is useful for horse doping
control [06332].
A method had been developed for determination of residues of 10 anabolic steroids (ASs) in
animal muscle tissues by liquid chromatography tandem mass spectrometry (LC/MS/MS).
After enzymolysis, the sample was extracted with tert-butyl methyl ether, cleaned up through
reverse solid-phase extraction and further determined by LC/MS/MS under multiple reaction
monitoring (MRM) mode. The limits of detection (LOD) of LC/MS/MS method used for testing
epitestosterone (ETS), nandrolone (17 beta-NT), 17 alpha-methyl-testosterone (MTS),
testosterone 17-propionate (PTS), medroxyprogesterone (MED), progesterone (PG), estrone
(ESN), 17 beta-estradiol (17 beta-ES), 17alpha-ethynylestradiol (EES) and estriol (EST) in
animal muscle ranged from 0.06 to 0.22 microg/kg, and the limits of quantification (LOQ)
were from 0.12 to 0.54 microg/kg. Experiments on spiked samples of pork, beef, chicken and
fish showed that at addition level of 1.0 microg/kg, the average recoveries of the ASs ranged
from 64 to 77 percent, and coefficients of variation from 7 to 20 percent, while at addition
level of 2.0 microg/kg, the average recoveries ranged from 70 to 89 percent, and coefficient
of variation from 7 to 19 percent [06241].
High- and low-resolution mass spectrometry
Within the European Union the use of anabolic steroids for promoting growth and improving
meat-to-fat ratio in food-producing animals has been banned since 1988. For the
unequivocal identification of hormone residues in a complex matrix such as meat we have
developed a rapid, specific and sensitive liquid chromatography/tandem mass spectrometry
(LC/MS/MS) method, in combination with a simple extraction procedure based on the matrix
solid-phase dispersion (MSPD). The performances of a triple quadrupole (QqQ) and a
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quadrupole/time-of-flight (QqTOF) were compared: the QqQ mass spectrometer was found
to be more sensitive for almost all studied analytes, but the selectivity was superior using the
QqTOF system; the full-scan spectra (acquired without losing sensitivity), mass accuracy and
resolution of the hybrid instrument enabled a more probatory analyte identification than that
obtained selecting two multiple-reaction monitoring (MRM) transitions with a QqQ. Average
recoveries ranged from 80 to 100 percent, and the detection capabilities (CCbetas) were less
than 1.06 ppb with the QqQ instrument and less than 5.20 ppb with the QqTOF instrument
for the bovine meat, which proved to be the most complex matrix [06242].
Laboratory testing of fluids
An analytical method for determining traditional and emerging drugs of abuse in particulate
matter, sewage sludge and sediment has been developed and validated. A total of 41 drugs
of abuse and metabolites including cocainics, tryptamines, amphetamines,
arylcyclohexylamines, cathinones, morphine derivatives, pyrrolidifenones derivatives,
entactogens, piperazines and other psychostimulants were selected. Samples were
ultrasound extracted with McIlvaine buffer and methanol, and the extracts were cleaned up
by solid phase extraction (SPE) using Strata-X cartridges. Drugs were eluted using methanol
and methanol-dichloromethane and determined by liquid chromatography tandem mass
spectrometry. The optimum solid-liquid extraction (SLE) conditions were: weight 1g of
sample and ultrasound assisted extraction (UAE) with 10mL of methanol-McIlvain buffer (1:1,
v/v, pH 4.5) for 10min. Recoveries for all compounds were ≥50 percent in the three matrices
with the exception of ephedrine (EPHE), 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine
(EDDP), ecgonine methyester (ECME), heroin (HER), 3,4-methylendioxyamphetamine
(MDA) and 4-acetoxy N,N'-dimethyltryptamine (4-AcO-DIPT) and methadone (MET). Data
acquisition was done by selective reaction monitoring (SRM), and the two most abundant
product ions were used for confirmation. Limits of detection were lower than 1.32 ng/g dry
weight (d.w.) and limits of quantification were between 0.12 and 3.96 ng/g (d.w.). The
method was applied to the analysis of particulate matter, where cocaine (COC),
benzoylecgonine (BECG), ecgoninemethylester (ECME), cocaethylene (COCET),
methadone (MET) and codeine (COD) were mostly detected. In the case of dehydrated
sludge, opioids are at higher concentration than cocainics and some emerging drugs such as
4-methoxyamphetamine (PMA), ketamine (KET) and bufotenine (BUF) were detected. In
sediment COC, 4-methoxyphencyclidine (4-MeO-PCP), MET and BECG were most relevant
compounds [150096].
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PREVENTION OF DOPING
The potential for adverse effects and the prevailing attitudes towards “unethical”
performance-enhancing substances have prompted various efforts to prevent anabolic
steroids use in adolescent athletes over the past 25 years. Principal among these has been a
punitive approach consisting of in- and out-of-competition doping controls and suspension or
expulsion for refusing testing or failed tests. Although these actions may have some impact
on elite-level adolescents athletes, they are of questionable utility for those at lower levels
where testing is not conducted and of no use for young AAS users not involved in organised
sport. Prevention programs founded on cognitive-behavioural or social environment
paradigms that do not focus on a fear-based philosophy may be more effective. Based on
their findings that a significant proportion of steroid users “have access to models that have
used or use anabolic steroids.” It has also been recommended interventions that focus on
“environments and groups where the general level of drug use is high.” Furthermore, as
these adolescents know users, the claims by authorities of the significant adverse effects of
steroid use “may be perceived as unsubstantiated.” Therefore, a different model than “fear
based” is needed to deter use among children and adolescents, whether involved in
organised sports or not. This view is supported by early prevention efforts when it was
compared a “balanced education program (potential risks and benefits)” to a “risks-only
(negative or scare tactics) program” of AAS use and found that while the balanced approach
resulted in significant improvements in the athletes’ attitudes to adverse AAS effects, there
was no change in the fear-based group [09023].
Anti-doping activities in sport have shifted from secondary prevention (intervening after
athletes have used) to educational strategies focused on primary prevention through
promoting abstinence. There is no empirical evidence to guide targeting of anti-doping
education initiatives. In one paper, a heuristic to guide education initiatives was derived by
re-analysing a series of interviews (n=20) with athletes, coaches, sports managers,
physiotherapists and sports nutritionists. The findings indicate primary prevention of doping
may be enhanced by timing it around periods of career instability where athlete vulnerability
to doping may increase as a function of winning or losing sponsorship. Sponsorship is
broadly defined as financial (e.g. salary stipend) and non-financial support (e.g. training
facilities). This provides a basis for targeting education interventions to promote abstinence.
Two options are offered to mitigate the need to time prevention activity around career
instability by lessening the effect of sponsorship on athlete doping. The first is liberalising
access to legitimate performance enhancing technologies (e.g. training techniques or
nutritional supplements). The second is to delay access to financial sponsorship (beyond
living expenses) until retirement, with monetary gains (e.g. prize money) deposited into an
account where penalties are debited if the athlete is caught doping [10304].
Anti-doping activities in sport have shifted from secondary prevention (intervening after
athletes have used) to educational strategies focused on primary prevention through
promoting abstinence. There is no empirical evidence to guide targeting of anti-doping
education initiatives. In this paper, a heuristic to guide education initiatives was derived by re-
analysing a series of interviews (n=20) with athletes, coaches, sports managers,
physiotherapists and sports nutritionists. The findings indicate primary prevention of doping
may be enhanced by timing it around periods of career instability where athlete vulnerability
to doping may increase as a function of winning or losing sponsorship. Sponsorship is
broadly defined as financial (e.g. salary stipend) and non-financial support (e.g. training
facilities). This provides a basis for targeting education interventions to promote abstinence.
Two options are offered to mitigate the need to time prevention activity around career
instability by lessening the effect of sponsorship on athlete doping. The first is liberalising
669
access to legitimate performance enhancing technologies (e.g. training techniques or
nutritional supplements). The second is to delay access to financial sponsorship (beyond
living expenses) until retirement, with monetary gains (e.g. prize money) deposited into an
account where penalties are debited if the athlete is caught doping [11025].
One study examined the extent to which the trajectory of participation in sports, athletics or
exercising (PSAE) covaried with substance use in early adulthood controlling for team sports
participation using parallel process latent growth curve modeling. Analysis of data collected
from a series of panel studies using a cohort-sequential design. Specifically, the analyses
used longitudinal data from 11 741 individuals from the graduating classes of 1986-2001, first
surveyed as seniors in American high schools. Up to four additional follow-up surveys were
administered to age 26 years. Data were collected using in-school and mailed self-
administered questionnaires. Level of PSAE, past-30-day alcohol, cigarette and marijuana
use frequency and any past-30-day use of illicit drugs other than marijuana (IOTM) were the
main processes of interest. Self-reported race/ethnicity, college status at age 19/20 years,
parental education, gender and team sports participation during high school were included as
covariates. Results indicate that higher initial levels of PSAE related to lower initial substance
use prevalence rates other than alcohol, and lower initial prevalence rates of substance use
then corresponded with lower substance use rates throughout early adulthood. Further, as
individuals increased PSAE levels throughout early adulthood, the frequency of their use of
cigarettes, marijuana and IOTM correspondingly decreased. It was concluded that increased
participation in sports, athletics or exercising (PSAE) is related to significantly lower
substance use frequency at modal age 18 and through significantly and negatively correlated
growth trajectories through early adulthood. Encouraging PSAE among adolescents and
early adults may relate to lower substance use levels throughout early adulthood [11026].
The US National Institute on Drug Abuse has called for increased research into the use of
physical activity in substance abuse prevention, specifically research into physical activity
type and context. One paper examines the relationships between secondary school student
substance use and exercise in general and school athletic team participation, and examines
such relationships over time. Nationally representative cross-sectional samples of 8th-, 10th-,
and 12th-grade students were surveyed each year from 1991 to 2009. Substance use
measures included past 2-week binge drinking and past 30-day alcohol, cigarette, smokeless
tobacco, marijuana, and steroid use. Analyses were conducted during 2009-2010. Across
grades, higher levels of exercise were associated with lower levels of alcohol, cigarette, and
marijuana use. Higher levels of athletic team participation were associated with higher levels
of smokeless tobacco use and lower levels of cigarette and marijuana use across grades and
to higher levels of high school alcohol and steroid use. Exercise helped suppress the
undesired relationship between team participation and alcohol use; exercise and athletic
team participation worked synergistically in lowering cigarette and marijuana use. Observed
relationships were generally stable across time. In conclusion, there appear to be substantive
differences between exercise and team sport participation in relation to adolescent
substance use. These findings from cross-sectional data suggest that interventions to
improve levels of general physical activity should be evaluated to determine if they help delay
or reduce substance use among youth in general as well as among student athletes [11027].
Performance-enhancing substances include dietary supplements, prescription medications,

and illicit drugs. Virtually no data are available on the efficacy and safety in children and
adolescents of widely used performance-enhancing substances. This statement is intended
to provide a generalized but functional definition of performance-enhancing substances. The
American Academy of Pediatrics strongly condemns the use of performance-enhancing
substances and vigorously endorses efforts to eliminate their use among children and
adolescents. Performance-enhancing substance use in young people is a concern to
670
pediatricians and society because of potential adverse health consequences and the effects
that such practices have on moral development of the individual and on fair athletic
competition for all. Health care professionals can play a valuable role in counseling the
young person using or contemplating use of performance-enhancing substances by
conveying factual information about the proven benefits and medical consequences of these
substances and providing advice about healthful eating and training. Attempts to discourage
use through scare tactics or by dismissing known performance-enhancing effects of these
substances may seriously damage the credibility of the physician and do little to diminish
use. Efforts to minimize use of performance-enhancing substances require the pediatrician to
have an understanding of the incentives for use, a comprehensive definition of performance-
enhancing substances, and familiarity with strategies for prevention [05010].
The first level of addressing the problem of drug abuse by athletes is prevention. Drug
screening is used in higher-level athletics both to deter athletes from using drugs and to
punish and offer opportunities for rehabilitation to those who are found to have done so.
Didactic education is another method aimed at prevention. On the one hand, some authors
and clinicians feel that among the most effective preventive strategies for drug abuse in
sports is frequent, accurate, very closely observed, truly random urine drug testing. However,
some view drug testing as ineffective at preventing use of doping drugs. The argument for
the latter is that these interventions target doping behavior rather than athlete attitudes.
Athletes ultimately focus on their performance, and thus may view doping as rational
behavior. Moreover, knowledge of the potentially dangerous consequences from doping
imparted via didactic education does not necessarily dissuade athletes. For example, in
1997, Bamberger and Yaeger surveyed 198 Olympic athletes. When asked if they would use
PEDs under the hypothetical conditions of knowing they would not be caught and knowing
their use would result in victory, 195 of 198 responded “yes”. Moreover, if the caveat was
added that they would die within 5 years, 61 percent of the athletes still said they would use
them [014612].
There is little research available to guide counseling and psychiatric approaches to treatment
of athletes who abuse drugs. However, motivational interviewing approaches have been
suggested for athletes with drug abuse or doping problems, since athletes may often present
in the precontemplation stage of change. Important elements of motivational interviewing
include [14612]:
1. Clinician empathy
2. Developing discrepancies between where the athlete wants to go in life after sport
and the impact that continued use of the substance might have on those goals.
During this process, the provider helps athletes to clarify conflict among their values,
motives, interest, and behaviors.
3. Rolling with resistance. When resistance inevitably occurs, providers should avoid
arguing with athletes, as that can exacerbate resistance to change. The provider may
“agree to disagree” on certain points with some athletes. Providers may propose or
“wonder about” certain alternative viewpoints or actions, but they do not impose or
insist upon them.
4. Encouragement of self-efficacy. Athletes may need to shift their viewpoint from one of
being willing to do whatever it takes to win, to acknowledging that they would use
PEDs only if ultimately incapable of succeeding without them (with the hope that
athletes will never get to that point). If an athlete is physically dependent on a drug,
then additional strategies may be needed. These may include pharmacologic
interventions such as naltrexone, acamprosate, or disulfiram for alcohol dependence,
or buprenorphine for opiate dependence. Additionally, providers should assess for
comorbid mental illness, since co-occurrence of physical dependence and mental
671
illness is commonplace. Any underlying mental illness should be treated. A recent
review paper on the epidemiology of mental illness in athletes noted that some
mental illnesses such as depression are probably as common in athletes as
nonathletes. Twelve-step facilitation, cognitive behavioral therapy, and network
therapy are also approaches that may be helpful for athletes who are abusing drugs,
although studies are preliminary
Recent doping affairs clearly demonstrate that all sports are concerned, leading to a
generalized suspicion concerning champions and their performance. Many athletes
struggling to reach their own personal goals have the unacceptable impression that they
cannot compete fairly with competitors who are presumably (or effectively) doped. This
situation may easily lead to deviant behavior. The sincerity of many high-level athletes when
they discuss doping is very questionable in light of the notoriety and financial aspect linked
with success. Public image is all important.Unfortunately, at this level is appears to be utopic
to assume preventive measures would be effective. Several factors are involved taking into
consideration these different elements, one could be rather pessimistic concerning the
eradication of doping in high-level athletes. There could even be a risk of seeing a trend
towards public acceptance of more or less "tolerated" doping among professional athletes,
similar to what is observed in the United States. It might be preferable to concentrate efforts
on education and prevention in the young population [02014].
A conceptual framework for achieving performance enhancing drug
There has been, and continues to be, widespread international concern about athletes' use
of banned performance enhancing drugs (PEDs). This concern culminated in the formation of
the World Anti-Doping Agency (WADA) in November 1999. To date, the main focus on
controlling the use of PEDs has been on testing athletes and the development of tests to
detect usage. Although athletes' beliefs and values are known to influence whether or not an
athlete will use drugs, little is known about athletes' beliefs and attitudes, and the limited
empirical literature shows little use of behavioural science frameworks to guide research
methodology, results interpretation, and intervention implications. Mindful of this in preparing
its anti-doping strategy for the 2000 Olympics, the Australian Sports Drug Agency (ASDA) in
1997 commissioned a study to assess the extent to which models of attitude-behaviour
change in the public health/injury prevention literature had useful implications for compliance
campaigns in the sport drug area. A preliminary compliance model was developed from three
behavioural science frameworks: social cognition models; threat (or fear) appeals; and
instrumental and normative approaches. A subsequent review of the performance enhancing
drug literature confirmed that the overall framework was consistent with known empirical
data, and therefore had at least face validity if not construct validity. The overall model
showed six major inputs to an athlete's attitudes and intentions with respect to performance
enhancing drug usage: personality factors, threat appraisal, benefit appraisal, reference
group influences, personal morality and legitimacy. The model demonstrated that a
comprehensive, fully integrated programme is necessary for maximal effect, and provides
anti-doping agencies with a structured framework for strategic planning and implementing
interventions. Programmes can be developed in each of the six major areas, with allocation
of resources to each area based on needs-assessment research with athletes and other
relevant groups [02015].
Culturally sensitive measures
672
Recent studies in different countries have shown an increase in anabolic steroid
consumption among young people and the harm caused by indiscriminate use. In Brazil,
research on steroid abuse is scarce. The present study examines the risk perception of
health problems associated with anabolic steroid consumption among young working-class
adults engaged in body-building practices in a poor neighborhood in the city of Salvador,
Bahia. The methodology involved an anthropological approach based on qualitative research
techniques consisting of ethnography, in-depth interviews, and a focus group with steroid
users. The data describe the most common substances consumed and highlight the lack of
information among interviewees concerning potential related health hazards, showing that for
many steroid consumers the quest for muscle-mass development to achieve an idealized
body supersedes the risk of harmful side effects. The results indicate the need for culturally
sensitive measures to prevent steroid abuse among youth [02016].
Epidemiological issues
Owing to a widespread diffusion, the consumption of banned and potentially harmful

substances in sports has become a problem for the public health. Current estimations of the
prevalence of doping in sports are rather uncertain, as most investigative tools do not reflect
an absolute statistical power. However, the emerging scenario reflects a concerning
underestimation by Structures and Institutions that should establish definitive rules and set
reliable controls. Owing to restricted resources, prevention and fight against doping must be
supported by meditated and rational strategies, with the aim to identify suitable contests and
accurate procedures, considering carefully ethical issues that may arise from the positivity of
the athletes to antidoping controls [14029].
Drug prevention programs
One paper investigates baseline by treatment interactions (BTI) of a randomized anabolic

steroid prevention program delivered to high school football players. Baseline by treatment
interactions occur when a participant's score on an outcome variable is associated with both
their pretreatment standing on the outcome variable and the treatment itself. The program
was delivered to 31 high school football teams (Control=16, Treatment=15) in Oregon and
Washington over the course of 3 years (Total n=3207). Although most interactions were
nonsignificant, consistent baseline by treatment interactions were obtained for knowledge of
the effects of steroid use and intentions to use steroids. Both of these interactions were
beneficial in that they increased the effectiveness of the program for participants lower in
knowledge and higher in intentions at baseline [05016].
Education
The World Anti-Doping Agency (WADA) defines in the World Anti-Doping Code acts that
constitute a code violation. These include ‘‘the presence of a prohibited substance or its
metabolite or markers in an athlete’s bodily specimen’’. The World Anti-Doping Code lists
these prohibited substances. A doping offence occurs when a sportsperson tests positive for
a banned substance, regardless of how it got there. Although there are undoubtedly
sportspeople who do deliberately cheat by taking banned drugs, there are also a number
who take something purely by accident. Many everyday products contain banned substances
– for example, cold remedies, analgesics, hay fever medication, herbal preparations, and
nutritional supplements. Over the years there have been several high profile cases of
673
sportspeople who, having been found to commit a doping offence, argue that it was
inadvertent. In some of these cases, the subsequent disciplinary committee hearing has
agreed that the cause of the offence was an accident. These cases highlight the problem
faced by sportspeople in preventing inadvertent doping offences. Products may have
compositions that differ depending on the country of origin, or which change without warning.
The labelling is often incomplete, written in a foreign language, and may be too complex for a
non-specialist to understand. Increasing numbers of sportspeople train and compete abroad,
and it may be difficult for them to easily access advice about a substance they wish to take.
There may also be person specific errors such as ignorance, naivety, and failure to check
thoroughly. To identify educational needs of elite sportspeople with respect to the doping
laws a questionnaire survey of 196 Olympic level sportspeople from the fields of athletics,
cycling, rowing, and sailing. The questionnaire addressed the date and source of the last
doping educational update, the usefulness of current resources, sources of help, and
possible ways of improving the system. The questionnaire also sought to estimate the use of
nutritional supplements in these sports. Seventy four (38 %) athletes responded to the
questionnaire. Over 90 percent of responders had received a doping educational update in
the last six months, and most agreed with the statement "I have received the information I
need to avoid getting into trouble with the doping laws". Despite this, more than half of
responders agreed with the statements "I should receive reminders more often" and "The
authorities should do more to educate sportspeople". In addition, there were four people who
admitted taking a banned substance by accident. Forty-one (55 %) reported taking
supplements. The team doctor was the most popular source if information on a substance or
product was required, with 62 and 66 percent of subjects stating that they would contact their
team doctor when based in the United Kingdom and abroad respectively. The UK Sports
website was often suggested in relation to ways of improving knowledge. It was concluded
that there is a need to alter the educational process, particularly with respect to contingency
planning for minor illness. The use of internet based resources for up to date information
about banned substances needs to be promoted, and access to the internet improved. The
educational needs of team doctors with respect to the doping laws need to be assessed
[05017].
The risk of being caught for doping
It has been estimated the probability of a doping athlete being caught by a single random test
at less than 3 percent because the average sensitivity of doping tests is about 40 percent,
and the window of opportunity for detecting illicit drug doping is narrow. They estimated that
in order to have a 100 percent chance of detecting drug cheats with existing methods, each
of the athletes would need to be tested up to 50 times a year, at costs which are by far larger
than current costs. The case of Lance Armstrong, tested on multiple occasions over many
years with no conviction, may have been the reason that even a report of a WADA working
Group admitted: “To date, testing has not proven to be particularly effective in detecting
dopers/cheats.” Numerous proposals were made for a better quality program, including more
random, front-loading, out-of-competition tests (i.e. more testing before competition rather
than after competition), and better anticipation of the athletes’ evasion techniques. It might be
added that a larger share of the budget should be devoted to researching new testing
procedures [14416].
Economic incentives
If doping-prone competitions were canceled from the Olympic Program and/or if the
674
broadcasters would abandon these transmissions, audience, and thus sponsor revenue,
would fall significantly. This does not affect the rationale of the potential doper – if he/she is
discovered he/she loses his/her income, independent of the broadcasting. However, this
might induce another effect. If every athlete knows, that his/her own income depends not
only on his/her own behavior, but also on the behavior of other athletes, the mentality and
milieu will change in the affected sport away from a mentality where doping is known (and
accepted), virtually every person knew – doctors, soigneurs, riders, team managers,
mechanics – toward a behavior where everyone feels responsible for each other. Each
athlete would have an incentive to intervene and to report the suspicion to an internal
ombudsman [14416].
Sponsors
Taken one step further, doping must be financed, which explains that almost all doping cases
are emerging in such “professional” sports. Given this background, it is astonishing that up
until now, the international antidoping strategies have not concentrated more on an approach
to tackle the problem by more efficient penalties. Existing penalties, which mostly consist of
temporary bans, are efficient to a limited extent. It is not surprising that many athletes, who
have doped or who are under suspicion of doping near the end of their careers, that such a
ban would not particularly affect them. Economically efficient penalties would consist – taking
the obviously cash-driven doping athletes into account – of fines, where the amount would
have to be high enough to turn the cost-benefit analysis outlined above into a negative. As a
“back on the envelope calculation,” and ignoring the nonpecuniary costs and benefits of
doping and assuming risk-neutrality, the lowest fine could be calculated by multiplying the
expected benefit from doping by the reciprocal value of the probability of discovery. If, for
example, prize money and sponsorship money from winning amount to a hundred thousand
dollars, and if the established probability of being detected in doping controls is one-third, the
fine should be at least Usd 300,000. Comparably, the fine for tax evasion, after conviction,
amounts to a multiple of the evaded tax in the fiscal laws of several nations. These penalties
should be subjected to the laws of contracts, which are not – despite all legal restrictions that
are binding here as well – (such as the competition bans) within the narrow limits of labor law
and personality rights. After all, some sports federations have begun to implement
contractual agreements under which doping offenders need to pay a fine, for example, in the
amount of their annual salary [14416].
Fines
Further refinements are also necessary. It may be argued that the probability of detection of
a doping offense (and thus the necessary amount of fine) differs from sport to sport, and
even within a sport from event to event, as well as it differs between training and competition
modes. In any case, it is difficult to calculate. However, this is not a serious objection. It only
matters when choosing the level of the fine such that the expected cost of doping does not
fall below the required minimum level. An under-estimated probability of detection (and thus
an “overly high” fine) is not a problem when viewed from an incentive-compatible
perspective. A pragmatic solution in the sense of a tatonnement process could be that if too
many athletes get caught, the fines need to be increased [14416].
It may be argued that such high penalties may not be recovered from the athletes. For
example, tennis player Petar Korda was not only banned from competition but was also
required to repay the prize money won by him as a result of a doping offense in 1999,
according to the IOC Anti-Doping Code. He has never made this payment. One solution to
the problem of the implementation of penalties could be a deferred compensation model, that
675
is, a large part of the sponsoring money would have to be paid into funds which would then
be paid out at the end of a doping free sporting career. The incentive not to cheat would be
largest for “long-serving” athletes such as those mentioned above, who have hardly been
affected by penalty bans (and, in any case, these athletes have an increased doping
incentive because it is difficult for them to keep pace with younger athletes) [14416].
.
Laboratory costs
Finally, the “cost structure” of the laboratories should be checked. Complexity of doping tests
in high performance sports might be of high complexity. The mentioned costs of some USD
600 per doping analysis are nevertheless in stark contrast to the costs for tests demanded by
USA schools and the Federal Drug-Free Workplace (USD 10 and USD 30 per test). In any
case, it is known from theoretical and empirical economics that a well-designed competition
may ensure high quality and nevertheless reduced costs, compared to monopolistic or
cartelized solutions. There are some 34 WADA-accredited laboratories throughout the world.
Even in the USA, there is only one (Los Angeles) – implying that the WADA-accredited
laboratories enjoy regional or even national monopolistic situation. A routine call for bids for
quality-standardized tests by WADA could reduce the financial burden for the sports family
(and thus increase the system′s efficiency) [14416].
Self-reporting
Self-reporting and whistle blowing should be considered to play a role in antidoping. WADA
Working Group in 2013 demands: “Whistle-blowing should be encouraged and mechanisms
established to make such activity possible and productive.” Australian National Olympic
Committee thinks about criminal penalties for athletes who refuse to cooperate with
investigations, for example, by withholding information. In golfing, each player always has the
obligation to report the rule violations of others. In the best case scenario, international
federations/institutions would no longer have to create their own antidoping policies (besides
credibly threatening to cancel the competition or its transmission). Instead, the participants,
coaches and other relevant staff, and organizers themselves, would develop efficient
countermeasures. Perhaps the mechanisms developed by them would even be innovative,
and could add to the measures developed for other sports [14416].
Mass media potential responsability
The responsibility of media is also true in another respect: Doping is lucrative because the
spread of the income from prize money, advertising, and other income between the winners
and the other participants is high. This effect can be explained with the “superstar” effect:
The (media) attention is focused on the winner, and only to a negligible extent on the other
participants. This focus may be regarded as a mirror of the global culture of sports and of
other areas of human activity in which winning at all costs and having “an edge” have been
“normed”, losing, and “losers” are stigmatized, and neither the health and wellbeing of the
athlete, nor fair play as a process and value, are manifested as being prime interests for a
range of sports managers, coaches, fans, etc [14416].
Prevention versus incentives for the use of performance-enhancing

substances
676
The temptation for young people to use performance-enhancing substances should be easily
understood by anyone who is familiar with high-level sports in our society. Success (that is,
winning) is considered by many to be the most important goal of sports. At the level of
professional sports, winning is the ultimate goal. This attitude permeates lower levels of
sports as well, down to youth sports. Society rewards success in sports with celebrity, status,
and favoritism. For athletes of all ages, the pursuit of excellence in sports is an endeavor to
be admired and encouraged. Success in sports involves obtaining an “edge” over the
competition. However, sometimes the drive for success can be so engrossing and so
compelling that a young person can easily lose sight of what is fair and right. Some
individuals may view the use of performance-enhancing substances as a substitute for hard
work. For others, performance-enhancing substances may be considered a necessary
adjunct to hard work or part of the price of success. From the user’s perspective, the
prospects for success in sports often outweigh the prospects for serious medical
complications from use of performance-enhancing substances. For some, winning has a
monetary incentive as well. The enormous salaries paid to professional athletes in the United
States and elsewhere are powerful inducements for a young person with outstanding athletic
talent to try anything to ensure continued athletic success. Adolescents may be uniquely
vulnerable to the lure of performance-enhancing substances. Many adolescents engage in
risk-taking behavior and experimentation at a time when they are coping with the
developmental tasks of adolescence, including defining their sexual identity, emancipating
themselves from their families, achieving a sense of mastery and self-efficacy, and finding a
peer group with which they can identify. The adolescent, by nature, feels invincible and often
shuns any suggestion that use of a substance for purposes other than legitimate therapy
might pose a danger to their health or their eligibility for sports. Adolescents are also
intensely preoccupied with body image. Personal rewards perceived from enhancing size,
strength, stamina, or body build can be strong motivators. A significant number of
adolescents who are not involved in competitive athletics use performance-enhancing
substances. The child athlete, particularly the adolescent, in today’s society is caught in a
struggle between ideals highly valued by society but often in direct conflict: the attitude of
winning at all costs and the values of fairness and wholesomeness [05010].
Assessment of physical and sports aptitudes, and prevention of doping

Present levels of training loads – which can exceed thirty hours a week for high level
sportsmen – expose to overwork then to overtraining syndrome and finally to temptation of
doping. At the same time testing techniques improve, particularly exercise tests with a
linearly increasing load, and the follow-up of training effects on physical fitness provides
more accurate data. The specialized literature has developed the notions of cardiac
frequency reserve and of oxygen intake reserve within the last ten years. These notions, as
those of produced power reserve, were applied here to assess the ventilatory threshold of
104 sportsmen (51 cyclists with high endurance and 53 team sportsmen with lower
endurance) and of 223 sedentary witnesses. They allow, when completed with absolute level
of aerobic endurance, to appreciate physical fitness of sportsmen all along sports season, to
predict their capabilities to progress by an increase of training load or to reinforce the
hypothesis of an over-working onset and doping [00017].
A novel antidoping and medical care delivery model in Nanjing 2014
677
Antidoping and medical care delivery programmes are required at all large international
multisport events. To document and critique the novel antidoping and medical care delivery
models implemented at the 2nd Summer Youth Olympic Games, Nanjing 2014 the
International Olympic Committee implemented two new models of delivery of antidoping and
medical care at the YOG. A review of these models as well as the public health programme
and two health educational initiatives in the Cultural and Educational Program was
undertaken by the International Olympic Committee. The implementation of the new
antidoping model was feasible in the setting of the YOG. The antidoping rules and
regulations of the International Olympic Committee were respected. This model enhanced
the educational initiative and provided financial as well as human resource savings. The
execution of the hospital-based venue model of medical care delivery at the YOG was also
feasible in this setting. This model provided a practical infrastructure for the delivery of
medical care at multisport events with the goal of providing optimum athlete healthcare. A
public health prevention programme was implemented and no public health risks were
encountered by the participants or the Nanjing citizens during the YOG. Finally, the
implementation of the athlete health educational programmes within the Cultural and
Educational Program provided athletes with an opportunity to improve their health and
performance. It was concluded that to achieve the goal of protecting athlete health, and of
employing effective doping control and education, new alternate models of antidoping and
medical care delivery can be implemented [150064].
To document and critique the novel antidoping and medical care delivery models
implemented at the 2nd Summer Youth Olympic Games, Nanjing 2014. The International
Olympic Committee implemented two new models of delivery of antidoping and medical care
at the YOG. A review of these models as well as the public health programme and two health
educational initiatives in the Cultural and Educational Program was undertaken by the
International Olympic Committee. The implementation of the new antidoping model was
feasible in the setting of the YOG. The antidoping rules and regulations of the International
Olympic Committee were respected. This model enhanced the educational initiative and
provided financial as well as human resource savings. The execution of the hospital-based
venue model of medical care delivery at the YOG was also feasible in this setting. This
model provided a practical infrastructure for the delivery of medical care at multisport events
with the goal of providing optimum athlete healthcare. A public health prevention programme
was implemented and no public health risks were encountered by the participants or the
Nanjing citizens during the YOG. Finally, the implementation of the athlete health educational
programmes within the Cultural and Educational Program provided athletes with an
opportunity to improve their health and performance. To achieve the goal of protecting
athlete health, and of employing effective doping control and education, new alternate
models of antidoping and medical care delivery can be implemented [150062].
Athletes Targeting Healthy Exercise and Nutrition Alternatives (ATHENA)
To implement and to assess the efficacy of a school-based, sport team-centered program to

prevent young female high school athletes' disordered eating and body-shaping drug use a
prospective controlled trial in 18 high schools, with balanced random assignment by school
to the intervention and usual-care control conditions was performed. It was enrolled 928
students from 40 participating sport teams. Mean age was 15 years, and follow-up retention
was 72 percent. The ATHENA (Athletes Targeting Healthy Exercise and Nutrition Alternative)
curriculum's 8 weekly 45-minute sessions were incorporated into a team's usual practice
activities. Content was gender-specific, peer-led, and explicitly scripted. Topics included
healthy sport nutrition, effective exercise training, drug use and other unhealthy behaviors'
678
effects on sport performance, media images of females, and depression prevention. It was
assessed participants by confidential questionnaire prior to and following their sport season.
Experimental athletes reported significantly less ongoing and new use of diet pills and less
new use of athletic-enhancing substances (amphetamines, anabolic steroids, and sport
supplements). Other health-harming actions also were reduced (less riding with an alcohol-
consuming driver, more seat belt use, and less new sexual activity). The ATHENA athletes
had coincident positive changes in strength-training self-efficacy and healthy eating
behaviors. Reductions occurred in intentions toward future use of diet pills, vomiting to lose
weight, and use of tobacco and muscle-building supplements. The program's curriculum
components were altered appropriately (controlling mood, refusal skills, belief in the media,
and perceptions of closest friends' body-shaping drug use). It was concluded that sport
teams are effective natural vehicles for gender-specific, peer-led curricula to promote healthy
lifestyles and to deter disordered eating, athletic-enhancing substance use, and other health-
harming behaviors [14030].
To explain, through mediation analyses, the mechanisms by which ATHENA (Athletes

Targeting Healthy Exercise and Nutrition Alternatives), a primary prevention and health
promotion intervention was designed to deter unhealthy body shaping behaviors among
female high school athletes, produced immediate changes in intentions for unhealthy weight
loss and steroid/creatine use, and to examine the link to long-term follow-up intentions and
behaviors. In a randomized trial of 1668 athletes, intervention participants completed coach-
led peer-facilitated sessions during their sport season. Participants provided pre-test,
immediate post-test, and 9-month follow-up assessments. ATHENA decreased intentions for
steroid/creatine use and intentions for unhealthy weight loss behaviors at post-test. These
effects were most strongly mediated by social norms and self-efficacy for healthy eating. Low
post-test intentions were maintained 9 months later and predicted subsequent behavior. It
was concluded that ATHENA successfully modified mediators that in turn related to athletic-
enhancing substance use and unhealthy weight loss practices. Mediation analyses aid in the
understanding of health promotion interventions and inform program development [09030].
Almost one half of male and female students participate in high school-sponsored athletics,
and high school also is a time when classroom health promotion curricula are less effective.
The Athletes Training and Learning to Avoid Steroids is a sport team-centered drug-use
prevention program for male high school athletes, which has been shown to reduce alcohol
and illicit drug use. Just as anabolic steroid use is associated with male athletes, female
sport participants may be at a greater risk for disordered eating and body-shaping drug use.
Extending sport team-centered programs to young women athletes required defining and
ranking factors related to developing those harmful behaviors. Survey results from a cross-
sectional cohort of female middle and high school student athletes were used to identify and
prioritize potential curriculum components, including mood and self-esteem, norms of
behavior, perceptions of healthy body weight, effects of media depictions of women, and
societal pressures to be thin. The derived sport team-centered program was prospectively
assessed among a second group of female student athletes from 18 high schools,
randomized to receive the intervention or the usual care control condition. The Athletes
Targeting Healthy Exercise and Nutrition Alternatives (ATHENA) intervention is a scripted,
coach-facilitated, peer-led 8-session program, which was incorporated into a team's usual
training activities. The ATHENA program significantly altered the targeted risk factors and
reduced ongoing and new use of diet pills and body-shaping substances (amphetamines,
anabolic steroids, and sport supplements). These findings illustrate the utility of a structured
process to define curriculum content, and the program's positive results also confirm the
sport team's potential as a vehicle to effectively deter health-harming behaviors [06030].
679
Prevention of doping amoung children and adolescents
Rationale for a statement on performance-enhancing substances and youth
In the last 2 decades, a considerable amount of research has been conducted with
performance-enhancing substances such as creatine, amino acids, androstenedione, and
dehydroepiandrosterone. Virtually no experimental research on either the ergogenic effects
or adverse effects of performance-enhancing substances has been conducted in subjects
younger than 18 years. The amount of scientific data from well-designed studies on the
effects of these substances in adults continues to accumulate at such a rate that systematic
reviews are soon made obsolete. This statement is not intended to provide a review of
currently available data on performance-enhancing substances. A list of resources for
detailed information on specific performance-enhancing substances is provided at the end of
this statement. Rather, this statement is intended to convey a more general policy on the
basis of the following 3 points. First, the intentional use of any substance for performance
enhancement is unfair and, therefore, morally and ethically indefensible. Second, use of any
substance for the purpose of enhancing sports performance, including over-the-counter
supplements, the composition and quality of which are not under federal regulation, may
pose a significant health risk to the young person. Third, use and promotion of performance-
enhancing substances tends to devalue the principles of a balanced diet, good coaching, and
sound physical training [05010].
Current definitions of performance-enhancing substances
Limitations of current definitions

Traditionally, sports organizations such as the International Olympic Committee and the
National Collegiate Athletic Association have defined performance-enhancing substances as
substances that create an unfair competitive advantage. These organizations have produced
lists of banned or prohibited drugs that include substances with known performance-
enhancing effects as well as substances used by athletes that have been associated with
adverse health effects. Detection of illegal or banned substances by drug testing is a critical
element of the enforcement and efficacy of these policies. However, current definitions of
performance-enhancing substances have contextual limitations. If the substance does not
have adverse medical consequences, if the substance is not detectable by drug testing, or if
testing for the drug is not performed (so that a potentially dangerous substance or unfair
practice may go undetected), then the substance in question would not be included in a list of
banned substances. To date, there is no definition of performance-enhancing substances
that applies to all potential users. A definition of a performance-enhancing substance that is
applicable to the pediatric age group should not exclude any individual who may have a
substance-abuse problem or any substance that cannot be readily detected. With the
prohibitive cost of testing and deficiencies associated with a detection-based banned list,
widespread drug testing of children and adolescents is unlikely to be effective or practical. A
definition of a performance-enhancing substance for the pediatric age group, therefore, must
be independent of whether testing of the substance is conducted in that age group. Because
new substances for performance enhancement as well as methods for masking the presence
of these substances are continually being discovered, a definition of performance-enhancing
substances must remain valid in a changing environment [05010].
General definition of performance-enhancing substances
680
A performance-enhancing substance is any substance taken in nonpharmacologic doses
specifically for the purposes of improving sports performance. A substance should be
considered performance enhancing if it benefits sports performance by increasing strength,
power, speed, or endurance (ergogenic) or by altering body weight or body composition.
Furthermore, substances that improve performance by causing changes in behavior, arousal
level, and/or perception of pain should be considered performance enhancing. Performance-
enhancing substances include the following [05010]:
- Pharmacologic agents (prescription or nonprescription) taken in doses that exceed

the recommended therapeutic dose or taken when the therapeutic indication(s) are
not present (e.g. using decongestants for stimulant effect, using bronchodilators when
exercise-induced bronchospasm is not present, increasing baseline methylphenidate
hydrochloride dose for athletic competition)
- Agents used for weight control, including stimulants, diet pills, diuretics, and laxatives,
when the user is in a sport that has weight classifications or that rewards leanness
- Agents used for weight gain, including over-the-counter products advertised as
promoting increased muscle mass
- Physiologic agents or other strategies used to enhance oxygen-carrying capacity,
including erythropoietin and red blood cell transfusions (blood doping)
- Any substance that is used for reasons other than to treat a documented disease
state or deficiency
- Any substance that is known to mask adverse effects or detectability of another
performance-enhancing substance
- Nutritional supplements taken at supraphysiologic doses or at levels greater than
required to replace deficits created by a disease state, training, and/or participation in
sports
Strategies for preventing use of performance-enhancing substances
The methods most widely used to prevent use of performance-enhancing substances,

namely drug bans and drug testing, are primarily punitive. Drug bans imposed by
organizations that regulate and oversee sports programs at various levels, from the
International Olympic Committee to the National Collegiate Athletic Association and state
high-school sports associations, effectively make the use of such substances “against the
rules.” Enforcement of drug bans has necessarily involved the use of drug testing, with
positive tests carrying stiff penalties or sanctions including loss of playing privileges, removal
of awards or championships from the entire team, loss of scholarships, and restrictions on
future regular-season and postseason play. Drug testing and legal sanctions are intended to
be deterrents but have little effect on most children and adolescents involved in sports.
Neither the use of drug bans nor the implementation of drug testing provides the young
athlete with any framework or guidelines for resolving the conflict between the drive to win
and the imperative to do the right thing. A variety of programs educating young athletes
about substance abuse in general and targeting specific performance-enhancing drugs such
as anabolic steroids have been tested at the international, collegiate, and even high-school
levels.4 It is unfortunate that few evaluations of these programs have included measurement
of continued drug use after the intervention, and programs appropriately studied have not
been highly successful in curbing use. One program that combined drug education with
training in personal skills to resist the social influences that drive the use of performance-
enhancing substances was successful in decreasing the intention to use anabolic steroids
among adolescent football players. Little effort has been made to target adults who are
responsible for collegiate, high-school, middle-school, and youth sports programs.
Permissiveness often has the same effect as active encouragement when it comes to using
performance-enhancing substances. A “don’t-ask” attitude should be as intolerable to
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parents as the provision of performance-enhancing substances to athletes by coaches would
be [05010].
Identification of the young person using performance-enhancing substances
Data from epidemiologic studies and case descriptions have provided information about
users of performance-enhancing substances that can help pediatricians to identify them.
Users of anabolic or androgenic compounds are more likely to be male; are more likely to be
involved in sports that demand high levels of strength, power, size, and speed; and are likely
to use other illegal substances such as tobacco and alcohol.5–7 Young people who participate
in sports that demand leanness are also more likely to use performance-enhancing
substances than are those involved in sports in which leanness is not essential. Young men
and women who are not competitive athletes but who are obsessed with body image and
who train intensely primarily to improve their physique are also more likely to use
performance-enhancing substances. Users of certain performance-enhancing substances
might be identified by outward signs such as virilization in females, testicular atrophy in
males, and mood changes produced by anabolic steroids. Unfortunately, most young people
who use performance-enhancing substances are not readily identified by outward signs.
Therefore, it is imperative that all adolescents be asked about use of performance-enhancing
substances in the assessment of high-risk behaviors that should be a part of every
adolescent health maintenance visit, including sports physicals, camp physicals, and all other
scheduled physician-adolescent encounters [05010].
Innovation leading to improved adolescent health
The use of drugs to enhance physical performance and appearance has been observed for
thousands of years. Today individuals, including adolescents, continue to employ a wide
variety of drugs in the hope of improving their athletic performance and looking better.
Unfortunately, beyond the assessment of anabolic-androgenic steroid (AAS) use, very little is
known regarding the use, safety and efficacy of other performance-enhancing drugs and
nutritional supplements among adolescents. Most studies report that 3-12 percent of
adolescent males admit to using an AAS at some time during their life. Among adolescent
females, studies find that 1-2 percent report having used steroids. The current strategy for
dealing with performance-enhancing drug use by adolescents is multi-faceted and primarily
involves education and prevention strategies, interdiction and drug testing programmes.
However, the demand for performance-enhancing drugs has been created by our societal
fixation on winning and physical appearance. In order to alter the current use of
performance-enhancing drugs by adolescents, we as a society must come to grips with our
addiction to sport and the importance we place on winning and appearance. We must
change our values [00016].
An innovative prospective controlled trial of 18 high schools, including 928 students from 40
participating sports teams, was designed to prevent young female high school athletes’
disordered eating and body-shaping drug use. Balanced random assignment was used to
assign the schools to the intervention (eight weekly 45 min sessions) or to usual-care control
conditions. Experimental athletes reported significantly less ongoing and new use of diet
pills, and less new use of performance-enhancing substances (amphetamines, anabolic
steroids and sports supplements). Other health-harming actions were also reduced (e.g.
fewer instances of riding with an alcohol-consuming driver, more seat belt use and less new
sexual activity). The experimental athletes had coincident positive changes in strength-
training self-efficacy and healthy eating behaviours. Thus, sports teams can be effective
natural vehicles for peer-led curricula to promote healthy lifestyles in adolescents and to
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deter disordered eating, performance-enhancing substance use and other health-harming
behaviours [05008].
Recommendations
To assist the pediatrician in dealing with users or potential users of performance-enhancing

substances, the American Academy of Pediatrics offers the following recommendations
[05010]:
1. Use of performance-enhancing substances for athletic or other purposes should be

strongly discouraged.
2. Parents should take a strong stand against the use of performance-enhancing
substances and, whenever possible, demand that coaches be educated about the
adverse health effects of performance-enhancing substances.
3. Schools and other sports organizations should be proactive in discouraging the use of
performance-enhancing substances, incorporating this message into policy and
educational materials for coaches, parents, and athletes.
4. Interventions for encouraging substance-free competition should be developed that
are more positive than punitive, such as programs that teach sound nutrition and
training practices along with skills to resist the social pressures to use performance-
enhancing substances.
5. Colleges, schools, and sports clubs should make use of educational interventions that
encourage open and frank discussion of issues related to the use of performance-
enhancing substances, with the aim of promoting decisions about personal drug use
based on principles of fair competition and character rather than on the fear of getting
caught.
6. Coaches at all levels, including youth sports, should encourage wholesome and fair
competition by emphasizing healthy nutrition and training practices, taking a strong
stand against cheating, and avoiding the “win-at-all-costs” philosophy.
7. Inquiries about the use of performance-enhancing substances should be made in a
manner similar to inquiries about use of tobacco, alcohol, or other substances of
abuse. Guidelines for patient confidentiality should be followed and explained to the
patient.
8. Athletes who admit using performance-enhancing substances should be provided
unbiased medical information about benefits, known adverse effects, and other risks.
When appropriate, additional testing may be necessary to investigate or rule out
adverse medical effects.
9. The pediatric health care professional providing care for an athlete who admits to
using a performance-enhancing substance should explore the athlete’s motivations for
using these substances, evaluate other associated high-risk behaviors, and provide
counseling on safer, more appropriate alternatives for meeting fitness or sports-
performance goals.
10. Nonusers of performance-enhancing substances should have their decisions
reinforced while establishing an open channel of communication if questions about
performance-enhancing substances arise in the future.
11. Pediatric health care professionals should promote safe physical activity and sports
participation by providing or making available sound medical information on exercise
physiology, conditioning, nutrition, weight management, and injury prevention and by
helping to care for sports-related medical conditions and injuries.
Funded research
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The IOC as the leader of the Olympic movement and the sport sector at large is committed to
the protection of clean athletes for the sake of the future of sport and for the protection of
athletes’ health. To this end, the IOC has committed USD 20 million to research the
protection of athletes and fair play, half of which will be used for programmes to combat
match fixing and illegal betting and half for research into anti-doping. WADA, jointly funded
by the IOC and governments, has successfully funded research over the past 10 years. To
further strengthen these efforts, governments who are WADA signatories have been invited
to match this extra IOC contribution to create a combined fund from the IOC and
governments that will be administered by WADA. Any IOC funds that are not matched by a
commitment from the governments will be distributed independently by the IOC Medical
Commission. The strategy for distribution of these independent funds is to complement but
not duplicate existing anti-doping research programmes. The priority is innovative and novel
research in all areas of anti-doping, which have the potential to lead to a significant change in
the way anti-doping programmes are carried out and will have a direct impact on the daily life
of the clean athlete. Anti-doping organisations agree that alternative strategies are needed,
so this investment aims at supporting new ideas and novel anti-doping practices. Particular
priority will be given to research that could lead to an enhancement of the life of clean
athletes, that focuses on prevention strategies, that evaluates the effectiveness of current
anti-doping programmes or that develops alternative approaches to anti-doping, such as the
use of information technology, intelligence and new media. Multicentre and collaborative
projects are encouraged, as well as proposals from scientists who have never before been
involved in anti-doping research. Grants will not normally be given to fund work of
commercial companies or where the research should be the responsibility of other bodies.
Areas that are not a priority for the IOC-independent research fund are the assessment of
ergogenic agents, gene doping or laboratory-based research to improve the sensitivity of
current analytical techniques. These areas are typically supported by WADA research grants.
The IOC plans to distribute all grants by 2016 for projects lasting up to 3 years. In this way,
the fund will have the best chance of making a difference by inspiring new ideas towards a
common goal with WADA and all stakeholders whose aim is the protection of clean athletes
[150059].
A Swedish health promotion programme to prevent misuse of anabolic

steroids
The aim of one study was to design an appearance programme in order to prevent the
misuse of androgenic anabolic steroids among adolescents and to evaluate the adolescents'
perception of this programme. The study was performed in all schools in a primary health
care area on the south west coast of Sweden. The intervention targeted all 16- and 17-year-
old males and females (n=921). The intervention and evaluation were completed by 451
boys. The strategy of the appearance programme was to create awareness of and to discuss
attitudes towards steroid hormones among these adolescents. Youth leaders and health
workers, who discussed these subjects with adolescents over a period of 2 years, carried out
the intervention programme. The perception of the programme was analysed anonymously
using questionnaires. Effects on the total population of youths were assessed by two cross-
sectional surveys. The intervention programme was well received by the adolescents. The
misuse of androgenic anabolic steroids had a tendency to decrease after the appearance
programme. We demonstrated a method for involving the community in an appearance
programme to reduce misuse of anabolic steroids and showed that youth were sensitive to
our discussions about appearance and attitudes. This study indicates that drug abuse among
adolescents can be decreased by health promotion activities, such as group discussions.
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Controlled studies are needed before the results of this appearance programme can be
generalized [04031].
An American anti-doping program
Use of alcohol and other illicit drugs by adolescent male athletes is a significant problem.
Participation in sports may encourage use of drugs that enhance athletic performance,
especially anabolic steroids (AS). Because no other intervention has successfully altered
substance abuse by athletes, it was developed and assessed the efficacy of a team-
centered, sex-specific education program designed to reduce adolescent athletes' intentions
to use and use of AS and alcohol and other illicit drugs. It was studied 31 high school football
teams that comprised 3207 athletes in 3 successive annual cohorts (1994-1996). The
intervention included interactive classroom and exercise training sessions given by peer
educators and facilitated by coaches and strength trainers. Program content included
discussion of sports nutrition, exercise alternatives to AS and sport supplements, and the
effects of substance abuse in sports, drug refusal role-playing, and the creation of health
promotion messages. Questionnaires assessing AS, the use of sport supplements and
alcohol and other illicit drugs, and potential risk and protective factors were administered
before and after the intervention (before and after the football season) and up to 1 year after
the program. At season's end, intentions to use and actual AS use were significantly lower
among students who participated in the study. Although AS reduction did not achieve
significance at 1 year, intentions to use AS remained lower. Illicit drug use (marijuana,
amphetamines, and narcotics) was reduced at 1 year, whether alcohol was included or
excluded from the index. Other long-term effects included fewer students reporting drinking
and driving, less sport supplement use, and improved nutrition behaviors. It was concluded
that use of alcohol and other illicit drugs and associated harmful activities can be prevented
with a sex-specific, team-centered education. School athletic teams provide an optimal
environment in which to provide drug prevention and health promotion education [00015].
SAFE (Safe and Fair Events)
A recent development in the attempt to boost performance is the use of erythropoietin (EPO)
and oxygen carriers. Because EPO can only be detected in the urine for approximately one
week after the last dosage, additional blood screening has been introduced into sports such
as cycling, skiing and skating. With the original blood screening hemoglobin and hematocrit
levels were measured and cut off values for no-start have been introduced. Presently one of
the most fair and advanced blood testing methods is the SAFE (Safe and Fair Events)
paradigm. With this method all athletes are screened with one blood sample taken the day
before the competition, and post competition samples taken only on those athletes who have
been selected for doping control. With this approach the entire field is checked for potentially
dangerous polycythemia prior to start. In addition, parameters of red blood cell formation
indicate the use of EPO, oxygen carriers, plasma expanders and some other manipulations.
In suspected cases the plasma can be spun, frozen and studied. In addition targeted urine
sampling for EPO detection can take place. Although the experience with this approach is
positive, further studies are needed to refine and validate the methodology. A scientific
journal is a proper medium to publish research and to discuss the matter scientifically.
Therefore, original investigations and review papers like the ones in the current issue, are
welcomed. Since some of the forbidden substances may be used in athletes while scientific
studies only include animal research, additional research on humans should have a high
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priority. Therefore, exercise scientists should take their responsibility in this matter, and
contribute to safe and fair sport [01014].
Harm minimisation – by the sports doctor
Needle exchange is one of the cornerstones of the UK policy for harm minimisation within the
drug-using population. As HIV and hepatitis B and C became major problems in the drug
using community, through the sharing of injecting equipment, needle exchanges allowed
drug users access to clean injecting equipment and advice. to minimise the risk to the
community of used equipment being discarded inappropriately. In the North East of England
it was estimated that 60 percent of people accessing needle exchanges were not heroin
users but were injecting anabolic steroids. Clearly, this group had realized that they were at
the same risk of blood-borne infections as those users of illicit drugs. Many of these patients
were informing the needle exchange co-ordinators that they felt isolated from medical care
with their own general practitioners often unwilling to offer any monitoring of their drug taking
or give advice other than to stop using anabolic steroids due to the “immense harm” they can
cause. Just as it was denied the efficacy of these drugs for performance enhancement, in
spite of the obvious performance effects that many athletes had noticed, in predicting
calamity we once more demonstrated a lack of insight into the complexities of this problem.
In truth the athlete had come to believe that we really didn’t know what we were talking
about, increasing their reliance on locker-room anecdotes and advice from other drug users.
In reaction to this information Health Authorities, such as Durham, felt the need to set up
specific clinics to address the issues being raised and institute a harm minimisation
programme also raising awareness of this situation within the community. Taking the lead
from people working with illicit drug users, a harm minimisation policy is a logical approach to
this problem. The first thing the physician must ascertain is the type of performance-
enhancing drug user they are dealing with to allow them to develop a strategy of care. As
with all medical examinations, one must take a history, perform an examination and then
decide on further investigations. There are four general groups of patients that use
performance-enhancing drugs:
- Those who are seriously involved in their sport and see the use of performance-
enhancing drugs as a tool to achieve their ultimate goal. They tend to have a definite
plan of use, have often read around the subject and feel they are making an informed
judgement. The physician’s role here is to gain trust and monitor their health. With
time one can then engage more fully in a harm minimisation strategy.
- Those who have recently become involved in sport or started attending the gym.
They may see performance-enhancing drug-taking as part of the culture that they
wish to subscribe to or as a short cut to their goal. Often with a lack of understanding
of effective diet, training techniques and effects of performance-enhancing drugs,
their use of these agents is more amenable to change.
- Occupational users such as doormen, police and prison warders. They have a
definite objective; often feeling threatened by aspects of their work they believe they
must increase their size and aggression both to threaten and protect others. This
group is very much like the first group.
- The “recreational user” using these drugs in an effort to enhance sex drive,
aggression, stamina and a sense of well-being. The use of illicit drugs is also
common in this group.
For the majority of people, advice on training and diet will bring about the desired effect in
their performance without the need for drugs. Exploring the patient’s aims and goals allows a
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dialogue to convey interest and an awareness of their informed decision. At worst this will
afford the opportunity of monitoring and advice if problems arise, at best it will persuade the
patient not to use drugs at all. Performance-enhancing drugs do work but the benefit they
provide is of little moment for the majority of people using them and this can often be
explained. For those patients intent on using performance-enhancing drugs one must explore
the drugs to be used, use of multiple drugs (stacking), style of drug taking (injecting and/or
oral usage) and proposed doses the patient intends to use. This will allow the physician to
seek areas of harm reduction. The ideal is not to use performance-enhancing drugs at all but
if the patient will not be swayed from using them then one should advise against high dose
and oral regimes, also encouraging the patient to use short courses (cycles) of drugs rather
than remaining on them continuously. An average cycle is for 6-12 weeks with a similar
duration off-cycle. For high-level bodybuilders some remain on cycles for up to 1 year,
potentially longer. Clearly, one should advise to use the lowest possible doses of drugs and
have long cycles off of the anabolic steroids to allow the hypothalamo–pituitary axis and
other systems time to recover [01015].
Ironically, it may be safer to advise the patient to inject non-17-alkylated drugs than continue
to use drugs orally. Oral drugs tend to be 17-alkylated to protect against hepatic metabolism
but this also renders them potentially more toxic to the liver. If advice is to be given on
injecting drug use the patients must have access to a needle exchange system to minimize
the risk of abscess formation, hepatitis B and C and HIV transmission. One should also
advise the patient not to inject individual muscle groups but to inject in the upper outer gluteal
region to minimise the risk of injection trauma. The injection of joints and injured muscle
tissue is to be dissuaded, as many of the patients erroneously believe that these drugs will
have similar effects to corticosteroids. As with any medical examination following the taking
of a history, a full examination should be performed with investigations for haematological
and biochemical monitoring and other tests such as an ECG to assess electrical criteria for
left ventricular hypertrophy and the QT interval. However, an awareness of the drugs used
and their potential side effects is of importance [01015].
The FIFA program
The Fédération Internationale de Football Association (FIFA) introduced doping controls in

1966 to ensure a fair competition prior and during the FIFA World Cup tournaments. The
doping controls of the 2014 FIFA World Cup in Brazil were managed by the FIFA Medical
Office and the FIFA Sports Medical Committee, as done for all the previous World Cups.
Antidoping procedures were applied in accordance with the FIFA antidoping regulations and
the respective WADA International Standards. Prior to the World Cup finals, an extensive
campaign of out-of-competition (OOC) antidoping tests was organised to collect blood and
urine samples from individual players from the 32 qualified countries, corresponding to a total
of more than 750 OOC samples. This collection was the first step to implement the athlete
biological passport (ABP), a new strategy in football. During the 64 matches played in Brazil,
a further 300 blood and urine samples were collected in competition (IC). The real effect on
the blood values for the biological passport obtained after a football game has not been really
investigated until now. The FIFA has implemented an important antidoping programme for
the 2014 FIFA World Cup. All qualified players from the 32 teams participating in the World
Cup were tested out-of-competition. During the World Cup, 2-8 players per match were
tested. Over 1000 samples were collected in total and analysed in the WADA accredited
Laboratory of Lausanne. The quality of the analyses was at the required level as described in
the WADA technical documents. The urinary steroid profiles of the players were stable and
consistent with previously published papers on football players. During the competition,
687
amphetamine was detected in a sample collected on a player who had a therapeutic use
exemption for attention deficit hyperactivity disorder. The blood passport data showed no
significant difference in hemoglobin values between out-of-competition and postmatch
samples. It was concluded that logistical issues linked to biological samples collection, and
the overseas shipment during the World Cup did not impair the quality of the analyses,
especially when used as the biological passport of football players [150063].
A medical and scientific multidisciplinary consensus meeting was held from 29 to 30

November 2013 on Anti-Doping in Sport at the Home of FIFA in Zurich, Switzerland, to
create a roadmap for the implementation of the 2015 World Anti-Doping Code. The
consensus statement and accompanying papers set out the priorities for the antidoping
community in research, science and medicine. The participants achieved consensus on a
strategy for the implementation of the 2015 World Anti-Doping Code. Key components of this
strategy include:
- sport-specific risk assessment

- prevalence measurement
- sport-specific test distribution plans
- storage and reanalysis
- analytical challenges
- forensic intelligence
- psychological approach to optimise the most deterrent effect
- the Athlete Biological Passport (ABP) and confounding factors
- data management system (Anti-Doping Administration & Management System
(ADAMS)
- education
- research needs and necessary advances
- inadvertent doping
- management and ethics: biological data
True implementation of the 2015 World Anti-Doping Code will depend largely on the ability to
align thinking around these core concepts and strategies. FIFA, jointly with all other engaged
International Federations of sports (IFs), the International Olympic Committee (IOC) and
World Anti-Doping Agency (WADA), are ideally placed to lead transformational change with
the unwavering support of the wider antidoping community. The outcome of the consensus
meeting was the creation of the ad hoc Working Group charged with the responsibility of
moving this agenda forward. The antidoping movement is now poised to take a major step
forward in the fight against doping in sport by implementing the new 2015 World Anti-Doping
Code and drawing on the major advances in science and medicine, much of which are
underpinned by research funded by WADA and the International Federations of sports (IFs).
As such, evidence-based, targeted, sport-specific and situation-specific strategies along with
more effective analysis and improved efficiency and intelligence are approaches envisaged
to lead to better deterrence of doping in sport. These strategies, combined with education
and the commitment of all antidoping organisations (ADOs) to implement evidence-based
programmes, should help protect the integrity of sport and all those athletes who do not dope
[14417].
The overall strategy in the fight against doping must be based on good scientific evidence,
statistical analysis reflecting the prevalence of doping cases and the monitoring of illicit
substances. This assessment must be sport specific as the risk and temptation to dope and
the doping strategy will depend largely on the type of sport. For example, within individual
688
sports, endurance athletes in track and field, cycling or cross-country skiing would choose
different substances and methods to illicitly improve their performance, in contrast to athletes
who depend primarily on strength and power, such as weight-lifters, wrestlers or athletes in
certain track and field disciplines. The situation in team sports is likely to be different as
results depend primarily on the collective team performance, albeit that individual athletes
could still be tempted to dope to improve performance prospects for team selection. The risk
assessment must also take into account the timing during the season when peak
performance is desirable and expected. The longer the preparation period for a particular
event – the so-called “out of competition” period – that may precede cycling tours or
marathon running, the greater the likelihood of eluding doping controls and the temptation to
illicitly augment performance during this training period. It is prudent, therefore, that the IFs
along with the organisations of athletes consider sport-specific risk assessments. Such
assessments should be reappraised regularly reflecting scientific evidence and information
derived from forensic intelligence. The IFs, national antidoping organisations (NADOs),
doctors, scientists and paramedic personnel, together with representatives from WADA and
the WADA-accredited laboratories, must design and formulate the risk assessment by
sharing information with the IFs, the IOC and other major event organisations. The role of
WADA in this respect is to coordinate information exchange and alert the IFs and all involved
stakeholders of new information on possible doping substances, methods or approaches that
could potentially help uncover doping [14417]
.
A recommended approach for defining the scope of the antidoping activity is to measure the
prevalence of doping. The use of appropriate epidemiological tools and the careful
interpretation of survey results with an understanding of the population examined will allow
the extent of the doping problem to be assessed. This can be achieved by conducting
questionnaire surveys as is commonly used in social sciences. Epidemiological studies can
be further enhanced by measuring putative biomarkers of doping in doping-control samples.
It is essential to carry out doping prevalence studies in a population of competitors, and to
appropriately interpret the data before defining and implementing a programme of
longitudinal follow-up. This is particularly important because of the specificity of different
sports, which can be influenced differently by doping depending on the physical and
physiological characteristics required for performance enhancement. In public health,
pandemics are not distributed evenly around the globe, and environmental, social and
economic factors play an important role in their distribution. The same is true in sport where
doping prevalence may vary as a function of the sport and the country in which it is practiced.
Research recently conducted in track and field demonstrated that the prevalence of
abnormal blood profiles can vary from 3 to 48 percent depending on the country of origin of
the athletes. Certain sports federations, sports disciplines and even countries may fear total
transparency in examining their “doping cultures” as acknowledgement may hurt their public
image. However, a transparent approach is necessary if the biological monitoring of athletes
is to become an effective tool in the fight against doping [14417].
The 2015 World Anti-Doping Code makes specific reference to the development of test
distribution plans and the necessity for thoughtful and strategic approaches to testing. Such
approaches will ensure that the effective, intelligent and most efficient testing strategies are
adopted. Fundamental to these considerations are the identification of areas of sport that
might be deemed to be high risk for doping practices. International Federations and NADOs
will have particular responsibilities in this respect. Several elements should feature
prominently in the development and preparation of a sport-specific testing programme.
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These include consideration of the unique subcultures of sport and the degree to which
doping may have been “normalised” within such subcultures, the history of doping practices
within a sport, the specific physiological demands of particular events, emerging training
practices, the competitive schedule, recovery from injury, an awareness of dramatic changes
in performance, an understanding of the supplement marketplace and a familiarity with what
is happening on the street as well as in the stadium. Continual, ongoing conversation with
athletes, coaches and others in the sport community can provide an enriched understanding
of the likelihood of doping behaviour and emerging problematic practices. Successful
antidoping programmes of the future will embody high-quality, intelligent testing practices
rather than high-quantity test volumes [14417].
The implementation of the new World Anti-Doping Code with the emphasis on more
intelligent testing affords a great opportunity for enhanced, more effective and more efficient
approaches to doping control. The Athlete Biological Passport (ABP) represents a further
opportunity to ensure strategic and more focused testing. Implicit in the new approaches to
doping control is the necessity for strategic relationships between and among ADOs at every
level. Agreements between IFs, NADOs and major event organisers addressing shared
approaches to results management and testing strategies – particularly as they apply to
competitors who are part of ABP programmes – will benefit the antidoping movement. The
perspectives, experiences and strategies of IFs and NADOs can be integrated so as to
permit more timely and cost-efficient testing, the sharing of intelligence regarding doping
practices and doping practitioners and heightened vigilance of customs and other civil
authorities with regard to the importation and distribution of prohibited substances particularly
at the time of major sporting events. These approaches will benefit from the developing
international “community of practice” represented by leading IFs and NADOs; the
development of that community will itself be stimulated by the growth of strategic
partnerships and cooperative antidoping activities [14417].
New peptides or designer drugs may be used by athletes who feel that there are currently no
reliable analytical tests available. However, the 2015 World Anti-Doping Code allows for the
storage of samples for up to 10 years, which markedly transforms the antidoping
environment. The deterrent effect of delayed testing with newly devised analytical methods is
substantial. It is important that ADOs implement this process; the new 2015 International
Standard on Testing and Investigations sets out the requirements for ADOs to test, store and
reanalyse samples. The IFs and other ADOs must prioritise which samples should be stored
from which competitions on the basis of their risk assessments. Such decisions should, to
the extent permissible under applicable laws, remain confidential to optimise the deterrent
and detection elements of this new approach to doping control. It is important that the
storage of samples be conducted in a manner that enables future analysis with methods that
may not yet be fully developed or operational. An example would be the future analysis of
molecular signatures of doping. It is also imperative that samples are stored in a manner that
protects the integrity of biological samples and the antidoping process, having due regard to
legal requirements surrounding handling of human biological materials and related data
[14417].
The continuously growing knowledge in medicine, molecular biology, biochemistry and

biotechnology has substantially expanded the options to pharmacologically manipulate the
performances of athletes. Unsurprisingly, this has resulted in the suspected and proven
misuse of a wide range of peptide hormones and substances such as insulin, insulin-like
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growth factor 1, human growth hormone (hGH), epoetins, chorionic gonadotrophins, gene
doping substances including RNA interference (RNAi), “designer drugs” (e.g.
tetrahydrogestrinone) as well as non-approved, emerging or discontinued compounds (e.g.
aminoimidazolecarboxamideriboside-AICAR, GW1516, selective androgen receptor
modulators, hypoxia-inducible factor stabilisers and erythropoietin (Epo)-mimetic agents).
These developments represent a considerable analytical challenge for antidoping scientists,
which require dedicated research and the development of new methodologies. Concerted
activities with civil authorities are also necessary to understand and combat the changing
scope of doping practices and products. Analytically, the issue of new, discontinued, or
‘tailored’ drug entities has been successfully tackled by applying non-target/open
approaches, biomarker-based assays (eg, haematological, steroidal and endocrinological
modules of the ABP), “omics” strategies and monitoring effects of drug (mis)use rather than
the administered drug. This has been carried out via proactive and retrospective monitoring
programmes. Some recent successes include the introduction of section S.0 to the WADA
Prohibited List, new detection methods for the determination of RNAi-based compounds in
blood and urine, the analysis of Epo microdosing, adverse analytical findings for the non-
approved drugs GW1516, andarine and ostarine, as well as new methods to detect the illicit
use of natural compounds such as AICAR [14417].
The importance of investigations is enhanced in the 2015 World Anti-Doping Code to further
encourage ADOs to pursue antidoping violations based on the strongest evidence possible.
While investigations as the primary means of proving doping violations are uncommon in
sport, investigations have been successful in highlighting doping practices and developing
novel approaches in the fight against doping in sport. High-profile investigations include the
Bay Area Laboratory Co-Operative (BALCO) investigation that was undertaken in the USA in
2003.This case resulted in the prosecution of several athletes for the use of hGH and new
designer steroids and led to new laboratory detection capabilities and changes to the World
Anti-Doping Code. In 2006, a Spanish investigation code-named Operation Puerto
highlighted serious doping practices involving Dr Eufemiano Fuentes and a number of elite
athletes. In 2013, the Australian Crime Commission uncovered links between organised
crime and professional sporting teams and the use of performance-enhancing ‘peptides’ and
other illicit substances. This investigation led to a team in the Australian Football League
being charged and subsequently sanctioned. At the heart of this case were the activities of
support staff. Therefore, it follows that close scrutiny of support staff is explicit in the 2015
World Anti-Doping Code [14417].
The successful conclusion of each of these highlighted cases relied on forensic science and
other modern investigative techniques now at the disposal of IFs and NADOs and in
accordance with the investigation aspects of the 2015 World Anti-Doping Code. A forensic
intelligence model of antidoping investigations has been proposed and included broader
exploitation of information held at different levels by antidoping partners such as the police,
borders agencies and postal services, strategic Internet monitoring, physical and chemical
drug profiling and doping script analysis within a forensic intelligence framework. Tactical use
of forensic intelligence tools relies largely on the exploitation of bioanalytical results,
documents linked to doping practice and seizures of prohibited substances. At the
operational level, the exploitation of such information serves to uncover trends related to the
abuse of prohibited substances, the existence of organised doping programmes including the
trafficking of doping agents, and helps identify their structure and mechanisms of operation.
Strategic Internet monitoring also allows the identification and monitoring of online sales
websites, forums, blogs, social networks and other online media, thus helping to create a
clearer picture of the market and emergence of new trends. Monitoring of the physical and
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chemical profiles of seized products further enhances the understanding of the organisational
structure of the trafficking of prohibited substances. Drug profiling as well as digital and other
data allow the building of ‘inference models’ that can link to product seizures, highlight
distribution networks and identify the sources of supply. Finally, script analysis can map the
complete sequence of activities before, during and after doping to identify the key stages and
possible intervention points where the doping process might be disrupted or even prevented
[14417].
The perception of the likelihood of detection, the severity of the penalty and the speed with
which sanctions will be applied all appear to deter doping behaviour. Better understanding of
doping deterrents will enhance doping control programmes and reinforce the need for testing
strategies to be carefully considered, strategically applied and robustly enforced. The sense
of right and wrong and the perception of normative behaviour within a sport community are
perhaps the most fundamental determinants of appropriate sporting behaviour. Doping
practices have been mostly embedded in sports in which it was widely understood that such
behaviours were part of the sport ‘culture’. Therefore, sport organisations should consistently
emphasise that drug-taking behaviour is fundamentally contrary to the principles and
precepts of sport, that is, against the spirit of sport. Sport can profoundly mould and modify
attitudes and beliefs. The clear, unequivocal expression of a set of expectations regarding
conduct and behaviour within a sport can have a powerful and enduring impact. The degree
to which these expectations are upheld by athletes can enhance their legitimacy in the eyes
of their fellow competitors and strengthen the perception that violations of such expectations
are wrong. The profound disapproval that follows the violation of broadly valued standards of
behaviour can be an immensely powerful sanction and the desire to avoid such disapproval
an equally potent deterrent. Therefore, the creation of what has been described as a ‘moral
cosmology’ and an associated ‘moral community’ is central to the development of a sporting
community in which doping practices are reduced to an absolute minimal level (accepting
that there will always be those who succumb in sport, as elsewhere, to the temptation to
cheat) [14417].
The ABP and confounding factors
Typical doping control based on the direct detection of a substance or its metabolites is an
effective approach. However, it has limitations particularly when an athlete may be using
substances on an intermittent and/or low-dose basis, which may therefore go undetected
under even the most robust In-of-Competition and Out-of-Competition Doping Control
programme. Furthermore, the availability of substances virtually identical to those produced
by the human body, such as the native form of Epo, testosterone and growth hormone
necessitated a new drug-testing paradigm.Longitudinal profiling, which eventually became
harmonised into the scientifically robust WADA ABP programme, is a complementary and
alternative means to traditional doping control. Doping leaves a characteristic “fingerprint” on
the biology of the athlete and the ABP is used to identify that fingerprint, and thus the
occurrence of doping. Once a biomarker of doping is implemented in the ABP, the potential
to detect those changes brought on by performance-enhancing drugs is increased; it may
prove possible to detect changes caused by substances that have not yet been identified.
The intelligent and timely interpretation of ABP data can lead to target testing for specific
substances. Alternatively, an atypical passport finding which is confirmed by an Expert Panel
can lead to an athlete being charged with an antidoping rule violation without a ‘positive’ test
(Adverse Analytical Finding). Thus, the ABP can be seen as an innovative and reliable
antidoping tool as reflected by the findings of Court of Arbitration for Sport panels in several
cases. The introduction of the ABP also provides a strong doping deterrent and a boost to
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the credibility of the fight against doping in any given sport. The ABP does not only involve
the monitoring of biological markers. Confounding factors such as age, gender and exposure
to higher altitude for the haematological module are also included in the passport for
improved decision-making. Several confounding factors are also described in the WADA
2014 endogenous anabolic steroids technical document (TDEAAS2014). Detailed
information regarding sample collection, transport and analysis is included in the technical
documents that accompany the WADA ABP Operating Guidelines [14417].
Data management system: ADAMS
Regardless of advancements in science and enhanced antidoping practice and policy, the
fight against doping in sport can only succeed if there is a coordinated effort to ensure that
the limited resources are used effectively. In this regard, the collection, analysis and sharing
of doping control-related information and intelligence are imperative. Only by using a single
database to collect and disseminate such information can the global antidoping community
intelligently coordinate their efforts. Anti-Doping Administration and Management System
(ADAMS) provided by WADA, adapts to support the ever-changing antidoping environment.
A single database also ensures consistency in protecting the rights of athletes vis-à-vis their
information and ever-emerging data protection best practices [14417].
Education
One of the objectives of successful antidoping education is to ensure that all those involved
in sport understand the harm caused by doping to the health of athletes and to the integrity
and essence of sport. As all sport-related stakeholders have a role to play to promote clean
sport, educational efforts must be inclusive of the broad sporting community including
athletes, coaches, physicians, teachers and parents. This objective will require the
commitment of the IOC, IFs, governments (to reach schools and community-level sport) and
NADOs, with WADA as the coordinator. Education should be ongoing and sustained; it must
take place throughout the entire sporting career of an individual and focus on values and
good decision-making skills as well as an appreciation of the roles and responsibilities of
athletes. The education of athletes has to start as early as possible, preferably prior to an
athlete's first national/international competition. For example, FIFA introduced a standard
educational programme to all participating teams of the FIFA U-17 World Cup (boys and
girls) 2012 that was overseen by doctors responsible for the competition. This is in addition
to other grassroot education programmes already implemented by organisations such as
UEFA that involve the education of more than 1000 young international football players each
year. For the success of these programmes, the message presented to athletes needs to be
clear and at the correct level. The use of posters, to decorate the typically unfriendly
surroundings, and advice cards could also facilitate such communication [14417].
Research needs and necessary advances
Approaches to detect doping have improved significantly in recent years but remain
imperfect and therefore new direct and indirect detection methods are required. New
integrative “Omics”-based solutions are being developed that have the potential to improve
the analytical performance of current detection methods. In particular, WADA is funding
studies to identify a molecular signature of recombinant human erythropoietin (RhumanEpo)
doping and preliminary results are promising. For example, in the first systematic study to be
conducted, the expression of hundreds of genes were found to be altered by RhumanEpo
with numerous gene transcripts being differentially expressed after the first injection and
further transcripts profoundly upregulated during and subsequently downregulated up to 4
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weeks postadministration of the drug, with the same transcriptomic pattern observed in all
participants. The identification of a blood “molecular signature” of RhumanEpo administration
is the strongest evidence to date that gene biomarkers have the potential to substantially
improve the analytical performance of current antidoping methods such as the ABP for
RhumanEpo detection. These encouraging results serve to strongly reinforce the feasibility
and need for this complex, expensive and technically demanding approach involving leading
industry partners for the detection of banned substances and methods. Therefore, research
using an “omics”-based approach involving genomics, transcriptomics, proteomics and
metabolomics should be greatly intensified in order to achieve improved detection of
Recombinant human Epo and other doping substances and methods difficult to detect such
as growth hormone and blood transfusions [14417].
Inadvertent doping
A major objective of the global fight against doping is the protection of the clean athlete, and
hence the need to inform athletes of the risks of inadvertent doping. In recent years,
antidoping research has identified contaminated nutritional supplements and food as the
principal sources of inadvertent doping. Nutritional supplements have been contaminated
with various stimulants, beta2-agonists, prohormones, classic anabolic steroids and non-
approved designer steroids. Similarly, the consumption of certain foods, especially meat
products, is of particular concern as they may result in an adverse analytical finding. Recent
investigations have shown that the anabolic agent clenbuterol is misused in some countries
as a growth promoter in cattle feeding. The consumption of meat from clenbuterol-treated
cattle may lead to adverse analytical findings and to poisoning cases. WADA has
communicated with specific governments to address this concern as it relates to doping and
sport. WADA and FIFA are also working on studies that may differentiate the source of
clenbuterol to detect whether the substance found in the body may be due to an indirect
ingestion from an animal product. First promising results have already been obtained. The
identification of such doping traps and the dissemination of the knowledge of such sources of
inadvertent doping to all stakeholders are important aspects in the protection of athletes
[14417].
The introduction of the ABP is a reason to consider expanding the role of the medical
profession in the fight against doping in sport. The ABP is a recently validated approach to
the identification and prosecution of doping rule violations. The routine implementation of the
ABP may result in the inadvertent identification of potential clinical situations and this will
need to be addressed by medical experts. When alerted to laboratory findings, physicians
are obliged to inform the athlete via the ADOs if there is a suspected pathology. ADOs,
particularly the IFs and the NADOs, must respond accordingly to ensure the appropriate
involvement of medical professionals in the process of individual case management. At
present, the mandate of ADOs is primarily to ensure that the health of the athlete is not
affected by entering a doping spiral rather than as a health check system, and the process
typically does not involve a physician. New rules would be required to address this issue.
Whenever doctors affiliated with ADOs are involved in results management, they must
ensure that anomalous results of potential clinical significance are investigated appropriately.
In such situations, communication with other physicians involved in the care of the particular
athlete outlining the findings, their implication and suggesting, when necessary, an approach
to their further investigation has been a common approach. It is unreasonable to expect that
non-physicians would have the training, experience or perspective to assume responsibility
for such a process. The management of such cases requires distinct medical knowledge and
clinical experience [14417].
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The introduction of the ABP has raised a number of questions that reflect a profound concern
for issues of confidentiality and the responsibility of those in receipt of biological information
to take action if and when information that may relate to the health of a competitor becomes
apparent. There may be significant national considerations to take into account in such
circumstances. In some jurisdictions, testing authorities and laboratories are not considered
or accredited as providing healthcare-related services and the disclosure of information
emanating from such facilities, subsequently used for clinical purposes, may jeopardise such
accreditation. Clearly, there is no expectation that analytical laboratories should see their role
as expanding into clinicopathological domains. The identification of anomalous findings by a
clinician should be seen as an en passant phenomenon occasioned by a clinician's review of
antidoping laboratory results and prompting further clinical-standard investigations. The
activities of clinicians in this respect should not be misinterpreted as evidence that antidoping
analyses reflect clinical activities on the part of the antidoping laboratory [14417].
However, clinicians would argue that they have a fundamental ethical responsibility to take
action when provided with information that may reflect an underlying pathological condition.
Physicians are obliged to alert the ADOs if they note anomalous results that are suspicious
of pathology. The assessment of results by laboratory and medical experts (as part of the
ABP) is carried out anonymously, therefore contact must be made via the ADO. Notably, in
urine samples from male athletes, an elevation of the levels of human choriogonadotropin
hormone (hCG) is quite common with approximately 90 cases a year according to WADA
statistics. Elevated hCG may be due to the intake of the exogenous hormone but could
reflect an underlying pathology – most typically testicular cancer. For all such cases, a
specialised medical examination must be recommended as soon as possible to ensure
appropriate investigation and treatment. Physicians experienced in providing oversight to
antidoping programmes are familiar with this scenario and understand the importance of their
intervention to ensure that proper clinical attention is given to the athletes concerned. WADA
has provided clear instructions to ADOs to contact athletes to seek further medical
investigation when elevated hCG levels are detected. There are a number of documented
cases where such early intervention has led to a complete cure of the underlying condition
and can be seen as an extremely positive aspect of doping control activities. hCG testing is
part of routine analytical doping tests and not part of the ABP [14417].
Equally challenging is the question of the right of competitors to have access to their test
results. This is a complex and highly problematic issue given that such access may allow
doping competitors to manipulate or modify their strategies so as to be more likely to escape
detection. Furthermore, national legislation may, in many jurisdictions, mandate the release
of such information; the question of the timing of such release may be critically important in
ensuring the integrity of the testing system. These challenges notwithstanding, the
importance to protect the data accumulated in the conduct of doping control programmes is
paramount. It is important for sport organisations and their officials and staff to understand
the robust and rigorous approaches that are used to safeguard personal health and related
biological information in other community settings [14417].
The FIFA Antidoping program during the World Cup in Brazil 2014
Clenbuterol
Among the 779 urine samples collected OOC, 4 samples, collected in Mexico and Spain,
showed traces of clenbuterol. Two of these samples were reported as negative because
clenbuterol concentrations were below the reporting limit of the laboratory (5 pg/mL) whereas
the other two samples contained clenbuterol concentration of about 50 pg/mL and were
reported as adverse analytical findings (AAF) [150063].
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Tramadol and formestane
Among the samples collected OOC, 7 contained tramadol parent compound (ranging from 16
to 5 µg/mL) and metabolite (ranging from 130 ng/mL to 3.5 microg/mL). One urine sample
contained an abnormally high concentration of formestane (73 ng/mL) [150063].
Glucocorticoids, hCG and LH

Among the 300 IC samples, 3 contained glucocorticoids (prednisone and prednisolone,
triamcinolone acetonide, budesonide metabolite) above the WADA minimum required
performance limit (MRPL) of 30 ng/mL and in another sample prednisolone was detected at a
concentration below the MRPL. Immulite screening quantification of hCG revealed that one
sample contained hCG at a concentration of 13.6 mIU/mL. Additionally, one sample
presented an elevated LH concentration (69 mIU/mL) [150063].
Amphetamine
One IC sample was reported as AAF for the presence of amphetamine at a concentration of
about 600 ng/mL. However, the presence of the substance was explained by a valid
therapeutic use exemption (TUE) for a treatment against attention deficit hyperactivity
disorder [150063].
Endogenous steroid profiles

Without correction, a high number of samples have a testosterone concentration in the low
range of 0-10 ng/mL. Considering the SG distribution more samples have an SG lower than
1.020, which then produced an increase of apparent testosterone concentration after
correction. Adjustment of testosterone concentrations was calculated using the formula
described in the technical document [150063].
Whole blood samples were collected from all the qualified players during the OOC tests and
all tested players after the games. The WADA procedures for the blood tests, as described in
the operational guidelines for the biological passport, were entirely applied, except for the 2 h
delay before blood collection after the match. In the operating guidelines, the appendix A is
dedicated to the blood sample collection requirements. If collection occurs after training or
Competition, test planning shall … ensure testing does not occur within two hours of such
activity…If for some reason, the Sample was taken within two hours of training or
competition, the nature, duration and intensity of the exertion shall be recorded.” For
logistical reasons, the 2 h wait after exercise was not feasible at the FIFA World Cup and,
therefore, was not applied. In fact, antidoping tests in football right after the game have
always been challenging because of the players. In principle, blood collection is not time-
consuming, in contrast to urine sampling. For this reason, the antidoping authorities of FIFA
collect the blood tests as soon as the player arrives in the antidoping collection room, directly
after the game. Depending on the location of the antidoping room in the stadium and the
duration of press interviews, the time after exercise can vary from 15 and 90 min, if the player
was still on the pitch at the end of the game. Furthermore, the nature and the duration of
participation in the competition were recorded. Even though the World Cup tournament is not
considered as a controlled study, the data obtained on the field can give a good indication of
the real effect of a football match on these parameters. The cumulative frequency distribution
curves of the hemoglobin concentrations and the reticulocyte percentage, the two main
components of the blood passport do not show any difference between OOC and IC
samples. Contrary to what was shown at Euro 2008, there was no significant effect of the
competition on the values of these critical blood parameters [150063].
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Telephone counseling
Drug abuse, most notably anabolic-androgenic steroid (AAS) use, in athletes is widespread.
As a result, athletes and exercise enthusiasts who abuse these drugs are troubled by the
side effects of these illicit drugs, especially AAS. In an attempt to improve this situation, since
1993, it was counseled athletes who abuse drugs and others with questions about AAS via
telephone and tabulated the results. Counseling sessions took place by telephone every
Monday between 19:00-23:00 h. The number of cases was tabulated each year and the
specific items discussed during each consultation were categorized by key words. Cases
consisted of both drug abusers and athletes who did not abuse drugs and were concerned
about the side effects or other various problems surrounding the use of AAS. From 1993 to
1996, there were about 50 cases yearly; thereafter, the number of consultations dropped to
about 30 to 40 cases each year. In 2002, consultations with drug abusers accounted for 52
percent of all consultations, compared with 46 percent of all consultations from 1993 to 2002.
It was found that abusers of hormones exist in Japan, as well as elsewhere. It was hoped
these results will demonstrate the necessity of employing public institutional counseling
systems for athletes who are drug abusers in Japan, similar to the successful system
instituted by the Swedish National Service [07029].
National policy against doping
Sweden
In 2003, the Swedish Parliament adopted a cross-sectorial national public health policy
based on the social determinants of health, with an overarching aim – to create societal
conditions that will ensure good health, on equal terms, for the entire population – and eleven
objective domains. At that time the policy was globally unique, and serves as guidance for
public health practice at the national, regional and local levels. The development of the public
health policy and the determinants of health are presented regularly in various reports by the
Swedish National Institute of Public Health. In order to provide a holistic approach to
analysing implemented measures and providing new recommendations within the eleven
objective domains of the Swedish national public health policy, we have divided these in
three strategic areas. These are: Good Living Conditions, Health-Promoting Living
Environments and Living Habits, and Alcohol, Illicit Drugs, Doping, Tobacco and Gambling,
each described in the respective introductions for Chapters 3-5. The production of the report
was supported by a common analytical model that clarified the societal prerequisites for
health in the eleven objective domains. These are factors that can be influenced by political
actions in order to create a change. Economic analyses have also been developed to provide
a priority basis for political decisions. Analyses of the development of public health
determinants were based on data from the National Public Health Survey and data delivered
from about 15 various national agencies. Measures that have been implemented between
2004 and 2009 are analysed in details, as the basis for new recommendations for future
measures [13060].
Switzerland
Biking
After the scandal at the Tour de France 1998, the fight against doping was intensified on
national as well as international levels. In particular, the foundation of the new World Anti-
Doping Agency (WADA) has been a landmark: for the first time in the history of the fight
against doping, there exists now an international structure that includes partners from the
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Olympic movement as well as from the governments. The importance of the WADA has
already been demonstrated during the Olympic Games 2000 in Sydney. First, it conducted
several thousands of un-announced doping tests internationally and second, members of the
WADA acted as independent observers to judge the carrying out of doping controls during
the games. In Switzerland, important new measures were taken in the past years: The
introduction of an independent attorney for the judgment of sanctions or the employment of
four professional doping control officers had a clear effect on the quality of doping controls. In
addition, research – e.g. a survey on the perception of doping among the population or the
development of new technologies for doping analysis – was intensified. In the fields of
information and prevention, the existing printed material was extended with a website on
doping (www.dopinginfo.ch). Furthermore, international cooperation to develop new
didactical material for schools is intended. On the legal level, Switzerland will introduce a
new law in mid 2001. It will enable the government to fight against the entourage of athletes
when it provides doping substances to athletes. The sanctioning of athletes using doping will
still be in the jurisdiction of the sports federations [01012].
Spain
Anabolic androgenic steroids (AAS) can cause serious adverse effects when used without a
therapeutic purpose. One article aimed to show that the AAS are susceptible to being sold on
the black market. It was also aimed to describe how certain limitations on the health
inspection services of the Galician health service to pursue these illegal actions prompted a
regulatory initiative demanding that additional actions be granted to community pharmacies
when dispensing AAS. Four pharmacy inspections detected the diversion of a total of 3118
packages of AAS, which led to the opening of four disciplinary proceedings. In two of these,
specialized police forces were called in as there was sufficient evidence of possible diversion
to gymnasiums, resulting in a police operation called Operation Fitness [150060].
Brazil
A retrospective study reports data obtained from the National Institute of Criminalistics of the
Brazilian Federal Police Department (DPF) on 3676 anabolic products seized between 2006
and 2011. Anabolic androgenic steroids (AAS) were declared on the labels of 96 percent of
the products. About one third of the products declared to be from Paraguay, and 14 percent
from Brazil. Stanozolol, testosterone and nandrolone were the substances most declared on
the labels. Package and qualitative chemical analyses (performed on 2818 products) found
that 32 percent of the seized products were counterfeit, with an increase in the counterfeit
detection rate during the period. Almost half of the fake products did not contain the declared
substances, and 28 percent had only non-declared substances. Testosterone and its esters
were responsible for 45 percent of the 582 cases of non-declared drug detection. Package
analysis alone was responsible for the identification of 5 percent of all counterfeit products.
These results indicate the need for a continuous effort by the government aimed at
decreasing the availability of these products in the country [13061].
The current Brazilian situation is such that it is difficult to obtain a worldwide evaluation of
failure in education, intervention, or prevention programs. How fragile Brazil's anti-doping
system is, its appropriateness as well as its relevance, with needed policy infrastructures for
achieving the selected goals, and how wide the gap is between education and prevention
program effectiveness between high-performance athletes and recreational practitioners who
just want to look good. An additional concern, and ever present flaw regarding Brazil's
"common sportsman" in day-to-day society is their not receiving known and necessary
"sports education," enabling the development of an "at-risk" population for self-harm.
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Reflections on public health policy are noted [150061].
USA
One study investigated the mediating mechanisms responsible for the effects of a program
designed to reduce intentions to use anabolic steroids, improve nutrition, and increase
strength training self-efficacy. Fifteen of 31 high school football teams (n=1,506 players at
baseline) in Oregon and Washington were assigned to receive the intervention. The
multicomponent program addressed the social influences promoting ergogenic drug use and
engaging students in healthy nutrition and strength training alternative behaviors. Although
the results differed across the three dependent variables, the program appeared to work by
changing team norms. Unlike prevention of other drugs, changes in knowledge and
perceived severity were mediators of program effects in this study [01013].
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OVERVIEWS OF GENERAL LABORATORY TECHNIQUES
A minority of athletes continues to use prohibited drugs in sports to enhance performance.

Athletes discovered using these drugs can be subject to severe penalties, often resulting in
media and public scrutiny, especially at major events such as the Olympic Games. The
International Olympic Committee (IOC) has set out the classes of the substances it bans in
the IOC Medical Code. In many cases "old" drugs such as anabolic steroids are still used,
and current testing regimes can test for these. Advances in the therapeutic treatment of
illness have resulted in new drugs or practices, many of which are difficult to detect and
which have been turned to the sinister role of performance enhancement. Detection of some
newly developed drugs which have been placed on the banned list offers a major challenge
to laboratories involved in sports dope testing. In some cases this requires research into new
applications of research techniques. These techniques involve the novel use of gas
chromatography/mass spectrometry (GC/MS) techniques, high-resolution mass spectrometry
(HRMS), carbon isotope ratio mass spectrometry, and immunoassay techniques [00023].
International Olympic Committee accredited laboratories play a key role in upholding the
principle of fair play and innate ability, as desired by the majority of sports competitors and
spectators. Not only does doping damage the image of sport, but it can also be harmful to
the individual. The great majority of samples test negative but, when an adverse finding is
declared, the analytical data must be of a sufficiently high standard to withstand legal
challenges by third parties. The most widely misused performance-enhancing drugs are the
anabolic-androgenic steroids, commonly referred to as “anabolic steroids”. One review
attempted to address the complex issues concerning anabolic steroids in sport by
considering the clinical, biochemical and analytical perspectives [03034].

alternative doping regimens or taking advantage of the limitations in testing methodologies.
One article reviewed the testing standards and analytical techniques underlying the
procedures used to identify banned substances in biological samples, setting the stage for
future summaries of the testing required to establish the use of steroids, stimulants, diuretics,
and other prohibited substances [150121].
The presence of a drug in a biologic specimen can be used to document exposure. The
detection of doping agents is a major challenge due both to the large number of compounds
involved and to the sensitivity thresholds required in a small volume of urine. The detection
might also be complicated by the variability in the dosages and the structures of the
androgens taken and their metabolites. The standard for drug testing is an immunoassay
screen conducted on a urine sample. However, the most successful technique is a
combination of gas-liquid chromatography (GG) and gas spectroscopy (GS) applied in urine
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speciments. The suspicious peaks are identified by GS and their identity is established by
mass spectroscopy. These techniques were applied for the first time at the 1972 Olympic
Games and since then have become very sophisticated [03002].
Mass spectrometry methods can be used to screen for xenobiotics at a certain sensitivity
level. Isotopic mass spectrometry appears to provide means for resolving the problem of
compounds with low molecular mass such as steroids. Remarkable advances in sensitive
analytic techniques have permitted the analysis of drugs in saliva or hair. Hair analysis by
GC/MS might also be useful for the detection of anabolic steroids, beta-adrenergic
compounds, ephedrine and other doping agents. It might be valuable to document the doping
practice and demonstrate repetitive exposure to anabolic compounds as a complementary
method to urinary analysis. Recently, liquid chromatography/tandem mass spectrometry
(LC/MS/MS), with a methanol-water gradient including 5 mM ammonium acetate and 0.01
percent acetic acid, was found suitable to detect free anabolic steroid fractions in urine at low
(ng/mL) levels [03002].
Unification of the screening protocols for a wide range of doping agents has become an
important issue for doping control laboratories. This study presents the development and
validation of a generic liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS)
screening method of 241 small molecule analytes from various categories of prohibited
substances (stimulants, narcotics, diuretics, beta2-agonists, beta-blockers, hormone
antagonists and modulators, glucocorticosteroids and anabolic agents). It is based on a
single-step liquid-liquid extraction of hydrolyzed urine and the use of a rapid-resolution liquid
chromatography/high-resolution time-of-flight mass spectrometric system acquiring
continuous full scan data. Electrospray ionization in the positive mode was used. Validation
parameters consisted of identification capability, limit of detection, specificity, ion
suppression, extraction recovery, repeatability and mass accuracy. Detection criteria were
established on the basis of retention time reproducibility and mass accuracy. The suitability
of the methodology for doping control was demonstrated with positive urine samples. The
preventive role of the method was proved by the case where full scan acquisition with
accurate mass measurement allowed the retrospective reprocessing of acquired data from
past doping control samples for the detection of a designer drug, the stimulant 4-methyl-2-
hexanamine, which resulted in re-reporting a number of stored samples as positives for this
particular substance, when, initially, they had been reported as negatives [10027].
The PubMed and Google Scholar search engines were used to identify publications
addressing various forms of doping, methods employed in their detection, and adverse
effects associated with their use. The list of drugs prohibited by the World Anti-Doping
Agency (WADA) has grown in the last decade. The newer entries into this list include
gonadotropins, estrogen antagonists, aromatase inhibitors, androgen precursors, and
selective androgen receptor modulators. The use of mass spectrometry has revolutionized
the detection of various compounds; however, challenges remain in identifying newer
designer androgens because their chemical signature is unknown. Development of high
throughput bioassays may be an answer to this problem. It appears that the use of anabolic
steroids continues to be associated with premature mortality (especially cardiovascular) in
addition to suppressed spermatogenesis, gynecomastia, and virilization. The attention that
androgen abuse has received lately should be used as an opportunity to educate both
athletes and the general population regarding their adverse effects. The development of
sensitive detection techniques may help discourage (at least to some extent) the abuse of
these compounds [10028].
The increasing number of samples and target substances in doping control requires
continuously improved screening methods, combining high-throughput analysis, simplified
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sample preparation, robustness and reliability. The issue of doping in sport is multifaceted.
New drugs not only with anabolic properties such as selective androgen receptor modulators,
synthetic insulins, blood doping with erythropoietins or homologous and autologous blood
transfusions but also with sample manipulation have necessitated sensitive, comprehensive
and specific detection assays allowing for the identification of cheats. New methods based
on mass spectrometry, flow cytometry and immunological techniques have been introduced
and improved in the past years to support and enhance the antidoping fight [08051].
The analysis of sports samples for prohibited substances began in the 1960s and has
developed since then using modern technologies close to the latest scientific discoveries. For
small molecules, apart from the routine use of GC-MS, the newer techniques include the use
of isotope ratio MS to detect testosterone and nandrolone administration and LC-MS/MS
(liquid chromatography-tandem MS) to detect diuretics. For large molecules, several
applications of LC-MS/MS are described as well as immunoprocedures for erythropoietin and
human growth hormone [08052].
The ability to measure isotope distribution at natural abundance with high accuracy and
precision has increased the application of gas chromatography-combustion-isotope ratio
mass spectrometry (GC-C-IRMS) to doping control in recent years. GC-C-IRMS is capable of
measuring the carbon isotope ratio (delta13C) of urinary steroids and confirm their synthetic
origin based on the abnormal 13C content. One tutorial describes some of the complexities
encountered by obtaining valid delta13C measurements from GC-C-IRMS and the need for
careful interpretation of all relevant information concerning an individual's metabolism in
order to make an informed decision with respect to a doping violation [08053].
Technical advances are being made in many areas of biotechnology and genetics that are
facilitating the detection of doping in sport. These improvements have been catalyzed by the
need to counter the ever-increasing sophistication of the community of athletes and their
retinues who are intent on the illicit use of physical, pharmacological and genetic tools and
methods to enhance athletic performance, in contravention of established international
ethical and legal standards and of international treaty. The methods described in one article
present a partial and general picture of only some of these advances [12047].
Doping control is a particularly demanding task for both analytical and interpretive reasons
[05020]:
- the extremely wide spectrum of doping substances, in terms of molecular weight,

polarity, PKa, and chemical/thermal stability
- the high sensitivity of detection required for many compounds that, being
administered long before competition, are expected to be present in urine at the low
micrograms-per-liter level at the time of competition
- the short term often required to give results, particularly during high-level sport events
- the discrimination of doping from other possible reasons for positive, such as the use
of drugs for recreational purposes or physiological/pathological alterations of
endogenous steroids levels.
Within the mosaic display of international anti-doping efforts, analytical strategies based on
up-to-date instrumentation as well as most recent information about physiology,
pharmacology, metabolism, etc, of prohibited substances and methods of doping are
indispensable. The continuous emergence of new chemical entities and the identification of
arguably beneficial effects of established or even obsolete drugs on endurance, strength,
and regeneration, necessitate frequent and adequate adaptations of sports drug testing
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procedures. These largely rely on exploiting new technologies, extending the substance
coverage of existing test protocols, and generating new insights into metabolism, distribution,
and elimination of compounds prohibited by the World Anti-Doping Agency (WADA) [14715].
Olympic laboratories
Summer and Winter Olympic anti-doping laboratories, accredited by the International

Olympic Committee in the past and the World Anti-Doping Agency in the present times,
acquire worldwide interest to apply all new analytical advancements in the fight against
doping in sports, hoping that this major human event will not become dirty by association
with this negative phenomenon. One article summarized the new analytical progresses,
technologies and knowledge used by the Olympic laboratories, which for the vast majority of
them are, eventually, incorporated into routine anti-doping analysis [12048].
2005 London was awarded the Games of the XXX Olympiad. Following the IOC motto of
“Citius, Altius, Fortius”, it was decided that this would be an appropriate strategy to ensure
faster analysis with higher sensitivity, and stronger proof in cases of doping. A number of
new analytical methods were developed. Faster analysis was obtained with excellent
sensitivity using a modified gas chromatography–tandem mass spectrometry (GC-MS/ MS)
screening method. After extensive development and optimisation, the run-times were
reduced from 40 min to 14 min using short capillary GC columns. This GC-MS/MS screen
was complemented with an ultra high pressure chromatography (UHPLC) with high
resolution mass spectrometry (HRMS) screen. For the first time, and after much research, it
was concluded that UHPLC with HRMS could reliably screen over 200 analytes. This needed
both positive and negative ionisation modes together with collision induced dissociation with
all three MS experiments occurring rapidly so as not to compromise chromatographic
performance. Working in full scan mode, the volume of data acquired was enormous and
review of the vast amount of data was a challenge. After extensive collaboration with the
manufacturer there was a software that will identify the targeted analytes in 2 min after a 10
min acquisition time. Any new designer substance can now be readily searched for since we
are working in full scan mode. Furthermore, now that multiple samples are collected from a
single athlete over time (the athlete biological passport) it can review such data to search for
any change in biomarkers. Indeed, as the electronic files, like the samples, are stored for 8
years, it will now be possible to review the data at a later date to confirm that no prohibited
substance had been taken at the time of sample collection. Although the qualitative
identification of a foreign substance is all that is required to provide such evidence, for some
substances, WADA rules establish a quantitative threshold. This requires the laboratory to
quantify the substance with less than a documented uncertainty and establish that the
concentration exceeds the reporting threshold. Proving the administration of a pseudo-
endogenous substance, i.e. one that is virtually identical to the endogenous substance, is an
even more difficult task. Combustion isotope ratio mass spectrometry is used to evidence
exogenous administration by identification of a foreign substance [12049].
Practical testing in Brazil
It was summarized the results obtained from the doping control analysis during the period of
the 2007 Pan American Games held in Rio de Janeiro, Brazil. Approximately 5600 athletes
from 42 different countries competed in the games. Testing was performed in accordance to
World Anti-Doping Agency technical note for prohibited substances. One 8 mL urine sample
was used for the analysis of five steroid metabolites with two separate analyses by gas
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chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). Urine samples
were submitted to GC/C/IRMS for confirmation analysis to determine the 13C/12C ratio of
selected steroids. Fifty-seven urine samples were analyzed by GC/C/IRMS and the delta 13C
values (per thousand) of androsterone, etiocholanolone, 5beta-androstane-3alpha, 17beta-
diol (5beta-diol), 5alpha-androstane-3alpha, 17beta-diol (5alpha-diol) and 5beta-pregnane-
3alpha, 20alpha-diol (5beta-pdiol), and the endogenous reference compound were
presented. One urine sample with a testosterone/epitestosterone (T/E) ratio of 4.7 was
confirmed to be positive of doping by GC/C/IRMS analysis. The delta values of 5beta-diol
and 5alpha-diol were 3.8 and 10.8, respectively, compared to the endogenous reference
compound 5beta-pdiol, which exceeded the WADA limit of 3 per thousand [09046].
The worldwide network of World Anti-Doping Agency (WADA)-accredited anti-doping

laboratories plays a fundamental role in supporting the global fight against doping in sport.
This role is dependent on the ability to provide accurate, reliable and comparable data in
identifying and measuring the presence of prohibited substances and methods. The
accredited laboratories participate in WADA's External Quality Assessment Scheme (EQAS)
program, which provides the structure to continuously assess and improve laboratory
performance in compliance to the requirements of the International Standard for Laboratories
and related Technical Documents. The WADA EQAS is comprised of various programs,
including a blind EQAS, a double-blind EQAS and an educational EQAS, each with specific
goals with regard to monitoring and improving laboratory competence. In this article, the anti-
doping rules and processes that govern granting and maintenance of WADA laboratory
accreditation, aimed at ensuring a high-quality of laboratory operations within the framework
of the global fight against doping in sport, are reviewed [12050].
Transportation
The transportation of urine samples, collected for doping control analysis, does not always
meet ideal conditions of storage and prompt delivery to the World Anti-Doping Agency
(WADA) accredited laboratories. Because sample collection is not conducted under sterile
conditions, microbial activity may cause changes to the endogenous steroid profiles of
samples. In one work, funded by WADA, a chemical mixture consisting of antibiotics,
antimycotic substances and protease inhibitors was applied in urine aliquots fortified with
conjugated and deuterated steroids and inoculated with nine representative microorganisms.
Aliquots with and without the chemical mixture were incubated at 37 degrees C for 7 days to
simulate the transportation period, whereas another series of aliquots was stored at -20
degrees C as reference. Microbial growth was assessed immediately after inoculation and at
the end of the incubation period. Variations in pH and specific gravity values were recorded.
Gas chromatography-mass spectrometry (GC-MS) analysis was performed for the detection
of steroids in the free, glucuronide, and sulfate fractions. The addition of the chemical
stabilization mixture to urine samples inhibited microorganism growth and prevented steroid
degradation at 37 degrees C. On the other hand, four of the nine microorganisms induced
alterations in the steroid profile of the unstabilized samples incubated at 37 degrees C
[09034].
Quality of doping testing
Large amount of efforts are wasted on anti-doping testing and courts, while the current anti-
doping practices are not deem to be infallible or thoughtfully foolproof according to one
author. In one article published in the Journal of Applied Physiology, eight human subjects
704
were studied for 7 weeks and treated with recombinant human erythropoietin (rHuEpo) for 4
weeks and a post period of 3 weeks. Urine samples were obtained during all periods and
sent to two WADA-accredited laboratories. Whereas one of the two laboratories determined
rHuEpo misuse in all subjects during the boosting period, the second laboratory found no
misuse, with one sample to be negative, and the remaining seven to be suspicious. More
interestingly, while one laboratory found only two of 24 samples to be positive and three to
be suspicious during maintenance and post period, the second laboratory found no positive
or suspicious samples. As in other areas of healthcare, errors might also occur in laboratory
diagnostics. Given the high amount of tests performed every day in clinical laboratories, even
a low prevalence still reflects meaningful numbers, harbouring important public health and
patient safety implications. For a variety of reasons, false-positive and false-negative results
can occur in any area of laboratory diagnostics, including anti-doping testing. One may
support the hypothesis that the International anti-doping system might fail or, incidentally, do
fail. As such, it might produce contradictory outcomes, because false-negative results may
reassure the athletes that some forms of doping are hardly detectable, whereas falsepositive
results might lead to adverse clinical, ethical and economical consequences for those found
guilty. It is also to mention that the high number of athletes testing positive during anti-doping
controls clearly attests that the current strategy is probably ineffective to prevent athletes to
dope and modify this otherwise upsetting trend. A more suitable and less expensive strategy
might be planned, where identification of abnormal deviations from reference individual
values, regardless of pathological or artificial (doping) sources, would allow to follow and
target the athlete by an armamentarium of conventional and relatively inexpensive laboratory
tests, which are affordable to Governments and healthcare systems, and are also available
to vast majority of clinical laboratories [09035].
Blood sampling and blood samples handling
Although not (yet) a frequent doping control specimen, blood samples are advantageous
over urine specimens in a doping control context in at least two ways
- they commonly contain the intact drug rather than metabolites, which represents a
work-around when new or entirely unknown (designer) compounds are misused and
metabolism studies are not (or not publicly) available
- they provide information on drug concentrations at the time of sampling, which is of
utmost importance concerning those drugs prohibited in-competition only
As a consequence, the option to expand doping controls from urine and (less frequently)
plasma or serum to whole blood shortly before or after competition was evaluated and
assays for the analysis of minimal-invasively collected dried blood spots (DBS) were reported
in 2011 and 2012. DBS, created from a volume of 25 microl, were excised from blood
collection cards and consecutively extracted into methanol/tert.-butyl-methyl ether and
acetone. The combined extracts were concentrated, reconstituted and analyzed on a C-18
UHPLC column (2.1 × 50 mm, 1.9 microm particle size) with 0.2 percent formic acid (solvent
A) and acetonitrile (solvent B) connected via ESI to a quadrupole-orbitrap hybrid mass
spectrometer. Here, various MS modes were successively used comprising scan-to-scan
polarity switching combined with accurate mass full scan MS and target analyte inclusion list
(for online single-event product ion scan experiments) as well as all-ion fragmentation.
Hence, the combined targeted qualitative and quantitative analysis was possible and data for
non-target substances for retrospective evaluation or homology searches based on
conserved and common molecular structures were recorded. The model assay included a
total of 24 substances covering the prohibited classes S1, S3-S6, S8, S9, and P2, and LODs
ranged from 0.05-0.5 ng/mL. Moreover, LOQs were determined for four model substances
705
(tetrahydrocannabinol, cocaine, clenbuterol, and salbutamol) and were found between 0.25
and 2 ng/mL, meeting the required sensitivity to measure physiologically relevant
concentrations of these drugs [13012].
Handling of urine
Reference materials: freeze-dried urine samples
The feasibility of freeze-dried urine samples containing doping agents to be used in

intercomparison exercises and/or as reference materials has been evaluated. Freeze-dried
urine samples containing caffeine, ephedrine derivatives (ephedrine, methylephedrine,
norephedrine, pseudoephedrine and norpseudoephedrine), amphetamine derivatives
(amphetamine, metamphetamine, 3,4-methylenedioxyamphetamine and 3,4-
methylenedioxymethamphetamine) and testosterone and epitestosterone glucuronides have
been evaluated. For preparation of the samples, blank urines previously subjected to filtration
for clarification were fortified with standard solutions of the corresponding compounds and
filtered under sterile conditions. Some aliquots of the sterile liquid samples were used for
homogeneity testing, others were stored at -80 degree C for reference purposes, and the rest
were subjected to lyophilisation. Freeze-dried urine samples were stored at 4-8 degree C
and their stability was assessed for a period up to 18 months. Results obtained showed
minimal differences (lower than 5%) between lyophilised and non-lyophilised aliquots (stored
at -80 degree C) at all time periods except for amphetamine (up to 18 %) and
norpseudoephedrine (up to 10 %). Nevertheless, such differences remained constant over
the entire period of study, indicating that the loss of analytes was due to the initial
lyophilisation process. The loss of analytes in freeze-dried samples was due to their volatility.
Furthermore, an increase in pH by 1 unit was observed following reconstitution of samples
prepared from drug-free urine of commercial origin [04045].
Stability of doping substances in urine
For a correct interpretation of analytical results in doping control, knowledge on the stability
of prohibited substances in the urinary matrix is a prerequisite. So far, limited data is
available on the stability of prohibited substances in unaltered urine because most of the
studies investigating the stability of drugs have used stabilized, sterilized, or filtered urine. In
this work, the long-term stability of ephedrine, methylephedrine, cathine, 19-norandrosterone
glucuronide, and a wide range of diuretics was determined over a period of 9 months at -20
degrees C, 4 degrees C, 22 degrees C, and 37 degrees C. Short-term stability, including the
influence of 6 freeze-thaw cycles and 15 h storage at 60 degrees C, was also investigated.
Often, a tolerance limit of 15 percent, similar to what is commonly used in the evaluation of
precision data during method validation, is used to evaluate stability. One paper described an
alternative approach, using measurement uncertainty data to evaluate long-term stability with
a probability of 95 percent, and proposes a simple alternative for investigating the stability for
non-threshold substances. The results indicate that all the investigated substances are stable
when stored at -20 degrees C and 4 degrees C, but that at higher temperatures significant
degradation effects can occur. The study also shows that degradation can be dependent on
the urinary matrix and that the results from stability studies using stabilized, filtered, or
sterilized urine can underestimate degradation effects [07041].
Transportation of doping control urine samples from the collection sites to the World Anti-
doping Agency (WADA) Accredited Laboratories is conducted under ambient temperatures.
When sample delivery is not immediate, microbial contamination of urine, especially in
706
summer, is a common phenomenon that may affect sample integrity and may result in
misinterpretation of analytical data. Furthermore, the possibility of intentional contamination
of sports samples during collection with proteolytic enzymes, masking the abuse of
prohibited proteins such as erythropoietin (EPO) and peptide hormones, is a practice that
has already been reported. Consequently, stabilization of urine samples with a suitable
method in a way that protects samples' integrity is important. Currently, no stabilization
method is applied in the sample collection equipment system in order to prevent degradation
of urine compounds. An overview of a study, funded by WADA, on degradation and
stabilization aspects of sports urine samples against the above threats of degradation. Was
presented. Extensive method development resulted in the creation of a mixture of chemical
agents for the stabilization of urine. Evaluation of results demonstrated that the stabilization
mixture could stabilize endogenous steroids, recombinant EPO, and human chorionic
gonadotropin in almost the entire range of the experimental conditions tested [11033].
Diluted urine
Excessive fluid intake can substantially dilute urinary drug concentrations and result in false-
negative reports for drug users. Methods for correction ("normalization") of drug/metabolite
concentrations in urine have been utilized by anti-doping laboratories, pain monitoring
programs, and in environmental monitoring programs to compensate for excessive hydration,
but such procedures have not been used routinely in workplace, legal, and treatment
settings. It was evaluated two drug normalization procedures based on specific gravity and
creatinine. These corrections were applied to urine specimens collected from three distinct
groups (pain patients, heroin users, and marijuana/ cocaine users). Each group was unique
in characteristics, study design, and dosing conditions. The results of the two normalization
procedures were highly correlated. Increases in percent positives by specific gravity and
creatinine normalization were small for heroin users (normally hydrated subjects), modest for
pain patients (unknown hydration state), and substantial (2- to 38-fold increases) for
marijuana/cocaine users (excessively hydrated subjects). Despite some limitations, these
normalization procedures provide alternative means of dealing with highly dilute, dilute, and
concentrated urine specimens. Drug/metabolite concentration normalization by these
procedures is recommended for urine testing programs, especially as a means of coping with
dilute specimens [09036].
Urinary screening
The general strategy to perform anti-doping analyses of urine samples starts with the
screening for a wide range of compounds. This step should be fast, generic and able to
detect any sample that may contain a prohibited substance while avoiding false negatives
and reducing false positive results. The experiments presented in one work were based on
ultra-high-pressure liquid chromatography coupled to hybrid quadrupole time-of-flight mass
spectrometry. Thanks to the high sensitivity of the method, urine samples could be diluted 2-
fold prior to injection. One hundred and three forbidden substances from various classes
(such as stimulants, diuretics, narcotics, anti-estrogens) were analysed on a C(18) reversed-
phase column in two gradients of 9 min (including two 3 min equilibration periods) for positive
and negative electrospray ionisation and detected in the MS full scan mode. The automatic
identification of analytes was based on retention time and mass accuracy, with an automated
tool for peak picking. The method was validated according to the International Standard for
Laboratories described in the World Anti-Doping Code and was selective enough to comply
with the World Anti-Doping Agency recommendations. In addition, the matrix effect on MS
response was measured on all investigated analytes spiked in urine samples. The limits of
detection ranged from 1 to 500 ng/mL, allowing the identification of all tested compounds in
urine. When a sample was reported positive during the screening, a fast additional pre-
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confirmatory step was performed to reduce the number of confirmatory analyses [09037].
Lots of the steroids are extensively metabolised in the human body. Thus, knowledge of
urinary excretion is extremely important for the sensitive detection of steroid misuse in
doping control. The methods routinely used in steroid screening mainly focus on substances,
that are excreted unconjugated or as glucuronides. Common procedures include
deconjugation using a beta-glucuronidase enzyme. Following extraction and concentration
the analytes are submitted to LC-MS(/MS) analysis and/or GC-MS(/MS) analyses. Besides
the classical steroids, more and more products appear on the market for "dietary
supplements" containing steroids that have never been marketed as approved drugs, mostly
without proper labelling of the contents. To cover the whole range of potential products
comprehensive screening tools have to be utilised in addition to the classical methods.
Endogenous steroids, e.g. testosterone, represent a special group of compounds. As
classical chemical methodology is incapable of discriminating synthetic hormones from the
biosynthesised congeners, the method of steroid profiling is used for screening purpose.
Additionally, based on isotope signatures a discrimination of synthetic and natural hormones
can be achieved [09038].
Direct injection of urine
Direct injection of urine has gained interest in the field of analytical toxicology, including
doping control analysis. However, implementation of a direct urinalysis method for the LC-
MS/MS detection of 34 diuretics and 9 other doping agents yielded several analytical
problems, which were not observed using a traditional liquid-liquid extraction. Therefore a
comparative study was made between liquid-liquid extraction and direct injection.
Comparison of validation results showed that the liquid-liquid extraction at pH 7 allows to
analyze samples without major drawbacks regarding matrix effects. Hence, good sensitivity
was observed and detection limits ranged between 1 and 250 ng/mL for all compounds. In
the direct injection approach shifted retention times were observed for several acidic and
basic compounds due to unwanted matrix effects. This shift was reduced by a 25-fold dilution
of the urine samples. Besides the improved retention time stability the diluted samples also
exhibited lower ion suppression than the undiluted ones. After 25-fold dilution, detection
limits ranged between 10 and 250 ng/mL for all compounds. Since these detection limits are
at or below the minimum required performance level, imposed by the World Anti-Doping
Agency, the method could be applied to routine anti-doping analysis. Samples, previously
declared positive, were reanalysed using both the liquid-liquid extraction and direct injection.
With both techniques all 26 samples were found to be positive, showing the applicability of
direct injection for the analysis of diuretics [09039].
Effects of exercise on the urinary proteome
Exercise-induced proteinuria has been observed and studied for more than a century. It was
found that different sport disciplines alter the urinary proteome in different ways. Moderate-
intensity exercise results in increased glomerular filtration, meaning that medium-sized
proteins are excreted in higher amounts, while high-intensity exercise of short duration also
increases the excretion of low molecular weight proteins as a result of tubular dysfunction.
Exhaustive exercise may lead to the excretion of hemoglobin or myoglobin, which changes
the urinary proteome considerably. Studies comparing protein maps of different sport types
compared to a control group showed that quality and quantity of urinary proteins are
interindividually different. In addition, urine samples collected before and after exercise
exhibit substantially different protein patterns even from the same person. Therefore, further
studies investigating the urinary proteome are desirable. As the variation of protein content
and composition in urine are generally much higher than in other matrices, respective studies
708
need to be well controlled and homogenous groups of volunteers should be chosen. In
addition to the sport-related physiological and biochemical interest, exercise-induced protein
changes also need to be considered for biomarker measurements from urine samples for
kidney or other diseases [150122].
A purified enzyme for the mild hydrolysis of steroid sulfates
The hydrolysis of sulfate ester conjugates is frequently required prior to analysis for a range
of analytical techniques including gas chromatography-mass spectrometry (GC-MS). Sulfate
hydrolysis may be achieved with commercial crude arylsulfatase enzyme preparations such
as that derived from Helix pomatia but these contain additional enzyme activities such as
glucuronidase, oxidase, and reductase that make them unsuitable for many analytical
applications. Strong acid can also be used to hydrolyze sulfate esters but this can lead to
analyte degradation or increased matrix interference. In this work, the heterologously
expressed and purified arylsulfatase from Pseudomonas aeruginosa is shown to promote the
mild enzyme-catalyzed hydrolysis of a range of steroid sulfates. The substrate scope of this
P. aeruginosa arylsulfatase hydrolysis is compared with commercial crude enzyme
preparations such as that derived from H. pomatia. A detailed kinetic comparison is reported
for selected examples. Hydrolysis in a urine matrix is demonstrated for dehydroepiandro-
sterone 3-sulfate and epiandrosterone 3-sulfate. The purified P. aeruginosa arylsulfatase
contains only sulfatase activity allowing for the selective hydrolysis of sulfate esters in the
presence of glucuronide conjugates as demonstrated in the short three-step chemo-
enzymatic synthesis of 5alpha-androstane-3beta,17beta-diol 17-glucuronide from
epiandrosterone 3-sulfate. The P. aeruginosa arylsulfatase is readily expressed and purified
(0.9 g per L of culture) and thus provides a new and selective method for the hydrolysis of
steroid sulfate esters in analytical sample preparation [150123].
Proteases in doping control analysis.
Urine manipulation in sports drug testing has become a serious problem for doping control
laboratories, and recent scandals in elite endurance sports have revealed the problem of
urine manipulation presumably using proteases, which will impede the detection of drugs
such as erythropoietin (EPO) or other peptide hormones. Using commonly accepted
analytical strategies, a protocol was developed enabling the determination of elevated
protease activities in doping control specimens followed by the visualization of protein
degradation and identification of proteases such as chymotrypsin, trypsin and papain.
Therefore, protease detection kits based on fluorescein isothiocyanate-labeled casein were
employed, and protease concentrations greater than 15 microg/mL of urine entailed
subsequent 1-dimensional gel electrophoretic visualization of urinary proteins. The presence
of 20 microg of proteases per mL of urine caused a complete degradation of proteins usually
observed in urinary matrices ("trace of burning"), while respective proteases were still
detected in spiked urine samples after 10 days of storage at + 4 and - 20 degrees C.
Identification of target proteases at respective molecular weights was accomplished using
bottom-up sequencing approaches based on in-gel digestion of separated enzymes followed
by capillary liquid chromatography-Orbitrap tandem mass spectrometry [07042].
Forensic toxicology
Forensic toxicology has developed as a forensic science in recent years and is now widely
used to assist in death investigations, in civil and criminal matters involving drug use, in
drugs of abuse testing in correctional settings and custodial medicine, in road and workplace
709
safety, in matters involving environmental pollution, as well as in sports doping. Drugs most
commonly targeted include amphetamines, benzodiazepines, cannabis, cocaine and the
opiates, but can be any other illicit substance or almost any over-the-counter or prescribed
drug, as well as poisons available to the community. The discipline requires high level skills
in analytical techniques with a solid knowledge of pharmacology and pharmacokinetics.
Modern techniques rely heavily on immunoassay screening analyses and mass spectrometry
(MS) for confirmatory analyses using either high-performance liquid chromatography or gas
chromatography as the separation technique. Tandem MS has become more and more
popular compared to single-stage MS. It is essential that analytical systems are fully
validated and fit for the purpose and the assay batches are monitored with quality controls.
External proficiency programs monitor both the assay and the personnel performing the
work. For a laboratory to perform optimally, it is vital that the circumstances and context of
the case are known and the laboratory understands the limitations of the analytical systems
used, including drug stability. Drugs and poisons can change concentration postmortem due
to poor or unequal quality of blood and other specimens, anaerobic metabolism and
redistribution. The latter provides the largest handicap in the interpretation of postmortem
results [10029].
One paper reviewed chromatographic screening procedures for simultaneous detection of

several drug classes relevant to clinical and forensic toxicology or doping control in urine or
blood using gas chromatography-mass spectrometry (GC-MS), liquid chromatography
coupled with a diode-array detector (LC-DAD) or a mass spectrometer (LC-MS). The pros
and cons of the different techniques and procedures are discussed leading to the following
conclusions and perspectives. GC-MS, especially in the electron ionization full-scan mode, is
still the method of choice for comprehensive screening providing best separation power,
specificity and universality, although requiring derivatization. LC-DAD is also often used for
screening, but its separation power and its specificity are still inferior to those of GC-MS.
Finally, LC-MS has shown to be an ideal supplement, especially for the detection of more
polar, thermolabile and/or low-dose drugs, especially in blood plasma. It may become the
gold standard in clinical and forensic toxicology and doping control if, at a later date, the
costs of the apparatus will be markedly reduced, the current disadvantages like
irreproducibility of fragmentation, reduction of ionization by matrix, etc. will be overcome, and
finally if one of the increasing number of quite different techniques will become the apparatus
standard [04048].
Testing in famous cases
Katrin Krabbe et al
Manipulation of urine sampling in sports drug testing is considered a violation of anti-doping

rules and is consequently sanctioned by regulatory authorities. In 2003, three identical urine
specimens were provided by three different athletes, and the identity of all urine samples was
detected and substantiated using numerous analytical strategies including gas
chromatography-mass spectrometry with steroid and metabolite profiling, gas
chromatography-nitrogen/phosphorus detector analysis, high-performance liquid
chromatography-UV fingerprinting, and DNA-STR (short tandem repeat) analysis. None of
the respective athletes was the donor of the urine provided for doping analysis, which proved
to be a urine sample collected from other unidentified individual(s). Samples were considered
suspicious based on identical steroid profiles, one of the most important parameters for
specimen individualization in sports drug testing. A database containing 14,224 urinary
steroid profiles of athletes was screened for specific values of 4 characteristic parameters
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(ratios of testosterone/epitestosterone, androsterone/etiocholanolone, androsterone/
testosterone, and 5alpha-androstane-3alpha,17beta-diol/5beta-androstane-3alpha,17beta-
diol) and only the three suspicious samples matched all criteria. Further metabolite profiling
regarding indicated medications and high-performance liquid chromatography-UV
fingerprinting substantiated the assumption of manipulation. DNA-STR analyses
unequivocally confirmed that the 3 urine samples were from the same individual and not from
the athletes who provided DNA from either buccal cell material or blood specimens. This
supportive evidence led to punishment of all three athletes according to the rules of the
World Anti-Doping Agency. Application of a new multidisciplinary strategy employing
common and new doping control assays enables the detection of urine substitution in sports
drug testing [07043].
Lance Armstrong
One article examined the metabolic performance of an elite cyclist, Lance Armstrong, before
and after his diagnosis with testicular cancer. Although a champion cyclist in 1-day events
prior to his diagnosis of testicular cancer at age 25, he was not a contender in multi-day
endurance cycle races such as the 3-week Tour de France. His genetic makeup and
physiology (high VO2max, long femur, strong heavy build) coupled with his ambition and
motivation enabled him at an early age to become one of the best 1-day cyclists in the world.
Following his cancer diagnosis, he underwent a unilateral orchiectomy, brain surgery and
four cycles of chemotherapy. After recovering, he returned to cycling and surprisingly
excelled in the Tour de France, winning this hardest of endurance events 7 years running.
This dramatic transformation from a 1-day to a 3-week endurance champion has led many to
query how this is possible, and under the current climate, has led to suggestions of doping as
to the answer to this metamorphosis. Physiological tests following his recovery indicated that
physiological parameters such as VO2max were not affected by the unilateral orchiectomy and
chemotherapy. It was proposed that his dramatic improvement in recovery between stages,
the most important factor in winning multi-day stage races, is due to his unilateral
orchiectomy, a procedure that results in permanent changes in serum hormones. These
hormonal changes, specifically an increase in gonadotropins (and prolactin) required to
maintain serum testosterone levels, alter fuel metabolism; increasing hormone sensitive
lipase expression and activity, promoting increased free fatty acid (FFA) mobilization to, and
utilization by, muscles, thereby decreasing the requirement to expend limiting glycogen
stores before, during and after exercise. Such hormonal changes also have been associated
with ketone body production, improvements in muscle repair and haematocrit levels and may
facilitate the loss of body weight, thereby increasing power to weight ratio. Taken together,
these hormonal changes act to limit glycogen utilization, delay fatigue and enhance recovery
thereby allowing for optimal performances on a day-to-day basis. These insights provide the
foundation for future studies on the endocrinology of exercise metabolism, and suggest that
Lance Armstrong's athletic advantage was not due to drug use [07044].
Parallel investigations of saliva and urine
Stimulants are banned by the World Anti-Doping Agency (WADA) if used "in competition".
Being the analysis of stimulants presently carried out on urine samples only, it might be
useful, for a better interpretation of analytical data, to discriminate between an early intake of
the substance and an administration specifically aimed to improve the sport performance.
The purpose of one study was to investigate the differences, in terms of
excretion/disappearance of drugs, between urine and oral fluid, a sample that can reflect
plasmatic concentrations. Oral fluid and urine samples were collected following oral
711
administration of the following stimulants: modafinil (100 mg), selegiline (10 mg),
crotetamide/cropropamide (50 mg each), pentetrazol (100 mg), ephedrine (12 mg),
sibutramine (10 mg), mate de coca (a dose containing about 3mg of cocaine); analysis of
drugs/metabolites was carried out by gas chromatography/mass spectrometry (GC/MS) in
both body fluids. The results show that both the absolute concentrations and their variation
as a function of time, in urine and in oral fluid, are generally markedly different, being the
drugs eliminated from urine much more slowly than from oral fluid. The results also suggest
that the analysis of oral fluid could be used to successfully complement the data obtained
from urine for "in competition" anti-doping tests; in all those cases in which the metabolite(s)
concentration of a substance in urine is very low and the parent compound is not detected, it
is indeed impossible, relying on urinary data only, to discriminate between recent
administrations of small doses and remote administrations of higher doses [07045].
Effects of sample storage condition on salivary hormones
Measurement of steroid hormones in saliva is increasingly common in elite sport settings.

However, this environment may enforce handling and storage practices that introduce error
in measurement of hormone concentrations. It was assessed the influence of storage
temperature and duration on reproducibility of salivary steroid levels. Nine healthy adults
provided morning and afternoon saliva samples on two separate occasions. Each sample
was divided into identical saliva aliquots which were stored long-term (i.e. 28 and 84 days) at
- 80°C or -20°C (testing day 1), and short-term (i.e. 1, 3, 7 and 14 days) at 4°C or 20°C
(testing day 2). Samples were analyzed for cortisol, testosterone and estradiol using ELISA.
In non-freezer conditions, there was a decrease from baseline to 7 days in testosterone (-26
± 15 %) and estradiol (-58 ± 17 %) but not cortisol concentrations. This decrease was larger
in samples stored at room temperature than in the refrigerator. There were small but
significant changes in measured concentrations of all hormones after 28 and/or 84 days of
storage in freezer conditions, but these were generally within 12 percent of baseline
concentrations, and may be partly explained by inter-assay variability. Whole saliva samples
to be analyzed for cortisol, testosterone and estradiol should be frozen at -20°C or below
within 24 h of collection, and analyzed within 28 days. Storage of samples for measurement
of testosterone and estradiol at temperatures above -20°C can introduce large error variance
to measured concentrations [13113].
Since 2011, this category (S0) of banned substances has been a part of WADA's prohibited
list and encompasses a virtually infinite number of compounds currently not covered by any
of the other sections (e.g. anabolic agents, peptide hormones, growth factors and related
substances). New representatives of this class of compounds are low molecular weight
luteinizing hormone (LMWLH) receptor agonists, the characterization and identification of
which was presented. Focusing on two series of drug candidates based on either pyrazole or
thienopyrimidine core structures, two model substances were synthesized and used to
establish a targeted/non-targeted screening method employing both diagnostic precursor-
product ion pair detection and precursor ion scanning. In the absence of metabolism study
data, the presence of the intact drug or at least a conserved nucleus must be present to
allow the detection using the proposed strategy [13012].
Screening methods
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One paper reviewed high throughput screening procedures for the simultaneous detection of
several drug classes relevant to clinical and forensic toxicology or doping control in urine or
blood using gas chromatography-mass spectrometry (GC-MS), liquid chromatography
coupled with a diode-array detector (LC-DAD) or mass spectrometry (LC-MS). Basic
information describing these systematic toxicological analysis (STA) procedures such as the
analytes, the biosample, work-up, separation column, mobile phase or separation buffer,
detection mode and detection limits are summarized in tables arranged according to the
analytical method. Examples of typical applications are presented in figures [00025].
A comprehensive screening method for the detection of prohibited substances in doping

control is described and validated. This method is capable of detecting over 150 components
mentioned on the list of the World Anti-Doping Agency including anabolic androgenic
steroids, stimulants and all narcotic agents that are currently analysed using different
analytical methods. The analytes are extracted from urine by a combined extraction
procedure using freshly distilled diethyl ether and tert-butyl methyl ether as extraction
solvents at pH 9.5 and 14 respectively. Prior to GC-MS analysis the residues are combined
and derivatised using a mixture of N-methyl-N-trimethylsilyltrifluoroacetamide, NH4I and
ethanethiol. The mass spectrometer is simultaneously operated in the full scan mode (mass
range varies along with GC-oven temperature program) and in the selected ion monitoring
mode. . Besides narcotics, stimulants and anabolic androgenic agents, this method is also
capable of detecting several agents with anti-estrogenic activity and some beta-agonists.
This comprehensive screening method reduces the amount of urine needed and increases
the sample throughput without a loss in sensitivity and selectivity [08054].
A comprehensive screening method for the detection of prohibited substances in doping

control is described and validated. This method is capable of detecting over 150 components
mentioned on the list of WADA including anabolic androgenic steroids, stimulants and all
narcotic agents that are currently analysed using different analytical methods. The analytes
are extracted from urine by a combined extraction procedure using freshly distilled diethyl
ether and tert-butyl methyl ether as extraction solvents at pH 9.5 and 14 respectively. Prior to
GC-MS analysis the residues are combined and derivatised using a mixture of N-methyl-N-
trimethylsilyltrifluoroacetamide, NH4I and ethanethiol. The mass spectrometer is
simultaneously operated in the full scan mode (mass range varies along with GC-oven
temperature program) and in the selected ion monitoring mode. The obtained limits of
detection are in compliance with the requirements set by the World Anti-Doping Agency.
Besides narcotics, stimulants and anabolic androgenic agents, this method is also capable of
detecting several agents with anti-estrogenic activity and some beta-agonists. This
comprehensive screening method reduces the amount of urine needed and increases the
sample throughput without a loss in sensitivity and selectivity [08055].
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A simple and rapid multicomponent screening method of 130 substances for direct injections
of urine samples has been developed. The fully automated method based on ultra-
performance liquid chromatography (UPLC) and tandem mass spectrometry (MS/MS) is
used for three different classes of doping agents: diuretics, central nervous system
stimulants and opiates. The samples are diluted with buffer containing internal standards (IS)
by a pipetting robot system into 96-well plates. Samples are injected on a reversed phase
sub 2-microm particle column connected to a fast polarity switching and rapid scanning
tandem mass spectrometer with an electrospray interface. The software used to evaluate the
results produced reports containing a small-sized window for each component and a data
table list with flags to indicate any adverse analytical findings in the sample. The report can
also be processed automatically using an application software, which interpret the data and
indicate if there is a suspicious sample. One 96-well plate can be analyzed within 16 hours
[08056].
A new doping control screening method has been developed, for the analysis of doping
agents in human urine, using HPLC/orbitrap with in-source collision-induced dissociation and
atmospheric pressure chemical ionization. The developed method allows the detection of 29
compounds, including agents with antiestrogenic activity, beta2agonists, exogenous anabolic
steroids, and other anabolic agents. The mass accuracy of this method is better at 2 ppm
using an external reference. The detection limit for all compounds tested was better than 100
pg/ml. The recoveries of most analytes were above 70 percent. The measured median
repeatability values for doping agents included in the method at concentrations of 1 and 10
ng/ml were 21 and 17 percent, respectively. The relative standard deviation (RSD) of the
intraday precision (n=6) ranged from RSD 16-22 percent, whereas the interday precision
(n=18), ranged from RSD 17-26 percent, depending on the solute concentration investigated
[08057].
One paper presented a general screening method, based on liquid chromatography/mass

spectrometry (LC/MS), for the simultaneous detection in human urine of 72 xenobiotics (21
diuretics, 16 synthetic glucocorticoids, 17 beta-adrenergic drugs, 10 stimulants, 5 anti-
oestrogens and 3 anabolic steroids), excreted free or as glucuro-conjugates in urine.
Although the method has been specifically designed and evaluated in view of its potential
application to anti-doping analyses, it can also be effective in other areas of analytical
toxicology. Sample preparation was based on two liquid/liquid separation steps (performed at
alkaline and at acid pH, respectively) of hydrolyzed human urine, and then an assay by
LC/MS-MS in positive and negative ionization mode using an electrospray ionization source
(ESI) and multiple reaction monitoring as the acquisition mode. The overall time needed for
an LC run was less than 15 minutes. All compounds showed good reproducibility in terms of
both the retention times (CV %<1) and the relative abundances of the diagnostic transitions
(CV %<10). The limits of detection were in the range of 1-50 ng/mL for glucocorticoids, anti-
oestrogens and steroids, and 50-500 ng/mL for diuretics, beta-adrenergic drugs and
stimulants, thus satisfying the minimum required performance limits (MRPL) set by the World
Anti-Doping Agency for the accredited anti-doping laboratories [08058].
A general analytical platform and strategy
An effective screening procedure to identify and quantify active pharmaceutical substances

in suspected illegal medicinal products is described. The analytical platform, consisting of
accurate mass determination with liquid chromatography time-of-flight mass spectrometry
(LC-QTOF-MS) in combination with nuclear magnetic resonance (NMR) spectroscopy
provides an excellent analytical tool to screen for unknowns in medicinal products, food
supplements and herbal formulations. This analytical approach has been successfully
applied to analyze thousands of samples. The general screening method usually starts with a
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methanol extraction of tablets/capsules followed by liquid chromatographic separation on a
Halo Phenyl-Hexyl column (2.7 microm; 100 mm×2.1 mm) using an acetonitrile/0.1 percent
formic acid gradient as eluent. The accurate mass of peaks of interest was recorded and a
search made against an in-house database containing approximately 4200 substances,
mostly pharmaceutical compounds. The search could be general or tailored against different
classes of compounds. Hits were confirmed by analyzing a reference substance and/or by
NMR. Quantification was normally performed with quantitative NMR (qNMR) spectroscopy.
Applications for weight-loss substances like sibutramine and orlistat, sexual potency
enhancement (PDE-5 inhibitors), and analgesic drugs are presented in this study. It was also
identified prostaglandin analogues in eyelash growth serum, exemplified by isopropyl
cloprostenate and bimatoprost. For creams and ointments, matrix solid-phase dispersion
(MSPD) was found to give a clean extracts with high recovery prior to LC-MS analyses. The
structural elucidation of cetilistat, a new weight-loss substance recently found in illegal
medicines purchased over the Internet, was also presented [14733].
Automated sample preparation
Automation of sample preparation procedures in a doping control laboratory is of great

interest due to the large number of samples that have to be analyzed, especially in large
events where a high throughput protocol is required to process samples over 24 h. The
automation of such protocols requires specific equipment capable of carrying out the diverse
mechanical tasks required for accomplishing these analytical methodologies, which include
pipetting, shaking, heating, or crimping. An automated sample preparation procedure for the
determination of doping-related substances by gas chromatography-mass spectrometry (GC-
MS) and gas chromatography-tandem mass spectrometry (GC-MS/MS) analysis, including
enzymatic hydrolysis, liquid-phase extraction and derivatization steps, was developed by
using an automated liquid handling system. This paper presents a description of the
equipment, together with the validation data for 72 doping-related compounds including
extraction efficiency, evaluation of carry-over, interferences, and robustness. Validation was
approached as a comparison between the results obtained using the manual protocol and
the transferred automated one. The described methodology can be applied for sample
preparation in routine anti-doping analysis with high sample throughput and suitable
Multi-analyte testing
Traditionally, doping control analytical assays have been drug-class dedicated and tailored to
address requirements concerning sample preparation and chromatography/mass
spectrometry resulting from specific physicochemical properties of target compounds.
Improved analytical instrumentation (particularly based on liquid chromatography-(tandem)
mass spectrometry, LC-MS(/MS)), have enabled the development of numerous cost-effective
and rapid alternatives, allowing for multi-class/multi-analyte test methods. The trend towards
comprehensive and preferably combined targeted/non-targeted screening procedures has
been motivated in part in the requirement for analytical approaches to meet the minimum
required performance levels (MRPLs) stipulated by WADA. Within 2012, several LC-
MS(/MS)-based approaches were published representing options to complement or expand
the currently employed methodologies of doping control laboratories. Employing targeted
multiple-reaction monitoring (MRM), the detection of a total of 61 analytes (plus two internal
standards) from urine covering seven classes of prohibited substances (S1–S7) and one
agent categorized under M1 was reported. The apparatus employed consisted of a
conventional LC equipped with a C-18 reversed-phase (RP) analytical column (2×50 mm,
3microm particle size) interfaced to a triple-quadrupole MS (QqQ) via electrospray ionization
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(ESI), operated with scan-to-scan polarity switching. Urine samples were prepared for
analysis by the addition of two internal standards. An aliquot of 5 µl was injected into the LC-
MS/MS system. Gradient elution was conducted with 5 mM ammonium acetate (pH 3.5,
adjusted with acetic acid, solvent A) and acetonitrile (solvent B), completing a single run
within 10.75 min. For all target compounds, limits of detection (LODs) were far below the
aforementioned MRPLs. The unique feature of this assay compared to other multi-analyte
screening methods is the capability to detect polysaccharide-derived plasma volume
expanders (e.g. hydroxyethyl starch and dextran) by combined in-source dissociation and
subsequent MRM of diagnostic breakdown products was described. Another approach
covering 62 analytes (plus two internal standards) and five classes of prohibited substances
(S1, S3, S4, S9, and P2) was reported. Urine samples are enzymatically hydrolyzed and the
liberated phase-I-metabolites (or intact drugs) are extracted into a mixture of pentane and
diethylether. After evaporation to dryness and reconstitution, LC-MS/MS is conducted on two
different systems, both of which use 5 mM ammonium acetate (solvent A) and acetonitrile
(solvent B). Assay 1 is dedicated to the analytes of the categories S1, S3, S7, and S9 and
utilizes gradient elution on a C-12 HPLC column (2 × 50 mm, 4 microm particle size) with a
short overall run time of 4 min. Assay 2 aims at the detection of substances of the category
S4 and employs isocratic chromatography on a C-8 HPLC column (2 × 150 mm, 5 microm
particle size) at 70 percent solvent A, requiring a total run time of approximately 6 min. Mass
spectrometry is conducted in both cases with QqQ instruments operated with positive ESI
and MRM; unfortunately, no information on LODs is provided. In one study by, the value of
hydrophilic interaction liquid chromatography (HILIC) tandem mass spectrometry compared
to the commonly used, reversed-phase chromatography (and mass spectrometry) was
investigated and various options concerning column temperature, solvent composition, and
stationary phase material were evaluated. Eventually, the use of a 2.1 × 150 mm HILIC
column (5 µm particle size) operated at 35°C with 5 mM ammonium acetate (pH 4.5, eluent
A) and acetonitrile (eluent B) was considered optimal to analyze 6, 17, 4, and 17 drugs
belonging to the categories S3, S6, S7, and P2, respectively. Urine samples were prepared
for analysis by liquid-liquid extraction (LLE), the extract was concentrated and analyzed by
gradient elution on the above mentioned HILIC system followed by ESI in positive mode and
subsequent MRM detection in a single run of 14 min. The estimated LODs sufficiently met
WADA's MRPLs and the method's fitness-for-purpose was demonstrated with the required
validation process; it remains to be clarified however if omitting any hydrolysis compromises
the detection capability concerning agents largely excreted as conjugates. An assay enabling
the determination of 23 diuretics (S5) and 23 stimulants (S6) from a single urine extract was
described employing solid-phase extraction (SPE) of 1 ml of urine. The LC used in this study
was equipped with a C-18 HPLC column (2.1 × 50 mm, 3 microm particle size) and employed
0.2% formic acid (eluent A) and methanol (containing 0.2% formic acid, eluent B) for gradient
elution. In contrast to earlier methods, two separate injections for positive and negative ESI-
MS/MS (in MRM mode) were required at run times of 17 and 16 min, respectively,
necessitating the non-competitive overall measurement time per sample of 33 min. Moreover,
only one internal standard that is preferably ionized in positive mode was apparently used,
which is questionable when two separate analyses are conducted. The procedure was
validated according to applicable guidelines and LODs were accomplished satisfying
WADA's requirements. Consequently, the methodology might be fit-for-purpose if the
sample/instrument ratio and thus required analysis and reporting turn-around times are met.
While these assays are all designed to specifically measure a multitude of target compounds
with dedicated precursor-/product-ion pairs and thus gate out all other information (for the
advantage o sensitivity and speed), a trend towards combined targeted/non-targeted
analytical methods has been recognized over the last few years. Here, particularly LC-
MS(/MS) approaches with high resolution/high accuracy mass analyzers such as time-of-
flight (TOF) and orbitrap as well as hybrids consisting of quadrupole or ion trap mass
selective devices and TOF or orbitraps have been used for a variety of reasons
716
comprehensively summarized and reviewed in recent articles. The benefit of analytical
information being recorded in utmost extent (limited essentially only by sample preparation
and/or ionization capability) has been especially recognized and appreciated. Ionization
capability was subject to investigation in the development of a complementary LC-high
resolution/high accuracy MS (LC-HRMS) method in 2012. Using the so-called wrong-way-
round ionization, a total of 137 analytes belonging to the prohibited substance classes S1,
S3, S4-S7, S9, and P2 were measured in a single run (17 min) with positive ESI and HRMS.
The LC consisted of a conventional C-18 RP ultrahigh performance liquid chromatography
column (UHPLC, 2.1 × 50 mm, 1.7 µm particle size) operated under alkaline conditions with
3 mM ammonium hydroxide (pH = 10.3, solvent A) and 90% methanol (containing 3 mM
ammonium hydroxide, solvent B). Despite the use of positive ESI, the alkaline milieu
supported the generation and sensitive detection of protonated molecular species, adduct or
product ions (hence “wrong-way-round” ionization) on an LTQ-Orbitrap mass spectrometer in
full scan mode (m/z 100-650, 60.000 resolution@400 Da). Prior to analysis, urine samples
underwent enzymatic hydrolysis and LLE and all target compounds were detected in this
initial test method below respective MRPLs. Although not explicitly discussed, the presented
assay should allow retrospective data evaluation concerning compounds that possess similar
physicochemical properties as the ones tested. Covering 120 target analytes (34 diuretics,
83 stimulants, and 3 other analytes), the utility of a benchtop orbitrap mass analyzer for the
combined targeted/non-targeted analysis of drugs relevant for doping controls was also
presented. Following a ten-fold urine dilution (with addition of two internal standards),
chromatography was conducted by means of a C-8 UHPLC column (2.1 × 50 mm, 1.8 microm
particle size) and 1 mM ammonium acetate/ 0.001 percent acetic acid (solvent A) and 1 mM
ammonium acetate/0.001 percent acetic acid in methanol (solvent B). Gradient elution was
used yielding an overall run time of 10 min and the effluent was directed via ESI with scan-to-
scan polarity switching to the orbitrap analyzer. The detector was operated in full scan mode
(m/z 100–2000, 50.000 resolution@200 Da), and with the exception of glycerol, all analytes
were detected at LODs between 5 and 500 ng/ml, thus fulfilling the MRPLs stipulated by
WADA. Although included in the study, no further information on the capability to determine
glycerol at (or below) the suggested threshold of 200 microg/ml was given. Also here it is
worth mentioning that the generated and recorded data enable retrospective data mining,
facilitating follow-up or prevalence studies concerning newly observed or potential future
prohibited substances [13012].
Automation of sample preparation procedures in a doping control laboratory is of great

interest due to the large number of samples that have to be analyzed, especially in large
events where a high throughput protocol is required to process samples over 24 h. The
automation of such protocols requires specific equipment capable of carrying out the diverse
mechanical tasks required for accomplishing these analytical methodologies, which include
pipetting, shaking, heating, or crimping. An automated sample preparation procedure for the
determination of doping-related substances by gas chromatography-mass spectrometry (GC-
MS) and gas chromatography-tandem mass spectrometry (GC-MS/MS) analysis, including
enzymatic hydrolysis, liquid-phase extraction and derivatization steps, was developed by
using an automated liquid handling system. One paper presents a description of the
equipment, together with the validation data for 72 doping-related compounds including
extraction efficiency, evaluation of carry-over, interferences, and robustness. Validation was
approached as a comparison between the results obtained using the manual protocol and
the transferred automated one. The described methodology can be applied for sample
preparation in routine anti-doping analysis with high sample throughput and suitable
During the past five years, various multi-class and multi-analyte test methods have been
developed for sports drug testing purposes, fuelled by the increasing demand of
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incorporating more and more substances (and respective metabolites) into routine doping
controls without sacrificing the sample volume, faster turnaround times (particularly in case of
great sport events), as well as the capability of modern mass spectrometers to provide the
required scan speed and/or resolving power to cover hundreds of analytes per analytical run.
Its fitness-for-purpose was impressively demonstrated by its use during the 2012 Olympic
Games in London, where over 5000 samples were analyzed within a period of one month.
Moreover, the generated data allow for the nowadays frequently requested re-evaluation
concerning additional compounds and metabolites that were not screened for at the time of
the Games [13009].
Aiming at faster and/or simpler analytical approaches for drug testing in general, the potential
utility of evolving ambient mass spectrometry techniques has continued to be discussed. In
proof-of-concept studies, the analyses of drugs of abuse and occasionally doping agents by
means of, for example, desorption electrospray ionization (DESI), extractive electrospray
ionization (EESI), or electric discharge-based methodologies such as direct analysis in real
time (DART) were presented. Despite their ease-of-use and rapid generation of results, the
lack in comprehensiveness and capability to allow for the often required separation of
isomeric compounds, which is often required in sports drug testing, has been a major
obstacle in introducing these techniques in routine doping controls. Similarly, the undisputed
swiftness of matrix-assisted laser desorption ionization (MALDI)-MS(/MS) in drug detection in
biological matrices has so far not been considered a viable means for sports drug testing,
mainly due to the limitations resulting from omitting chromatographic separation of analytes
and, in selected cases, insufficient detection limits [13009].
”Alternative” specimens
The use of alternative specimens in the field of toxicology was first described in 1979, when
hair analysis was used to document chronic drug exposure. Since then, the use of this
”alternative” samples has gained tremendous importance in forensic toxicology, as well as in
clinic toxicology, doping control and workplace drug testing. It is not surprising, therefore, that
a large number of papers dealing with the determination of several classes of drugs in saliva,
sweat, meconium and hair have been published ever since, owing to the fact that
chromatographic equipment is becoming more and more sensitive, mass spectrometry (and
tandem mass spectrometry) being the most widely used analytical tool, combined with gas or
liquid chromatography. ”Alternative” specimens present a number of advantages over the
traditional samples normally used in toxicology (e.g. blood, urine and tissues), namely the
fact that their collection is not invasive, their adulteration is difficult, and they may allow
increased windows of detection for certain drugs. The main disadvantage of this kind of
samples is that drugs are present in very low concentrations, and therefore high-sensitivity
techniques are required to accomplish the analysis [08059].
Drug identification
Except for proteins, such as EPO, most prohibited drugs are identified by GC-MS, the
workhorse of doping-control laboratories. LC-MS is used increasingly for diuretics, some
anabolic steroids, and corticosteroids. Doping-control scientists identify a substance, in the
laboratory and in court, by matching chromatographic retention time and mass spectra
between unknown and standard. They need an authentic reference standard – a sample of
the substance, certified to be correct. The standard may be a white powder or an excretion
urine from a volunteer who took the drug. Chromatography coupled with MS makes it
possible to identify not just drug classes, but specific chemicals, with absolute certainty.
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Pharmaceuticals include some synthetic compounds that do not occur naturally (e.g the
anabolic steroid stanozolol) and some that do (e.g. testosterone). Unfortunately, GC-MS and
LC-MS cannot distinguish natural, endogenous testosterone from pharmaceutical,
exogenous testosterone; however, normal human urine samples contain a testosterone
isomer with no known function, epitestosterone. The urinary ratio of testosterone to
epitestosterone (T/E ratio) is roughly 1:1 in most normal men, and it increases upon
testosterone administration. Since the 1984 Olympics, the T/E ratio has been used to screen
for testosterone use. Adverse analytical findings are defined by a T/E cut-off, which currently
is 4. The two problems with any cut-off are that rare, drug-free individuals might have a
naturally elevated T/E and that T/E may never exceed the cut-off in some users, either
because their T/E is not responsive to administration or because they use small doses and
titrate themselves. To distinguish users from nonusers, longitudinal profiling consists of
plotting T/E and other urinary androgen parameters over time, expecting stability for
nonusers and a spike for users. In the 1990s a new approach was introduced: isotope ratio
MS (IRMS) [07046].
Blood tests
Serum can be tested by LC-MS-MS to detect hemoglobin-based oxygen carriers and by

immunoassay to detect recombinant human growth hormone (GH). Natural GH is a family of
isoforms, including a major one of 22 kd (22,000 atomic mass units) and some non–22-kd
isoforms, whereas recombinant GH is 100 percent 22-kd isoforms. Administration of
recombinant GH suppresses endogenous GH production. The current approach to
recombinant GH detection in serum is based on estimating the ratio of the 22-kd isoform to
non–22-kd isoforms by immunoassay; it can detect administration for 3 hours after the last
dose [07046].
Laboratory report interpretation
The laboratory urine drug test can determine what substance is present in the urine sample,
not the brand, formulation, route of administration, dose, or how long before urine collection
the drug was taken. Reasons why a urine drug test is negative include the drug is not
prohibited by this program; the drug was never used; the drug was used long enough ago to
have been eliminated completely; the drug is present below the cut-off; the drug is present
below the limit of detection of the test; the drug is a prohibited (designer) drug that the
laboratory does not look for; the sample was manipulated; and the sample was not real urine.
The latter can be revealed by steroid screen data devoid of natural steroids in cases that
would be missed by commercial adulteration tests and dipsticks. Many factors determine test
retrospectivity, or how long after the end of administration the test can detect the drug in
urine: among them is the dose, body burden, elimination pharmacokinetics, and test
sensitivity. Anabolic steroids can be detected for as little as only a few days or as long as
many months after the user stops taking them, depending on the type used (e.g. short-acting
pill or long-acting oily injection), how much was used, and for how long. In addition, some
steroids are easier to detect than others because of chemical differences. Individuals who
have been in a drug-testing program for some time are less likely to use long-acting, easy-to-
detect steroids.
Prohibited drug Period of detectability after the last dose
Stimulants A few hours to a few days

Anabolic steroid A few days (short acting, water soluble, small doses)
to many months (long-acting oily injections, large
719
dose)
Diuretics A few hours to a few days
Marijuana Some weeks
rEPO A few days
The test results on a follow-up sample collected some times after an initial, positive sample
needs to be interpreted in light of the above. If the follow-up test is positive for the same
drug, it may be because the drug was not completely eliminated yet or because the athlete
used the drug again in the meantime. Comparing the laboratory data from both tests may or
may not provide an indication of which is the case. The follow-up test is expected to be
negative if the drug was eliminated completely. This is why a negative follow-up test is not
relevant to determining the accuracy or inaccuracy of a positive result on a sample collected
previously. Conversely, a negative follow-up test is a valid check that the athlete has stopped
using the drug. Drug users who expect to be tested at events try to time their discontinuation
to pass the test; this is why no-notice, out-of-competition testing was implemented in the
1980s [07046].
Accuracy of testing
It is said that the test is blind to designer steroids, because the test is targeted and finds only
what it looks for. Typically, WADA-accredited laboratories screen for most anabolic steroids
by GC-MS in the more sensitive SIM mode, monitoring only a few ions per target compound
(e.g. an ion of 415 atomic mass units). A designer steroid could differ from a known one by
only two extra hydrogens, give an ion of 417 atomic mass units upon fragmentation, and
escape detection because the test monitors 415, not 417. Or the designer steroid could
fragment to ions that happen to be monitored, in which case data readers would see
suspicious signals and investigate further. The first reported designer steroid (norbolethone)
was a pharmaceutical abandoned decades before, during clinical trials. It resurfaced upon
further investigation of an athlete's urine sample devoid of normal endogenous androgens, a
telltale sign of endocrine suppression, which is expected after androgen administration
because of negative feedback. The second designer steroid (THG) was discovered because
a coach turned in a used syringe. THG simply is not detected in the standard steroid screen,
probably because its chemical properties are such that it disintegrates along the way.
Different modifications of the screen now allow its detection [07046].
Are the tests accurate? What are the risks of “false positive” or “false negative”? Both
phrases can have widely different meanings in common language compared with antidoping
jargon. In common language, a “false positive” might be any adverse analytical finding that
does not result in a sanction, perhaps because the athlete had a therapeutic use exemption,
because a courier's signature was missing on a shipping document, or because the
prohibited drug was a supplement contaminant. A case in which on appeal, an arbitrators'
panel had purely legal reasons to exonerate the athlete, might casually be called a “false
positive.” But for the laboratory, a false positive is only the case in which the laboratory
reports the presence of a drug and it is later proven scientifically that the drug was not
present. The same misinterpretation or mis-semantic is for a “false negative,” in common
language that might be a case where the athlete used a drug but passed the test. This could
be because the metabolite was accurately detected just below the cut-off – a perfectly
accurate negative result [07046].
Chromatography
720
Chromatography is an analytical chemistry technique used to separate (resolve) the
chemical compounds in a mixture. Gas chromatography (GC) is done in the gas phase. A
gas chromatograph has three parts: a sample introduction system (injector), an oven
containing a chromatography column to achieve separation, and a detector. Typically, a
microliter of liquid urine extract is automatically injected into the injector, a chamber at a high
temperature. The sample is vaporized and swept along a hair-thin glass tube (capillary
column, many meters long, flexible enough to be rolled up in a coil) by a carrier gas (mobile
phase), such as helium. Different compounds travel at different speeds because of the
differences in boiling point, polarity, and relative solubility in the carrier gas versus the
coating of the inner wall of the column (stationary phase). The compounds emerge from the
column outlet at different times after injection (the chromatographic retention time),
separated from each other. Under identical operating conditions, the retention time is
characteristic of each chemical compound. If two compounds have the same retention time,
they may be identical (e.g. testosterone). If two compounds have different retention times,
they certainly are different (e.g. testosterone and methyltestosterone). Matching retention
times between an unknown and a reference standard is one element of identification. A
graph of the amount of substance as a function of the retention time is a chromatogram. Two
common GC detectors in antidoping laboratories are the nitrogen-phosphorus detector
(NPD) and the mass spectrometer. The NPD detector is ideal for detecting nitrogen-
containing compounds, such as stimulants [07046].
Mass spectrometry
One review introduced fundamental aspects of mass spectrometry (MS) based proteomics
and illustrates how MS is an effective tool for the analysis of glycoprotein hormones. Matrix-
assisted laser desorption ionization time-of-flight (MALDI-TOF) and electrospray ionization
(ESI) MS are complementary approaches that have been applied for the analysis of
gonadotropins, e.g. to characterize differences in the oligosaccharide distribution of
commercial human chorionic gonadotropin preparations, for isolated nicked beta-subunit,
and identification of a metabolite of placental transforming growth factor in pharmaceutical
hCG preparations. Immunoaffinity trapping and concentration of digested sample extract
prior to MS analysis confers analytical sensitivity akin to immunoassay. A desirable objective
would be to develop for clinical purposes a rapid procedure for MS detection and
characterization of gonadotropins. Refinement of on-target immobilization and digestion for
subsequent ionization by MALDI may eventually help to provide this capability. The advent of
hybrid mass spectrometers will further advance the characterization of these complex
molecules [06036].
Mass spectrometry (MS) is an analytical chemistry technique used for structure elucidation of
unknowns or identification of known compounds. A mass spectrometer has three parts: an
ion source where the compound is ionized to form a molecular ion and fragmented into
smaller ions; a mass filter that separates ions by mass-to-charge ratio (m/z); and a detector.
The graph of ion abundance as a function of m/z is a mass spectrum. The fragmentation
pattern is determined by weak bonds and other physicochemical characteristics; therefore,
fragmentation is reproducible and characteristic of the molecular structure, and the mass
spectrum is like a fingerprint of the compound. Matching mass spectra between an unknown
and a reference standard is another element of identification. Significant ions are so
characteristic that matching only three ions (e.g. 143, 345, 360) and their percent abundance
relative to the most intense of the three (e.g 143) has long been widely accepted as proof of
chemical identification [07046].
721
The identification power of mass spectrometry has enabled the determination of hundreds of
prohibited drugs in doping-control analysis. A few years ago, its utility was extended to
peptide hormones such as erythropoietins, synthetic insulins and corticotrophins detectable
in blood or urine. New assays have been established to improve the fight against doping,
employing highly selective and sensitive detection methods based on chromatographic and
tandem mass spectrometric techniques [07047].
Owing to the sensitive, selective, and unambiguous nature of mass spectrometric analyses,
chromatographic techniques interfaced to various kinds of mass spectrometers have become
the most frequently employed strategy in the fight against doping. To obtain utmost
confidence in analytical assays, mass spectrometric characterization of target analytes and
typical dissociation pathways have been utilized as basis for the development of reliable and
robust screening as well as confirmation procedures. Methods for qualitative and/or
quantitative determinations of prohibited low and high molecular weight drugs have been
established in doping control laboratories preferably employing gas or liquid chromatography
combined with electron, chemical, or atmospheric pressure ionization followed by analyses
using quadrupole, ion trap, linear ion trap, or hyphenated techniques. The versatility of
modern mass spectrometers enable specific as well as comprehensive measurements
allowing sports drug testing laboratories to determine the misuse of therapeutics such as
anabolic-androgenic steroids, stimulants, masking agents or so-called designer drugs in
athletes' blood or urine specimens, and a selection of recent developments was summarized
in one review [07048].
Fast and unequivocal drug detection is of considerable importance in numerous fields of

analytical chemistry, and today mass spectrometry-based approaches are often the method
of choice due to their sensitive and specific nature. Mass spectrometry is in constant flux with
innovations and thus supports the development of new, complementary assays for rapid
determination of drugs and toxins as well as their metabolic products in clinical, forensic, and
doping control laboratories. Examples of such innovations that have greatly aided the
worldwide bioanalytical efforts are the modern improvements in ion trap, for example, Fourier
transform-ion cyclotron resonance (FT-ICR) mass spectrometer – Orbitrap, and time-of-flight
(TOF) mass analyzers when coupled with sensitive ionization techniques such as
electrospray ionization. In this perspective, the utility of state-of-the-art mass spectrometers
and recent instrumental developments such as new and/or improved hybrid analyzers were
discussed and selected applications presented [12066].
Since the installation of anti-doping rules and regulations and their international enforcement
in the mid-1960s, mass spectrometry has been an integral part of doping control procedures.
Although its utility was limited in the first decade, instrumental improvements and method
optimizations have made mass spectrometry, in all its facets, an indispensable tool in
modern sports drug testing. In this review, milestones in doping control analysis
accomplished in Germany and reaching from the early developments to the current use of
hyphenated mass spectrometric techniques concerning low- and high molecular mass
analytes are presented. The considered drug classes include anabolic agents, peptidic
drugs, nucleotide-derived therapeutics, approved and non-approved organic as well as
inorganic analytes, and particular focus is put on drug class- and instrument-driven strategies
[150124].
Drug confirmation by mass spectrometry
Drug confirmation by mass spectrometry coupled with chromatography is essential to
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toxicology, doping control, pain management, and workplace drug testing. High confidence in
this technology is due to its superior specificity and sensitivity. However, there are challenges
associated with drug confirmation, and proper setup and validation of these assays are
important in assuring high-quality results. In one article, assay parameters required for drug
confirmation are summarized based on recent scientific publications, various established
guidelines, and our own practical experience. Factors affecting the result quality and correct
results interpretation are critically reviewed. Several emerging technologies and their
potential applications are briefly explored [150125].
Small molecules
Recent developments in MS for the detection of small molecules in the context of doping
control analysis are reviewed. Doping control analysis is evolving together with MS, which is
the technique of choice in order to accomplish the analytical requirements in this field. Since
these analytical requirements for the detection of a doping agent depend on the substance,
in the first section we review the different scenarios. The commonly established approaches,
together with their achievements and drawbacks are described. New developments in
hyphenated MS techniques (both GC-MS/MS and LC-MS/MS) concerning interfaces and
analyzers are mentioned. The use (or potential use) of these developments in order to
minimize the limitations of the commonly established approaches in the doping control field is
discussed. Finally, a brief discussion about trends and remaining limitations is presented
[12067].
Small peptides in the urine

Although significant progress has been achieved during the past few years with the
introduction of new assays and analytical methodologies, the detection and quantification of
protein analytes, in particular of peptide hormones, continues to pose analytical challenges
for the World Anti-Doping Agency-accredited anti-doping laboratories. In one article, the
latest achievements in the application of MS-based methodologies and specific biochemical
and immunological assays to detect some of the prohibited substances listed in section S2 of
the World Anti-Doping Agency List of Prohibited Substances and Methods are reviewed. In
addition, it was looked towards the future by focusing on some of the most promising
analytical approaches under development for the detection of so-called biomarkers of doping
[068].
In one study, a screening assay was developed comprising 11 prohibited peptides (<1.5 kDa)
that are sufficiently purified from urine using weak cation exchange with subsequent
determination of all substances by means of nanoUHPLC separation coupled to high
resolution tandem mass spectrometry. These peptides included Gonadorelin (LH-RH),
Desmopressin and 9 growth hormone releasing peptides (GHRP-1, -2, -4, -5, -6, Hexarelin,
Alexamorelin, Ipamorelin and a GHRP-2 metabolite); however, the procedure is expandable
to further target analytes or metabolites. The method was validated with a main focus on
qualitative result interpretation considering the parameters specificity, linearity (0-500 pg/mL),
recovery (45-95 %), precision (<20 % at 100 pg/mL), limits of detection (2-10 pg/mL),
robustnesss and ion suppression. The proof-of-principle was shown by analysing excretion
study urine samples for LHRH, desmopressin and GHRP-2 [12065].
For most peptide hormones prohibited in elite sports the concentrations in plasma or urine
are very low (pg/mL). Accordingly, hyphenated purification and enrichment steps prior to
mass spectrometric detection are required to obtain sufficient doping control assays.
Immunoaffinity purification in combination with nano-scale liquid chromatography coupled to
high resolution/high accuracy mass spectrometry was found to have the potential of
723
providing the necessary sensitivity and unambiguous specificity to produce reliable results.
With the presented methodology 12 prohibited peptides (porcine insulin, Novolog, Apidra,
Lantus DesB30-32 metabolite, Humalog and human insulin, Synacthen (synthetic ACTH
analogue), luteinizing hormone-releasing hormone (LH-RH), growth hormone releasing
hormone (GH-RH(1-29)) and CJC-1295 (GH-RH analogue), LongR(3)-IGF-1 and IFG-1)
were simultaneously purified from plasma/serum or urine. With limits of detection for each
target compound ranging in the low pg/mL level (urine), the method enables the
determination of urinary peptides at physiologically relevant concentrations. For each class of
peptides an appropriate antibody and a respective internal standard was implemented
ensuring robust analysis conditions. Due to the fast and simple sample preparation
procedure (about 25 samples per day) and the fact that all materials are commercial
available, the implementation of the methodology to laboratories from other analytical fields
(forensics, pharmacokinetic sciences, etc.) is enabled [12070].
Detection of misuse of peptides and proteins as growth promoters is a major issue for sport
and food regulatory agencies. The limitations of current analytical detection strategies for this
class of compounds, in combination with their efficacy in growth-promoting effects, make
peptide and protein drugs highly susceptible to abuse by either athletes or farmers who seek
for products to illicitly enhance muscle growth. Mass spectrometry (MS) for qualitative
analysis of peptides and proteins is well-established, particularly due to tremendous efforts in
the proteomics community. Similarly, due to advancements in targeted proteomic strategies
and the rapid growth of protein-based biopharmaceuticals, MS for quantitative analysis of
peptides and proteins is becoming more widely accepted. These continuous advances in MS
instrumentation and MS-based methodologies offer enormous opportunities for detection and
confirmation of peptides and proteins. Therefore, MS seems to be the method of choice to
improve the qualitative and quantitative analysis of peptide and proteins with growth-
promoting properties. This review aims to address the opportunities of MS for peptide and
protein analysis in veterinary control and sports-doping control with a particular focus on
detection of illicit growth promotion. An overview of potential peptide and protein targets,
including their amino acid sequence characteristics and current MS-based detection
strategies is, therefore, provided. Furthermore, improvements of current and new detection
strategies with state-of-the-art MS instrumentation are discussed for qualitative and
quantitative approaches [13091].
Isotope ratio mass spectrometry or carbon isotope ratio
It so happens that there is a measurable difference in carbon-13 content between

endogenous and pharmaceutical testosterone. Most carbon atoms in nature are carbon-12,
with a nucleus containing six protons and six neutrons. Radiocarbon dating relies on the rare
carbon-14, an unstable, radioactive isotope, with a nucleus containing six protons and eight
neutrons, which decays over time. Between the two is carbon-13, a stable isotope with six
protons and seven neutrons. Roughly 1.1 percent of carbon in nature is carbon-13.
Pharmaceutical testosterone contains a few parts per thousand less carbon-13 than does
natural testosterone. This is because they arise from biosynthetic pathways that are
sufficiently different. Humans make endogenous testosterone from cholesterol, itself made
from acetate or coming from the diet. Pharmaceutical companies make testosterone by
semisynthesis from plant sterols. All carbon in living beings is ultimately derived from
atmospheric carbon dioxide (CO2), fixed in plants by photosynthesis. Different plants make
the first multicarbon intermediates and downstream biosynthetic compounds differently.
Animals eat plants, humans eat plants and animals, and “we are what we eat”. At every
biosynthetic step, carbon-13 is left behind. This is because of the isotopic effect: chemical
reactions go faster with lighter compounds; the molecule with a carbon-12 reacts sooner than
724
the molecule with a carbon-13 instead. Because the pathways from atmospheric CO2 to
endogenous or pharmaceutical testosterone are different enough, carbon-13 is depleted to
different extents; the difference happens to be measurable [07011].
Full-capillary sample injection combined with sweeping CE stacking
One study described an on-line stacking CE approach by sweeping with whole capillary
sample filling for analyzing five anabolic androgenic steroids in urine samples. The five
anabolic steroids for detection were androstenedione, testosterone, epitestosterone,
boldenone, and clostebol. Anabolic androgenic steroids are abused in sport doping because
they can promote muscle growth. Therefore, a sensitive detection method is imperatively
required for monitoring the urine samples of athletes. In this research, an interesting and
reliable stacking capillary electrophoresis method was established for analysis of anabolic
steroids in urine. After liquid-liquid extraction by n-hexane, the supernatant was dried and
reconstituted with 30 mM phosphate buffer (pH 5.00) and loaded into the capillary by
hydrodynamic injection (10 psi, 99.9 s). The stacking and separation were simultaneously
accomplished at -20 kV in phosphate buffer (30 mM, pH 5.0) containing 100 mM sodium
dodecyl sulfate and 40 percent methanol. During the method validation, calibration curves
were linear over a range of 50-1,000 ng/mL for the five analytes. In the evaluation of
precision and accuracy for this method, the absolute values of the RSD and the RE in the
intra-day (n=3) and inter-day (n=5) analyses were all less than 6.6 percent. The limit of
detection for the five analytes was 30 ng/mL (S/N = 5, sampling 99.9 s at 10 psi). Compared
with simple MECK, this stacking method possessed a 108- to 175-fold increase in sensitivity.
This simple and sensitive stacking method could be used as a powerful tool for monitoring
the illegal use of doping [12064].
Benchtop quadrupole-orbitrap hybrid mass spectrometry
Sensitive and unequivocal determination of analytes/contaminants in complex matrices is a

challenge in the field of food safety control. In this study, various acquisition modes (Full
MS/AIF, Full MS+tMS/MS, Full MS/dd MS/MS and tSIM/ddMS/MS) and parameters of a
quadrupole-orbitrap hybrid mass spectrometer (Q Exactive) were studied in detail. One of the
main conclusions has been that, reducing the scan range for Full MS (using the quadrupole)
and targeted modes give higher signal-to-noise (S/N) ratios and thereby better detection
limits for analytes in matrix. The use of Q Exactive in a complex case, for the confirmatory
analysis of hormones in animal urine is presented. A targeted SIM data dependent MS/MS
(tSIM/ddMS/MS) acquisition method for determination of eight synthetic hormones
(trenbolone, 17α ethinylestradiol, zeranol, stanozolol, dienestrol, diethylstilbestrol, hexestrol,
taleranol) and a naturally occurring hormone (zearalenone) in animal urine were optimized to
have sensitive precursors from targeted SIM mode and trigger MS/MS scans over the entire
chromatograph peak. The method was validated according to EC/657/2002. CCalpha
(decision limit) for the analytes ranged between 0.11 microg/L and 0.69 microg/L and CCbeta
(detection capability) ranged between 0.29 microg7L and 0.90 microg/L [13094].
Chromatographic-mass spectrometry
Urine samples have been the predominant matrix for doping controls for several decades.
However, owing to the complementary information provided by blood (as well as serum or
plasma and dried blood spots (DBS)), the benefits of its analysis have resulted in
continuously increasing appreciation by anti-doping authorities. On the one hand, blood
samples allow for the detection of various different methods of blood doping and the abuse of
erythropoiesis-stimulating agents (ESAs) via the Athlete Biological Passport; on the other
725
hand, targeted and non-targeted drug detection by means of chromatographic-mass
spectrometric methods represents an important tool to increase doping control frequencies
out-of-competition and to determine drug concentrations particularly in in-competition
scenarios. Moreover, blood analysis seldom requires in-depth knowledge of drug
metabolism, and the intact substance rather than potentially unknown or assumed metabolic
products can be targeted. In this review, the recent developments in human sports drug
testing concerning mass spectrometry-based techniques for qualitative and quantitative
analyses of therapeutics and emerging drug candidates are summarized and reviewed. The
analytical methods include both low and high molecular mass compounds (e.g., anabolic
agents, stimulants, metabolic modulators, peptide hormones, and small interfering RNA
(siRNA)) determined from serum, plasma, and DBS using state-of-the-art instrumentation
such as liquid chromatography (LC)-high resolution/high accuracy (tandem) mass
spectrometry (LC-HRMS), LC-low resolution tandem mass spectrometry (LC-MS/MS), and
gas chromatography-mass spectrometry (GC-MS) [13096].
Miniaturized competitive immunoassays on a protein chip
Gas chromatography-mass spectrometry (GC-MS) serves as an effective reference method,

but it is limited by low throughput and is therefore not suitable for large-scale screening. Use
of protein chips for high-throughput screening of all athletes for prohibited substances could
become an important complementary tool to GC-MS. It was developed a protein chip based
on an aldehyde-activated glass slide containing 10 physically isolated arrays. The chip was
used to screen urine from 1347 athletes for prohibited substances and to screen a negative
control group consisting of 200 females and 120 males. Urine samples from 66 individuals
known to be abusers, provided by the China Doping Control Center (CDCC), and 129
standard prohibited substances were tested as positive controls. All 1347 urine samples
screened by means of the protein chips were also subjected to reference analysis by GC-MS
at the CDCC. There was no qualitative difference between the results obtained with the two
methods. The correlation coefficient for the quantitative results obtained with the protein chip
and GC-MS was 0.991. It was concluded that the protein chip could be used to screen for a
series of 16 prohibited drugs in urine samples. This system has the potential to become an
effective screening method to test substances prohibited by the International Olympic
Committee [04046].
A unification of doping-control screening procedures of prohibited small molecule substances

may conceptually be achieved by the use of a combination of one gas chromatography-time-
of-flight mass spectrometry method and one liquid chromatography-time-of-flight mass
spectrometry method. In one work a quantitative screening method using high-resolution
liquid chromatography in combination with accurate-mass time-of-flight mass spectrometry
was developed and validated for determination of glucocorticosteroids, beta2-agonists,
thiazide diuretics, and narcotics and stimulants in urine. To enable the simultaneous isolation
of all the compounds of interest and the necessary purification of the resulting extracts, a
generic extraction and hydrolysis procedure was combined with a solid-phase extraction
modified for these groups of compounds. All 56 compounds are determined using positive
electrospray ionisation with the exception of the thiazide diuretics for which the best
sensitivity was obtained by using negative electrospray ionisation. The results show that, with
the exception of clenhexyl, procaterol, and reproterol, all compounds can be detected below
the respective minimum required performance level and the results for linearity, repeatability,
within-lab reproducibility, and accuracy show that the method can be used for quantitative
726
screening. If qualitative screening is sufficient the instrumental analysis may be limited to
positive ionisation, because all analytes including the thiazides can be detected at the
respective minimum required levels in the positive mode. The results show that the
application of accurate-mass time-of-flight mass spectrometry in combination with generic
extraction and purification procedures is suitable for unification and expansion of the window
of screening methods of doping laboratories. Moreover, the full-scan accurate-mass data
sets obtained still allow retrospective examination for emerging doping agents, without re-
analyzing the samples [10034].
Whereas GC is done in the gas phase, liquid chromatography (LC) is done in the liquid
phase. This is a crucial difference because it works for thermolabile compounds (destroyed
by GC) and polar compounds (cannot be vaporized). The separation principles are the same.
A typical high-pressure or high-performance LC (HPLC) column is a steel tube the size of a
fat marker pen, packed with microbeads on the surface of which is the stationary phase. The
mobile phase is a liquid solvent, often a mixture whose composition is programmed to
change during the run (gradient elution). Two common HPLC detectors are the diode-array
detector (DAD) and the mass spectrometer. The DAD monitors UV absorption over a range
of wavelengths or at selected wavelengths; it detects only those compounds that absorb UV
light. When the HPLC is connected to an MS, the instrument is called LC-MS. The most
advanced type of LC-MS can do tandem MS by one of several choices of conceptual and
hardware approaches. It is called LC-MS-MS or LC-tandem MS. For a given class of drugs,
such as diuretics, the LC-tandem MS screen is far superior to the GC-MS screen. Sample
preparation time can be well less than 1 hour, down from a full day's work, because unlike
GC, LC does not require the removal of water or salts, deconjugation or derivatization.
Typically, the instrumental analysis run-time is two to three times shorter, well under 10
minutes per sample, because LC-MS-MS is blind to interferences; therefore,
chromatographic resolution is not required, and LC run times can be shortened [07046].
The technique of liquid chromatography used in concert with (tandem) mass spectrometry
(LC-MS/MS) has complemented sports drug testing strategies ever since soft ionization
interfaces such as electrospray or atmospheric pressure chemical ionization (ESI or APCI,
respectively) became commercially available. Numerous applications have been developed
that allow the determination of prohibited therapeutics that are barely detectable or
undetectable with conventional gas chromatographic-mass spectrometric techniques (GC-
MS). Due to the progressive nature of doping controls, the continuously changing demands
originating from the dynamic pharmaceutical market, new illegal approaches that presumably
increase athletic performance, and modifications to the lists of prohibited compounds of
regulative authorities such as the World Anti-Doping Agency (WADA), numerous new
applications and drug-testing strategies based on LC-MS(/MS) are frequently developed in
order to improve the portfolios of drug-testing laboratories. Liquid chromatography-(tandem)
mass spectrometry (LC-MS/MS) has therefore revolutionized the detection assays used in
doping control analysis. New methods have enabled the determination of drugs that were
formerly difficult to detect or undetectable at preceding sample concentrations, and complex
and/or time-consuming procedures based on alternative chromatographic-mass
spectrometric or immunochemical principles have been replaced by faster, more
comprehensive and robust assays. Analytical assay sensitivity and throughput are key
factors for the LC-MS(/MS) approaches used in sports drug testing. Considerable
improvements in liquid chromatography (such as using monolithic or UPLC columns) have
resulted in shorter analysis times and significantly narrower peaks, which in turn lead to
enhanced signal-tonoise ratios, better detection limits, and increased productivity. However,
these fast analytical runs require very short mass spectrometric cycle times in order to obtain
sufficient data points per compound and corresponding peak. In order to incorporate newly
developed methods into sports drug testing systems, identification criteria are required,
727
which have only been established for low molecular weight drugs. Several aspects are
applicable and transferable to peptide and protein analysis, but specific issues – such as
molecular weight determination from multiply charged species or bottom-up sequencing
[07050].
Doping control analytical laboratories for human sports predominantly employ nowadays
chromatographic-mass spectrometric test methods for routine, high throughput screening
and confirmation assays concerning low and high molecular mass analytes. Liquid
chromatography-(tandem) mass spectrometry (LC-MS(/MS)) and particularly ultrahigh
pressure liquid chromatography (UHPLC)-MS/MS instruments have become devices of
choice due to their indispensable capabilities that compensate for limitations inherent to other
commonly used strategies such as immunological and gas chromatography-(tandem) mass
spectrometry (GC-MS(/MS))based detection methods. UHPLC-MS/MS-based assays at low
mass spectrometric resolution have been established allowing for fast and sensitive targeted
analyses focusing on pre-selected target analytes with diagnostic precursor-product ion
pairs. Combining UHPLC to high resolution/high accuracy MS(/MS) further expanded the
targeted approach (i.e., plotting extracted ion chromatograms of protonated or deprotonated
molecules as well as product ions measured with accurate masses) toward non-targeted
analyses enabling also retrospective data mining. In one review, recent applications of
UHPLC-MS/MS in sports drug testing procedures published between 2008 and 2012 were
presented and advantages as well as limitations in a short- and long-term perspective are
discussed [12057].
Drug testing for sports doping control programs is extensive and includes numerous classes
of banned compounds including anabolic androgenic steroids, beta2-agonists, hormone
antagonists and modulators, diuretics, various peptide hormones, and growth factors. During
competition, additional compounds may also be prohibited such as stimulants, narcotics,
cannabinoids, glucocorticosteroids, and beta-blockers depending both on the sport and level
of competition. Each of these classes of compounds can contain many prohibited substances
that must be identified during the testing procedure. Various methods that have been
designed to detect a large number of compounds in different drug classes are highly
desirable as initial screening tools. Liquid chromatography/tandem mass spectrometry (LC-
MS/MS) is widely used by anti-doping testing laboratories for this purpose and several rapid
methods have been described to simultaneously detect different classes of compounds.
Here, we describe a simple urine sample cleanup procedure that can be used to detect
numerous anabolic androgenic steroids, beta2-agonists, hormone antagonists and
modulators, glucocorticosteroids, and beta-blockers by LC-MS/MS [12058].
Hydrophilic interaction liquid chromatography
The chromatographic behaviour of 44 polar compounds (23 beta-adrenergic agents, 11

stimulants, 4 narcotics and 6 phenolalkylamines) included in the list of prohibited substances
and methods of the World Anti-Doping Agency, has been investigated under hydrophilic
interaction liquid chromatography conditions by application of different mobile phase
compositions (percentage of the organic solvent, type and amount of mobile phase additive
and ionic strength) and column temperatures. Detection of analytes was performed by a
triple quadrupole mass spectrometer in positive ionization mode and selected reaction
monitoring acquisition mode after liquid/liquid extraction. Data collected using as stationary
phase type-B silica materials from different producers, showed that the best chromatographic
conditions in terms of peak shape, selectivity and chromatographic retention were obtained
using an initial percentage of acetonitrile of 90 percent, a column temperature of 35 °C, a
mobile phase pH of 4.5 and ammonium acetate (5 mM) and acetic acid (0.1 %) as mobile
728
phase additives. The selected chromatographic conditions were used to develop screening
and confirmation analytical procedures to detect polar compounds in human urine for
antidoping purpose. The developed methods were validated in terms of specificity, matrix
effect, linearity, precision, accuracy, sensitivity, robustness and repeatability of retention
times and relative ion abundances. Such methods offer attractive alternatives and
considerable advantages over traditional approaches especially for the analysis of the
phenolalkylamines [11566].
Ultra-high-performance liquid chromatography
Doping control analyses are generally performed in urine, a matrix that provides a prolonged
detection time window, and less often in blood, serum, plasma, hair, saliva, and nails. To
identify the chemical structures of anabolic steroids the use of mass spectrometry detection
is very advantageous. Gas chromatography-mass spectrometry (GC-MS) techniques allowed
for the development of comprehensive screening methods. GC-MS methods are sensitive
and robust but present the disadvantages of time-consuming sample pretreatment, that is
often based on hydrolysis and derivatisation reactions. Liquid chromatography-mass
spectrometry (LC-MS) methods have been successfully used to identify and determinate
steroids in different matrices, as well as to study their metabolisms. Nowadays, automatic
rapid ultra high performance liquid chromatography (UHPLC) tandem mass spectrometry has
become the technique of choice for steroid analysis. Due to its generally higher speed,
sensitivity, reproducibility and specificity with respect to HPLC, it can be used to
simultaneously separate and determinate multi component steroid mixtures. The technique is
of huge interest to separate conjugates anabolic androgenic steroids, as it allows efficiency
enhancement due to the small particle (sub-2μm) column packing, which provides high peak
capacity within analysis times even 5-10 fold shorter than conventional HPLC methods.
Modern multiplex instruments can analyze thousands of samples per month so that,
notwithstanding the generally high instrumental costs, the cost of the individual assay is
affordable. In addition, the improved specificity and resolution offered by time-of-flight or
quadrupole time-of-flight mass spectrometry allow their application in doping control analysis
or in steroid profiling for accurate and sensitive full mass range acquisition. Aim of one
review was to consider, compare and discuss the applications of the UHPLC/MS methods
present in literature for the identification and determination of forbidden steroids and their
metabolites in human biological matrices [12001].
Liquid chromatography/mass spectrometry (LC/MS) has been successfully applied to the

detection of anabolic steroids in biological samples. However, the sensitive detection of
saturated hydroxysteroids, such as androstanediols, by electrospray ionisation (ESI) is
difficult because of their poor ability to ionise. In view of this, chemical derivatisation has
been used to enhance the detection sensitivity of hydroxysteroids by LC/MS. One paper
described the development of a sensitive ultra-high-performance liquid chromatography/
tandem mass spectrometry (UHPLC/MS/MS) method for the screening of anabolic steroids
in horse urine by incorporating a chemical derivatisation step, using picolinic acid as the
derivatisation reagent. The method involved solid-phase extraction (SPE) of both free and
conjugated anabolic steroids in horse urine using a polymer-based SPE cartridge. The
conjugated steroids in the eluate were hydrolysed by methanolysis and the resulting extract
was further cleaned up by liquid-liquid extraction. The resulting free steroids in the extract
were derivatised with picolinic acid to form the corresponding picolinoyl esters and analysed
by UHPLC/MS/MS in the positive ESI mode with selected-reaction-monitoring. Separation of
the targeted steroids was performed on a C18 UHPLC column. The instrument turnaround
time was 10.5 min inclusive of post-run equilibration. A total of thirty-three anabolic steroids
(including 17beta-estradiol, 5(10)-estrene-3beta,17α-diol, 5α-estrane-3beta,17alpha-diol,
17alpha-ethyl-5alpha-estran-3alpha,17beta-diol, 17alpha-methyl-5alpha-androstan-3,17beta-
729
diols, androstanediols, nandrolone and testosterone) spiked in negative horse urine at the
QC levels (ranging from 0.75 to 30 ng/mL) could be consistently detected. The intra-day and
inter-day precisions (% RSD) for the peak area ratios were around 7-51 percent and around
1-72 percent, respectively. The intra-day and inter-day precisions (% RSD) for the relative
retention times were both less than 1 percent for all analytes, except the inter-day precision
for boldione at 1.2 percent. The extraction recoveries for all targets were not less than 48
percent. With exceptional separation achieved by the UHPLC system, matrix interferences
were minimal at the expected retention times of the selected transitions. As detection was
performed with an UHPLC system coupled to a fast-scanning triple quadrupole mass
spectrometer, the method could easily be expanded to accommodate additional steroid
targets. This method has been validated for recovery and precision, and could be used
regularly for doping control testing of anabolic steroids in horse urine samples [12060].
It was described a sensitive, comprehensive and fast screening method based on liquid
chromatography-high resolution mass spectrometry for the detection of a large number of
analytes in sports samples. UHPLC coupled to high resolution mass spectrometry with
polarity switching capability is applied for the rapid screening of a large number of analytes in
human urine samples. Full scan data are acquired alternating both positive and negative
ionisation. Collision-induced dissociation with positive ionisation is also performed to produce
fragment ions to improve selectivity for some analytes. Data are reviewed as extracted ion
chromatograms based on narrow mass/charge windows (± 5 ppm). A simple sample
preparation method was developed, using direct enzymatic hydrolysis of glucuronide
conjugates, followed by solid phase extraction with mixed mode ion-exchange cartridges.
Within a 10min run time (including re-equilibration) the method presented allows for the
analysis of a large number of analytes from most of the classes in the World Anti-Doping
Agency (WADA) Prohibited List, including anabolic agents, beta2-agonists, hormone
antagonists and modulators, diuretics, stimulants, narcotics, glucocorticoids and beta-
blockers, and does so while meeting the WADA sensitivity requirements. The high
throughput of the method and the fast sample pre-treatment reduces analysis cost and
increases productivity. The method presented has been used for the analysis of over 5000
samples in about one month and proved to be reliable [13092].
Fast liquid chromatographic/mass spectrometric screening
A fast liquid chromatographic/mass spectrometric (LC/MS/MS) screening method for the

detection, in urine, of synthetic glucocorticoids, stimulants (formoterol, modafinil and
mesocarb), anti-oestrogens (finasteride, exemestane, anastrozole, letrozole and formestane)
and synthetic anabolic steroids (stanozolol, gestrinone and tetrahydrogestrinone) is
described. All these drugs (and/or their urinary metabolites) can be simultaneously extracted
by a single liquid/liquid extraction step, at alkaline pH, after enzymatic hydrolysis with beta-
glucuronidase, and assayed in 7 min by LC/MS/MS using electrospray ionization in positive
ion mode and multiple reaction monitoring as the acquisition mode. All compounds show
good reproducibility of both the retention times (CV% <2%) and the relative abundances
(CV% <10 %). The limits of detection for the anti-oestrogens, glucocorticoids and steroids
are in the range of 1-30 ng/mL, and for the stimulants are in the range of 100-200 ng/mL,
thus satisfying the minimum required performance limits of the World Anti-Doping Agency
[06037].
Although gas chromatography-mass spectrometry (GC-MS) is currently still the standard

technique in anti-doping analysis, because of its robustness and high level of
standardization, liquid chromatography-mass spectrometry (LC-MS) has a number of
features that can be effectively exploited in sports drug testing or that offer good perspectives
730
of application in the near future. First, LC-MS allows minimal sample preparation, thus
increasing the sample throughput by means of:
- direct analysis of conjugated metabolites

- chromatographic separation of polar compounds with no need for derivatization
- on-line sample preparation, because of the compatibility between aqueous sample
and analytical system, at least in the reversed-phase (RP) mode.
Furthermore, LC-MS makes it possible to detect the whole metabolic profile of drugs, from
the parent compound to very polar conjugated metabolites, thus possibly providing an aid in
the interpretation of resuits, as in the case of high testosterone-to-epitestosterone ratios or in
the discrimination between doping and recreational use of drugs such as stimulants or
narcotics. LC-MS can be helpful also in the analysis of chemically unstable and/or volatile
drugs, particularly when the evaporation/reconstitution step can be avoided. LC-MS allows
the extension of the upper mass limit to dozens of thousands of Daltons by means of
electrospray ionization (ESI), which is able to easily produce multiply charged ions, or the
most recently developed time-of flight (TOF) mass analyzers, thus permitting the detection of
peptide hormones [05020].
One paper reviewed liquid chromatographic-mass spectrometric (LC-MS) procedures for the
screening, identification and quantification of doping agents in urine and other biological
samples and devoted to drug testing in sports. Reviewed methods published approximately
within the last five years and cited in the PubMed database have been divided into groups
using the same classification of the 2004 World Anti-Doping Agency (WADA) Prohibited List.
Together with procedures specifically developed for anti-doping analysis, LC-MS applications
used in other fields (e.g. therapeutic drug monitoring, clinical and forensic toxicology, and
detection of drugs illicitly used in livestock production) have been included when considered
as potentially extensible to doping control. Information on the reasons for potential abuse by
athletes, on the requirements established by WADA for analysis, and on the WADA rules for
the interpretation of analytical findings are provided for the different classes of drugs [05020].
Most of the LC-MS applications specific to the anti-doping field available in the literature refer
to drugs not amenable to GC separation (e.g. conjugated steroids, peptide hormones), or to
substances suitable for GC after derivatization (e.g, diuretics, beta2-agonists, beta-blockers,
and corticosteroids), or to specific target compounds. However, also LC-MS methods
developed for other purposes (e.g. therapeutic drug monitoring, clinical and forensic
toxicology, and detection of drugs illicitly used in livestock production) have been considered
as potentially extensible to doping control [05020].
The application of modern and powerful analytical instruments consisting of liquid

chromatographs (LCs), sophisticated atmospheric pressure ion sources, and sensitive mass
analyzers has improved quality as well as speed of doping control analyses markedly during
the last 5 years. Numerous compounds such as beta-receptor blocking agents or diuretics
require derivatization prior to gas chromatographic (GC) and mass spectrometric (MS)
measurement, which is the reason for extended sample preparation periods. In addition,
several substances demonstrate poor GC-MS properties even after chemical modification,
and peptide hormones such as cross-linked hemoglobins cannot be analyzed at all by means
of GC-MS. With the availability of electrospray ionization and robust tandem MSs (e.g., triple-
stage quadrupole or ion trap instruments) many new or complementary screening and
confirmation assays have been developed, providing detailed qualitative and quantitative
information on prohibited drugs. With selected categories of compounds (ephedrines, beta-
blockers, beta2-agonists, diuretics, and bovine hemoglobin-based oxygen therapeutics) that
are banned according to the rules of the World Anti-Doping Agency and International
731
Olympic Committee, the advantages of LC-MS-MS procedures over conventional GC-MS
assays are demonstrated, such as enhanced separation of analytes, shorter sample
pretreatment, and identification of substances that are not identified by GC-MS [05021].
High-performance liquid chromatography

High-performance liquid chromatography (HPLC) separation of drugs at elevated pressure
with 1.7 microm hybrid C18 stationary phase columns was investigated. This technique,
which uses instrumentation engineered to handle the narrow peaks and high back pressures
generated by 1.7 microm particle columns, provided significantly better resolution and/or
faster analysis than conventional HPLC and capillary electrophoresis (CE). The use of 2 mm
internal diameter columns of 3-10 cm length has been evaluated for the separation of basic
and neutral drugs, drug profiling, and general screening (including acidic drugs). For these
applications, compared to conventional HPLC and CE, it provided up to 12x and 3x faster
analyses, respectively. Precision was excellent for both isocratic and gradient analyses. For
retention time and peak area, RSDs of <0.1 percent were obtainable. Fifteen anabolic
steroids and esters were well separated in a 2.5 min gradient. For drug profiling, compared to
HPLC and CE, approximately twice as many peaks were resolved. HPLC at elevated
pressure is also well suited as a general screening technique. Twenty-four solutes of varying
drug classes including narcotic analgesics, stimulants, depressants, hallucinogens, and
anabolic steroids were fully separated in a 13.5 min gradient [05022].
WADA identification criteria

WADA has established criteria for the identification of a compound by combined
chromatographic-mass spectrometric analysis. Regarding LC separation, the retention time
of the analyte is required not to "differ by more than 2 percent from that of the same
substance" in the reference material. As for MS detection, when a single-mass full or partial
scan is acquired, all diagnostic ions with a relative abundance greater than 10 percent in the
reference spectrum must be present in the unknown. In addition, the relative abundance of
three "diagnostic ions" (i.e. "molecular ion or fragment ions whose presence and abundance
are characteristic of a substance and thereby may assist in its identification") shall not differ
by more than a certain percentage-depending on the abundance of the ion relative to the
base ion in the reference spectrum from the relative intensities of the same ions in the
reference material. Background subtraction can be applied if necessary, but it must be used
consistently throughout the batch of samples. Computer-based library searching is also
allowed but criteria for identification have to be established in advance by the laboratory and
all the matches have to be reviewed by a qualified scientist as "the match factor for a reverse
search does not guarantee identification.” When acquisition is performed in selected ion
monitoring (SIM), at least three diagnostic ions must be acquired. The signal-to-noise ratio of
the least intense diagnostic ion must be greater than 3:1. The relative intensities of any of the
ions (preferably determined from the peak area or height of the integrated mass
chromatogram) shall not differ from the relative intensities of the same ions acquired in the
reference material of the analyte should be "comparable" in the sample and in the reference
material. Tandem MS can also be used for identification either in full scan or selected
reaction monitoring (SRM) mode. Collision conditions have to be selected in order to ensure
that the precursor ion is present in the product ion scan or in the SRM acquisition. In some
cases, a single reaction (one precursor ion – about one product ion) may be sufficiently
unique to be definitive, although in such case the mass resolution of the first mass analyzer
should be set to unity. When monitoring more than one product ion, the relative intensities of
any of the ions shall not differ from those of the same ions in the reference material
"analyzed contemporaneously". The signal-to-noise ratio of the least intense diagnostic ion
must be greater than 3:1. Both in SIM and in tandem MS detection, when a diagnostic ion
shows a relative abundance of less than 5 percent in the reference, the condition that has to
be satisfied for positive identification is the mere presence of the ion in the unknown. Finally,
732
if a sufficient number of diagnostic ions is not available, a derivative of the compound, or a
second ionization or fragmentation technique (able to provide different diagnostic ions)
[05020].
Orbitrap
A new doping control screening method for the analysis of diuretics and stimulants using
ultra high pressure liquid chromatography-high resolution Orbitrap mass spectrometry has
been developed. The screening was performed in full scan MS with scan-to-scan polarity
switching which allowed to detect more than 120 target analytes. Sample preparation was
limited to 10-fold dilution of the urine into the internal standard solution followed by injection.
Total run time per sample was 10 min. Validation of the method yielded detection limits for
diuretics between 25 and 250 ng/mL and for stimulants between 5 and 500 ng/mL. The
screening method has been implemented in routine doping control [12071].
Liquid chromatography-tandem mass spectrometry
A screening method for the urinary detection of 34 exogenous anabolic steroids has been
developed. The method involves an enzymatic hydrolysis, liquid-liquid extraction and
detection by liquid chromatography-tandem mass spectrometry. The use of some adducts
such as (M+NH4+, M+CH3COO- and M+H+MeOH+) was necessary in order to detect some
analytes at the required level (lower than 10 ng/ml). Two transitions were selected for each
analyte. Different concentration factors have been studied in order to increase the sensitivity.
A concentration factor of 50 was selected for the screening method although the high ion
suppression observed under these conditions can hamper its application as a quantitative
method. The method was validated and limits of detection were obtained by spiking ten
different blank urine samples at five different concentration levels. Up to 29 analytes were
detected in all spiked urines at the required level. Limits of detection between 1 and 10 ng/ml
were obtained for most analytes which fulfil current requirements. The applicability of the
method was shown by analysing positive samples [07051].
A simple and accurate liquid chromatography/tandem mass spectrometry (LC/MS/MS)

method has been developed and validated for the quantitative determination of ephedrine,
pseudoephedrine, methylephedrine, cathine, salbutamol, morphine and epitestosterone in
human urine. Urine samples were spiked with internal standard and diluted with acetonitrile.
After centrifugation, the supernatants were directly analyzed by LC/MS/MS using the
selected reaction monitoring (SRM) mode. The linearity, intra- and inter-day precision,
accuracy, limit of detection (LOD) and limit of quantification (LOQ) were evaluated and the
method was found to be accurate and reproducible for the quantitation of threshold
substances. When the method was applied to the analysis of blind urine samples for the
proficiency test, the results were close to the nominal concentrations, within 88-107 percent
of nominal values, suggesting that the developed methods can be successfully applied to
routine doping analyses [11429].
Liquid chromatography-(tandem) mass spectrometry (LC-MS(/MS) has become an integral

part of modern sports drug testing as it offers unique capabilities complementing
immunological and gas chromatography-(tandem) mass spectrometry (GC-MS(/MS)-based
detection methods for prohibited compounds. The improved options of fast and sensitive
targeted analysis as well as untargeted screening procedures utilizing high resolution/high
accuracy mass spectrometry have considerably expanded the tools available to anti-doping
laboratories for initial testing and confirmation methods. One approach is to focus on pre-
selected target analytes that are measured with utmost specificity and sensitivity using
733
diagnostic precursor-product ion pairs in low resolution tandem mass spectrometers. The
other scenario is to measure and plot extracted ion chromatograms of protonated or
deprotonated molecules as well as product ions as recorded in the full scan mode with high
resolution/high accuracy mass spectrometry. Examples of recent applications of sports drug
testing procedures published between 2007 and 2010 were presented and discussed,
outlining the particular advantages of the selected approaches as well as their limitations in a
short- and long-term perspective [11036].
Sample preparation is a critical step in large-scale multiclass analysis such as sport drug
testing. Due to the wide heterogeneity of the analytes and the complexity of the matrix, the
selection of a correct sample preparation method is essential, looking for a compromise
between good recoveries for most of the analytes and cleanliness of the extract. In the
present work, seven sample preparation procedures based on solid-phase extraction (SPE)
(with 5 different cartridges), liquid-liquid extraction (LLE) and sorbent-supported liquid
extraction (SLE) were evaluated for multiclass sport drug testing in urine. The selected SPE
sorbents were polymeric cartridges Agilent PLEXA™ and Oasis HLB™, mixed mode cation
and anion exchange cartridges Oasis MAX™ and MCX™, and C18 cartridges. LLE was
performed using tert-butyl methyl ether and SLE was carried out using Agilent Chem Elut™
cartridges. To evaluate the proposed extraction procedures, a list of 189 compounds were
selected as representative from different groups of doping agents, including 34 steroids, 14
glucocorticosteroids, 24 diuretics and masking agents, 11 stimulants, 9 beta-agonist, 16
beta-blockers, 6 Selective Estrogen Receptors Modulators (SERMs), 24 narcotics and 22
other drugs of abuse/sport drugs. Blank urine samples were spiked at two levels of
concentration, 2.5 and 25 microg/L and extracted with the different extraction protocols (n=6).
The analysis of the extracts was carried out by liquid chromatography electrospray time-of-
flight mass spectrometry. The use of solid-phase extraction with polymer cartridges provided
high recoveries for most of the analytes tested and was found the more suitable method for
this type of application given the additional advantages such as low sample and solvent
consumption along with increased automation and throughput [14633].
Matrix effects (ion suppression/enhancement) are a well-observed phenomenon in analyses

of biological matrices by high-performance liquid chromatography-mass spectrometry (LC-
MS). However, few simple solutions for detecting and minimizing these adverse effects have
been described so far in multianalyte analysis, especially in the field of doping control. This
study describes an exhaustive characterization of matrix effects in one hundred urine
samples fortified with 41 analytes (glucocorticoids and diuretics). It introduces a novel marker
to identify samples in which the reliability of the results is compromised because of acute ion
suppression. This new strategy strengthens the rigor of the analysis for screening purposes.
Once the matrix effect is identified, a selective sample preparation is introduced to minimize
the adverse ion suppression effect. That selective extraction together with the use of a
deuterated internal standard permits enhancing the ruggedness of the estimation of
glucocorticoid concentration in urine [12059].
Ultrahigh pressure liquid chromatography-(tandem) mass spectrometry
Doping control analytical laboratories for human sports predominantly employ nowadays
chromatographic-mass spectrometric test methods for routine, high throughput screening
and confirmation assays concerning low and high molecular mass analytes. Liquid
chromatography-(tandem) mass spectrometry [(LC-MS(/MS)] and particularly ultrahigh
pressure liquid chromatography (UHPLC)-MS/MS instruments have become devices of
choice due to their indispensable capabilities that compensate for limitations inherent to other
commonly used strategies such as immunological and gas chromatography-(tandem) mass
spectrometry [(GC-MS(/MS)]-based detection methods. UHPLC-MS/MS-based assays at low
734
mass spectrometric resolution have been established allowing for fast and sensitive targeted
analyses focusing on pre-selected target analytes with diagnostic precursor-product ion
pairs. Combining UHPLC to high resolution/high accuracy MS(/MS) further expanded the
targeted approach (i.e., plotting extracted ion chromatograms of protonated or deprotonated
molecules as well as product ions measured with accurate masses) toward non-targeted
analyses enabling also retrospective data mining. In one review, applications of UHPLC-
MS/MS in sports drug testing procedures published between 2008 and 2012 are presented
and advantages as well as limitations in a short- and long-term perspective were discussed
[13092].
Coupled with tandem mass spectrometry: analysis of biological samples

The potential and applicability of UHPSFC-MS/MS for anti-doping screening in urine samples
were tested for the first time. For this purpose, a group of 110 doping agents with diverse
physicochemical properties was analyzed using two separation techniques, namely UHPLC-
MS/MS and UHPSFC-MS/MS in both ESI+ and ESI- modes. The two approaches were
compared in terms of selectivity, sensitivity, linearity and matrix effects. As expected, very
diverse retentions and selectivities were obtained in UHPLC and UHPSFC, proving a good
complementarity of these analytical strategies. In both conditions, acceptable peak shapes
and MS detection capabilities were obtained within 7 min analysis time, enabling the
application of these two methods for screening purposes. Method sensitivity was found
comparable for 46 percent of tested compounds, while higher sensitivity was observed for 21
percent of tested compounds in UHPLC-MS/MS and for 32 percent in UHPSFC-MS/MS. The
latter demonstrated a lower susceptibility to matrix effects, which were mostly observed as
signal suppression. In the case of UHPLC-MS/MS, more serious matrix effects were
observed, leading typically to signal enhancement and the matrix effect was also
concentration dependent, i.e. more significant matrix effects occurred at the lowest
concentrations [150130].
Coupled with tandem mass spectrometry:investigation of mobile phase and MS conditions

The conditions for the analysis of selected doping substances by UHPSFC-MS/MS were
optimized to ensure suitable peak shapes and maximized MS responses. A representative
mixture of 31 acidic and basic doping agents was analyzed, in both ESI+ and ESI- modes.
The best compromise for all compounds in terms of MS sensitivity and chromatographic
performance was obtained when adding 2% water and 10mM ammonium formate in the
CO2/MeOH mobile phase. Beside mobile phase, the nature of the make-up solvent added
for interfacing UHPSFC with MS was also evaluated. Ethanol was found to be the best
candidate as it was able to compensate for the negative effect of 2% water addition in ESI-
mode and provided a suitable MS response for all doping agents. Sensitivity of the optimized
UHPSFC-MS/MS method was finally assessed and compared to the results obtained in
conventional UHPLC-MS/MS. Sensitivity was improved by 5-100-fold in UHPSFC-MS/MS vs.
UHPLC-MS/MS for 56 percent of compounds, while only one compound (bumetanide)
offered a significantly higher MS response (4-fold) under UHPLC-MS/MS conditions. In the
second paper of this series, the optimal conditions for UHPSFC-MS/MS analysis will be
employed to screen >100 doping agents in urine matrix and results will be compared to those
obtained by conventional UHPLC-MS/MS [150131].
Ultra-high-performance supercritical-fluid chromatography hyphenated MS/MS

Profiling conjugated urinary steroids to detect anabolic-steroid misuse is recognized as an
efficient analytical strategy in both chemical-food-safety and anti-doping fields. The relevance
and robustness of such profiling rely on the analysis of glucuronide and sulfate steroids,
which is expected to have properties including accuracy, specificity, sensitivity, and, if
possible, rapidity. In this context, the ability of ultra-high-performance supercritical-fluid
chromatography (UHPSFC) hyphenated tandem mass spectrometry (MS-MS) to provide
735
reliable and accurate phase II analysis of steroids was assessed. Four stationary phases
with sub-2 microm particles (BEH, BEH 2-ethyl-pyridine, HSS C18 SB, and CSH
fluorophenyl) were screened for their capacity to separate several conjugated steroid
isomers. Analytical conditions including stationary phase, modifier composition and
percentage, back pressure, column temperature, and composition and flow rate of make-up
solvent were investigated to improve the separation and/or the sensitivity. Thus, an analytical
procedure enabling the analysis of eight glucuronide and 12 sulfate steroids by two different
methods in 12 and 15 min, respectively, was optimized. The two procedures were evaluated,
and UHPSFC-MS-MS analysis revealed its ability to provide sensitive (limits of quantification:
0.1 ng/mL and 0.5 ng/mL for sulfate and glucuronide steroids, respectively) and reliable
quantitative performance through the use of suitable labeled internal standards. Comparison
with UHPLC-MS-MS was performed, and UHPSFC-MS-MS obtained better performance in
terms of sensitivity. Finally, as a proof of concept, this so-called green technology was used
in a chemical-food-safety context to profile steroid conjugates in urine samples from bovines
treated with estradiol. Graphical Abstract Glucuronide and sulfate steroids analysis in urine
by ultra-high performance supercritical fluid chromatography hyphenated tandem mass
spectrometry [150167].
Multi-targeted liquid chromatography-mass spectrometry screening procedure
One work presented an analytical method for the simultaneous analysis in human urine of 38
pharmacologically active compounds (19 benzodiazepine-like substances, 7 selective
serotonin reuptake inhibitors, 4 azole antifungal drugs, 5 inhibitors of the phosphodiesterases
type 4 and 3 inhibitors of the phosphodiesterase type 5) by liquid-chromatography coupled
with tandem mass spectrometry. The above substances classes include both the most
common "non banned" drugs used by the athletes (based on the information reported on the
"doping control form") and those drugs who are suspected to be performance enhancing
and/or act as masking agents in particular conditions. The chromatographic separation was
performed by a reverse-phase octadecyl column using as mobile phases acetonitrile and
ultra-purified water, both with 0.1 percent formic acid. The detection was carried out using a
triple quadrupole mass spectrometric analyser, positive electro-spray as ionization source
and selected reaction monitoring as acquisition mode. Sample pre-treatment consisted in an
enzymatic hydrolysis followed by a liquid-liquid extraction in neutral field using tert-butyl
methyl-ether. The analytical procedure, once developed, was validated in terms of sensitivity
(lower limits of detection in the range of 1-50 ng/mL), specificity (no interferences were
detected at the retention time of all the analytes under investigation), recovery (≥60 % with a
satisfactory repeatability, CV % lower than 10), matrix effect (lower than 30 %) and
reproducibility of retention times (CV % lower than 0.1) and of relative abundances (CV %
lower than 15). The performance and the applicability of the method was evaluated by
analyzing real samples containing benzodiazepines (alprazolam, diazepam, zolpidem or
zoplicone) or inhibitors of the phosphodiesterases type 5 (sildenafil or vardenafil) and
samples obtained incubating two of the phosphodiesterases type 4 studied (cilomilast or
roflumilast) with pooled human liver microsomes. All the parent compounds, together with
their main phase I metabolites, were clearly detected using the analytical procedures here
developed [150126].
Metabolite-based liquid chromatography-mass spectrometry
Today, immunoassays and several chromatographic methods are in use for drug screening
in clinical and forensic toxicology and in doping control. For further proof of the authors' new
metabolite-based liquid chromatography-mass spectrometry (LC-MSn) screening concept,
the detectability of drugs of abuse and their metabolites using this screening approach was
736
studied. As previously reported, the corresponding reference library was built up with MS 2
and MS3 wideband spectra using a LXQ linear ion trap with electrospray ionization in the
positive mode and full scan information-dependent acquisition. In addition to the parent drug
spectra recorded in methanolic solution, metabolite spectra were identified after protein
precipitation of urine from rats after administration of the corresponding drugs and added to
the library. This consists now of data of over 900 parent compounds, including 87 drugs of
abuse, and of over 2,300 metabolites and artifacts, among them 436 of drugs of abuse.
Recovery, process efficiency, matrix effects, and limits of detection for selected drugs of
abuse were determined using spiked human urine, and the resulting data have been
acceptable. Using two automatic data evaluation tools (ToxID and SmileMS), the intake of 54
of the studied drugs of abuse could be confirmed in urine samples of drug users after protein
precipitation and LC separation. The following drugs classes were covered: stimulants,
designer drugs, hallucinogens, (synthetic) cannabinoids, opioids, and selected
benzodiazepines. The presented LC-MSn method complements the well-established gas
chromatography-mass spectroscopy procedure in the authors' laboratory [11035].
High-resolution/accurate-mass LC-MS
Detection of androgenic-anabolic steroid abuse in equine sports requires knowledge of the

drug's metabolism in order to target appropriate metabolites, especially where urine is the
matrix of choice. Studying “designer” steroid metabolism is problematic since it is difficult to
obtain ethical approval for in vivo metabolism studies due to a lack of toxicological data. In
this study, the equine in vitro metabolism of eight steroids available for purchase on the
Internet is reported; including androsta-1,4,6-triene-3,17-dione, 4-chloro,17alpha-methyl-
androsta-1,4-diene-3,17beta-diol, estra-4,9-diene-3,17-dione, 4-hydroxyandrostenedione,
20-hydroxyecdysone, 11-keto-androstenedione, 17alpa-methyldrostanolone, and tetrahydro-
gestrinone. In order to allow for retrospective analysis of sample testing data, the use of a
high-resolution (HR) accurate-mass Thermo LTQ-Orbitrap liquid chromatography-mass
spectrometry (LC-MS) instrument was employed for metabolite identification of underivatized
sample extracts. The full scan LC-HRMS Orbitrap data were complimented by LC-HRMS/MS
and gas-chromatography-mass spectrometry (GC-MS) experiments in order to provide
fragmentation information and to ascertain whether GC-MS was capable of detecting any
metabolite not detected by LC-HRMS. With the exception of 20-hydroxyecdysone, all
compounds were found to be metabolized by equine liver S9 and/or microsomes. With the
exception of 17alpha-methyldrostanolone, which produced metabolites that could only be
detected by GC-MS, the metabolites of all other compounds could be identified using LC-
HRMS, thus allowing retrospective analysis of previously acquired full-scan data resulting
from routine equine drug testing screens. In summary, while in vitro techniques do not serve
as a replacement for more definitive in vivo studies in all situations, their use does offer an
alternative in situations where it would not be ethical to administer untested drugs to animals
[11044].
New designer drugs in post-mortem blood using high-resolution mass spectrometry

An analytical method was developed and validated for the purpose of detecting and
quantifying 37 new designer drugs including cathinones, hallucinogenic phenethylamines
and piperazines. Using only 100 microL whole blood, a salting-out-assisted liquid-liquid
extraction with acetonitrile was performed to isolate target compounds followed by
chromatographic separation using a Waters ACQUITY ultra performance liquid
chromatograph coupled to a Waters XEVO quadrupole time-of-flight mass spectrometer.
Mephedrone-d3 was used as an internal standard. A gradient elution was used in
combination with a Waters ACQUITY HSS C18 column (2.1 × 150 mm, 1.8 microm).
Samples were analyzed using the detector in positive electrospray ionization mode with MSE
737
acquisition. All compounds of interest were resolved in a 15 min run time and positively
identified based on accurate mass of the molecular ion, two product ions and retention time.
All analyte calibration curves were linear over the range of 0.05-2 mg/L with most correlation
coefficient (r2) values >0.98. The limits of detection were within the range of 0.007-0.07 mg/L
and limits of quantification within 0.05-0.1 mg/L. All analytes were stable 48 h after extraction
and most were stable in blood after 1 week stored in a refrigerator and 3 freeze-thaw cycles.
No carryover was observed up to 10 mg/L and no interferences from common therapeutic
drugs or endogenous compounds. Recoveries ranged from 71 to 100 percent and matrix
effects were assessed for blank, post-mortem and decomposed blood. All bias and percent
coefficient of variation values were within the acceptable values of ±15 and ≤15 percent,
respectively (±20 and ≤20 % at lower limit of quantification). The method was applied to
several forensic cases where the subject exhibited behavior characteristic of designer drug
intoxication and where routine screening for a panel of drugs was negative [14741].
With information dependent acquisition
Diuretics are a class of compounds largely used for either therapeutic or illegal (doping)
purposes. Probably owing to the substantial variety of their chemical structures, which makes
them hardly extractable from a biological matrix in a single procedure, a quite short list of
screening methods can be retrieved in the literature. One work presented a screening
procedure for 24 diuretics based on the direct injection of urine (after 50 folds dilution) in a
LC-ESI-MS/MS system (Applied Biosytems 4000 QTrap). Two information dependent
acquisitions (IDA), one in positive, one in negative ionization, allowed the acquisition of one
selected reaction monitoring transition for each compound, which, when a significant peak
was found, triggered the acquisition of the enhanced product ion (EPI) spectrum. EPI spectra
were stored in a library and the procedure was able to recognize by library matching various
diuretics in real positive samples. The limits of detection were comprised between 0.002 and
0.25 ml/L and ion suppression was not found to significantly influence the analysis [07052].
Liquid chromatography/time-of-flight mass spectrometry
The development of comprehensive methods able to tackle with the systematic identification
of drug metabolites in an automated fashion is of great interest. In this article, a strategy
based on the combined use of two complementary data mining tools is proposed for the
screening and systematic detection and identification of urinary drug metabolites by liquid
chromatography full-scan high resolution mass spectrometry. The proposed methodology is
based on the use of accurate mass extraction of diagnostic ions (compound-dependent
information) from in-source CID fragmentation without precursor ion isolation along with the
use of automated mass extraction of accurate-mass shifts corresponding to typical
biotransformations (non compound-dependent information) that xenobiotics usually undergo
when metabolized. The combined strategy was evaluated using LC-TOFMS with a suite of
nine sport drugs representative from different classes (propranolol, bumetanide, clenbuterol,
ephedrine, finasteride, methoxyphenamine, methylephedrine, salbutamol and terbutaline),
after single doses administered to rats. The metabolite identification coverage rate obtained
with the systematic method (compared to existing literature) was satisfactory, and provided
the identification of several non-previously reported metabolites. In addition, the combined
information obtained helps to minimize the number of false positives. As an example, the
systematic identification of urinary metabolites of propranolol enabled the identification of up
to 24 metabolites, 15 of them non previously described in literature, which is a valuable
indicator of the usefulness of the proposed systematic procedure [13098].
738
In one article, a screening method for the determination of 200 sport drugs in human urine
has been developed using liquid-chromatography electrospray time-of-flight mass
spectrometry (LC-TOFMS). The chromatographic separation of the targeted doping agents
was carried out by fast liquid chromatography using a C18 column (4.6×50 mm) with 1.8
microm particle size. Accurate mass measurements of the selected ion (typically [M+H](+)
and [M-H](-)) along with retention time matching was used for the screening and detection of
the targeted species. The proposed methodology comprised also a simple sample treatment
stage based on solid-phase extraction (SPE) with polymeric cartridges. The SPE method
displayed satisfactory recoveries rates (between 70 and 120 %) for the majority of the
compounds at both concentration levels tested (2.5 and 25 microg/L). The overall
performance of the method was satisfactory with all 200 compounds fulfilling WADA
minimum required performance levels (MRPLs), with limits of quantitation lower than 1
microg/L for 80 percent of the compounds, and showing an appropriate linearity in most
cases. Additionally, the ability of "in-source" collision induced dissociation (CID) for
confirmatory purposes was examined using as criterion the presence of two high-resolution
ions with relevant abundances for unambiguous confirmation. This stringent criterion was
fulfilled for 75 percent of the species using in-source CID fragmentation. The use of an
improved approach based on CID performed on a dedicated collision cell without precursor
ion selection (using a Q-TOF) provided at least two ions in all cases with the exception of 2-
aminoheptane. Finally, based on the use of diagnostic fragment ions, a workflow for the
comprehensive screening and identification of non-targeted compounds (viz. compounds
with no primary standards or retention time information available, such as metabolites) has
been also examined using rat urine samples. The proposed screening method has proved to
be effective for the analysis of targeted compounds, and also for the identification of
metabolites, expanding easily the search for doping agents not only limited to specific
banned parent compounds but also to derivate compounds with similar structure as well as
metabolites [150127].
A general screening method based on solid phase extraction (SPE) and liquid
chromatography/time-of-flight mass spectrometry (LC/TOFMS) was developed and
investigated with 124 different doping agents, including stimulants, beta-blockers, narcotics,
beta2-adrenergic agonists, agents with anti-estrogenic activity, diuretics and cannabinoids.
Mixed mode cation exchange/C8 cartridges were applied to SPE, and chromatography was
based on gradient elution on a C18 column. Ionization of the analytes was achieved with
electrospray ionization in the positive mode. Identification by LC/TOFMS was based on
retention time, accurate mass and isotopic pattern. Validation of the method consisted of
analysis of specificity, analytical recovery, limit of detection and repeatability. The minimum
required performance limit (MRPL), established by World Anti-Doping Agency (WADA), was
attained to 97 doping agents. The extraction recoveries varied between 33 and 98 percent
and the median was 58 percent. Mass accuracy was always better than 5 ppm,
corresponding to a maximum mass error of 0.7 mDa. The repeatability of the method for
spiked urine samples, expressed as median of relative standard deviations (RSD%) at
concentrations of MRPL and 10 times MRPL, were 14 and 9 percent, respectively. The
suitability of the LC/TOFMS method for doping control was demonstrated with authentic urine
samples [06038].
The development of comprehensive methods able to tackle with the systematic identification
of drug metabolites in an automated fashion is of great interest. In this article, a strategy
based on the combined use of two complementary data mining tools is proposed for the
screening and systematic detection and identification of urinary drug metabolites by liquid
chromatography full-scan high resolution mass spectrometry. The proposed methodology is
based on the use of accurate mass extraction of diagnostic ions (compound-dependent
information) from in-source CID fragmentation without precursor ion isolation along with the
739
use of automated mass extraction of accurate-mass shifts corresponding to typical
biotransformations (non compound-dependent information) that xenobiotics usually undergo
when metabolized. The combined strategy was evaluated using LC-TOFMS with a suite of
nine sport drugs representative from different classes (propranolol, bumetanide, clenbuterol,
ephedrine, finasteride, methoxyphenamine, methylephedrine, salbutamol and terbutaline),
after single doses administered to rats. The metabolite identification coverage rate obtained
with the systematic method (compared to existing literature) was satisfactory, and provided
the identification of several non-previously reported metabolites. In addition, the combined
information obtained helps to minimize the number of false positives. As an example, the
systematic identification of urinary metabolites of propranolol enabled the identification of up
to 24 metabolites, 15 of them non previously described in literature, which is a valuable
indicator of the usefulness of the proposed systematic procedure [12062].
A screening method for the determination of 200 sport drugs in human urine has been
developed using liquid-chromatography electrospray time-of-flight mass spectrometry (LC-
TOFMS). The chromatographic separation of the targeted doping agents was carried out by
fast liquid chromatography using a C18 column (4.6×50mm) with 1.8 microm particle size.
Accurate mass measurements of the selected ion (typically [M+H]+ and [M-H]- along with
retention time matching was used for the screening and detection of the targeted species.
The proposed methodology comprised also a simple sample treatment stage based on solid-
phase extraction (SPE) with polymeric cartridges. The SPE method displayed satisfactory
recoveries rates (between 70 and 120 %) for the majority of the compounds at both
concentration levels tested (2.5 and 25 microg/L). The overall performance of the method
was satisfactory with all 200 compounds fulfilling WADA minimum required performance
levels (MRPLs), with limits of quantitation lower than 1 microg/L for 80 percent of the
compounds, and showing an appropriate linearity in most cases. Additionally, the ability of
"in-source" collision induced dissociation (CID) for confirmatory purposes was examined
using as criterion the presence of two high-resolution ions with relevant abundances for
unambiguous confirmation. This stringent criterion was fulfilled for 75 percent of the species
using in-source CID fragmentation. The use of an improved approach based on CID
performed on a dedicated collision cell without precursor ion selection (using a Q-TOF)
provided at least two ions in all cases with the exception of 2-aminoheptane. Finally, based
on the use of diagnostic fragment ions, a workflow for the comprehensive screening and
identification of non-targeted compounds (viz. compounds with no primary standards or
retention time information available, such as metabolites) has been also examined using rat
urine samples. The proposed screening method has proved to be effective for the analysis of
targeted compounds, and also for the identification of metabolites, expanding easily the
search for doping agents not only limited to specific banned parent compounds but also to
derivate compounds with similar structure as well as metabolites [14736].
High performance liquid chromatography retention time of small molecules
Quantitative structure-retention relationship (QSRR) is a technique capable of improving the

identification of analytes by predicting their retention time on a liquid chromatography column
(LC) and/or their properties. This approach is particularly useful when LC is coupled with a
high-resolution mass spectrometry (HRMS) platform. The main aim of one study was to
develop and describe appropriate QSRR models that provide usable predictive capability,
allowing false positive identification to be removed during the interpretation of metabolomics
data, while additionally increasing confidence of experimental results in doping control area.
For this purpose, a dataset consisting of 146 drugs, metabolites and banned compounds
from World Anti-Doping Agency (WADA) lists, was used. A QSRR study was carried out
separately on high quality retention data determined by reversed-phase (RP-LC-HRMS) and
740
hydrophilic interaction chromatography (HILIC-LC-HRMS) systems, employing a single
protocol for each system. Multiple linear regression (MLR) was applied to construct the linear
QSRR models based on a variety of theoretical molecular descriptors. The regression
equations included a set of three descriptors for each model: ALogP, BELe6, R2p and
ALogP(2), FDI, BLTA96, were used in the analysis of reversed-phase and HILIC column
models, respectively. Statistically significant QSRR models indicate a strong correlation
between retention time and the molecular descriptors. An evaluation of the best correlation
models, performed by validation of each model using three tests (leave-one-out, leave-many-
out, external tests), demonstrated the reliability of the models [13088].
Nano-liquid chromatography/benchtop quadrupole orbitrap tandem-mass

spectrometry
In one study, a screening assay was developed comprising 11 prohibited peptides (<1.5 kDa)
that are sufficiently purified from urine using weak cation exchange with subsequent
determination of all substances by means of nanoUHPLC separation coupled to high
resolution tandem mass spectrometry. These peptides included Gonadorelin (LH-RH),
Desmopressin and 9 growth hormone releasing peptides (GHRP-1, -2, -4, -5, -6, Hexarelin,
Alexamorelin, Ipamorelin and a GHRP-2 metabolite); however, the procedure is expandable
to further target analytes or metabolites. The method was validated with a main focus on
qualitative result interpretation considering the parameters specificity, linearity (0-500 pg/mL),
recovery (45-95 %), precision (<20 % at 100 pg/mL), limits of detection (2-10 pg/mL),
robustnesss and ion suppression. The proof-of-principle was shown by analysing excretion
study urine samples for LHRH, desmopressin and GHRP-2 [12065].
Liquid and gas chromatography time-of-flight mass spectrometry
A new combined doping control screening method for the analysis of anabolic steroids in
human urine using liquid chromatography/electrospray ionization orthogonal acceleration
time-of-flight mass spectrometry (LCoaTOFMS) and gas chromatography/electron ionization
orthogonal acceleration time-of-flight mass spectrometry (GCoaTOFMS) was developed in
order to acquire accurate full scan MS data to be used to detect designer steroids. The
developed method allowed the detection of representative prohibited substances, in addition
to steroids, at concentrations of 10 ng/mL for anabolic agents and metabolites, 30 ng/mL for
corticosteroids, 500 ng/mL for stimulants and beta-blockers, 250 ng/mL for diuretics, and 200
ng/mL for narcotics. Sample preparation was based on liquid-liquid extraction of hydrolyzed
human urine, and the final extract was analyzed as trimethylsilylated derivatives in
GCoaTOFMS and underivatized in LCoaTOFMS in positive ion mode. The sensitivity, mass
accuracy, advantages and limitations of the developed method were presented [07053].
Gas chromatography
The application of comprehensive two-dimensional gas chromatography coupled to time-of-

flight mass spectrometry (GCxGC-TOFMS) for the analysis of six anabolic agents (AAs) in
doping control is investigated in this work. A non-polar-polar column configuration with 0.2
microm film thickness second dimension column was employed, offering much better spread
of the components on second dimension when compared to the alternative 0.1 microm
column. The proposed method was tested on the "key" AAs that the World Anti-Doping
Agency (WADA) had listed at the low ngm/L levels (clenbuterol, 19-norandrosterone,
epimethendiol, 17alpha-methyl-5alpha-androstane-3alpha,17beta-diol, 17alpha-methyl-
741
5beta-androstane-3alpha,17beta-diol and 3'-OH-stanozolol). The compounds were spiked in
a blank urine extract obtained by solid-phase extraction, hydrolysis and liquid-liquid
extraction; prior to analysis they were converted to the corresponding trimethylsilyl (TMS)
derivatives. The limit of detection (LOD) was below or equal to the minimum required
performance limit (MRPL) of 2ngmL(-1) defined by WADA, and the correlation coefficient
was in the range from 0.995 to 0.999. The method allows choosing an ion from the full mass
spectra which shows the least interference from the matrix and/or the best sensitivity; this
can only be done if full scan mass spectral data are available. The advantage of GCxGC
over classical one-dimensional GC, in terms of separation efficiency and sensitivity, is
demonstrated on a positive urine control sample at a concentration of 5ngm/L. The obtained
similarity to the in-house created TOFMS spectra library at this level of concentration was in
the range from 822 to 932 (on the scale from 0 to 999). Since full mass spectral information
are recorded, the method allows the retro-search of non-target compounds or new "designer
steroids", which cannot be detected with established GC-MS methods that use selected ion
monitoring mode [10035].
Urine samples have been the predominant matrix for doping controls for several decades.
However, owing to the complementary information provided by blood (as well as serum or
plasma and dried blood spots (DBS)), the benefits of its analysis have resulted in
continuously increasing appreciation by anti-doping authorities. On the one hand, blood
samples allow for the detection of various different methods of blood doping and the abuse of
erythropoiesis-stimulating agents (ESAs) via the Athlete Biological Passport; on the other
hand, targeted and non-targeted drug detection by means of chromatographic-mass
spectrometric methods represents an important tool to increase doping control frequencies
out-of-competition and to determine drug concentrations particularly in in-competition
scenarios. Moreover, blood analysis seldom requires in-depth knowledge of drug
metabolism, and the intact substance rather than potentially unknown or assumed metabolic
products can be targeted. In this review, the recent developments in human sports drug
testing concerning mass spectrometry-based techniques for qualitative and quantitative
analyses of therapeutics and emerging drug candidates are summarized and reviewed. The
analytical methods include both low and high molecular mass compounds (e.g., anabolic
agents, stimulants, metabolic modulators, peptide hormones, and small interfering RNA
(siRNA)) determined from serum, plasma, and DBS using state-of-the-art instrumentation
such as liquid chromatography (LC)-high resolution/high accuracy (tandem) mass
spectrometry (LC-HRMS), LC-low resolution tandem mass spectrometry (LC-MS/MS), and
gas chromatography-mass spectrometry (GC-MS) [13086].
Employing gas chromatography – triple quadrupole (QqQ) tandem mass spectrometry (GC-
MS/MS), a complementary methodology was presented in 2012, allowing for the qualitative
and partly quantitative identification of 173 analytes including representatives of anabolic
agents, beta2-agonists, hormone and metabolic modulators, masking agents, stimulants,
narcotics, beta-blockers, and cannabinoids. By means of targeted multiple reaction
monitoring (MRM) and 11 ISTDs (10 of which were isotopically labelled), a robust and
sensitive assay was established enabling the routine screening for over 150 xenobiotic
compounds plus the provision of the individual's steroid profile from a total of 1 mL of urine.
In agreement with earlier approaches, the urine was subjected to enzymatic hydrolysis,
liquid-liquid extraction (LLE) and finally trimethylsilylation prior to gas chromatography-
tandem mass spectrometry (GC-MS/MS) analysis [13019].
742
Comprehensive two-dimensional gas chromatography (GC x GC) now occupies a niche
within the GC technology regime. The technique is undeniably unique in the manner in which
the experiment is conducted, the way results are presented and the interpretive opportunities
offered. For the 1000th volume of this journal it is appropriate to expand upon these features,
and review the progress made in GC x GC to date. Firstly, brief general comment is made on
multidimensional procedures, and to review key aspects of GC x GC. The use of the targeted
multidimensional GC method allows absolute retentions in the second dimension of a GC x
GC experiment to be estimated, and also offers a novel way to obtain enhanced response for
resolved solutes. Then, to illustrate the utility of the technique, the application of GC x GC to
the screening of drugs and their metabolites in biological fluids is described using prolintane
metabolites in canine urine as an example, with samples taken at four time intervals after
administration. This example illustrates the first application of GC x GC in the field of forensic
toxicology, an area traditionally dominated by GC-MS. Most drug compounds were found to
be retained on the 0.8-m second column for a greater time than the modulation period (3 s)
used for initial analysis, under the conditions described. Hence a 0.4-m D2 BPX50 (50 %
phenyl methyl polysilphenylene) column was then used throughout, with most compounds
retained less than 4 s. For the standard drug mixture, three overlapping drugs on the first
dimension column (BPX5) were subsequently baseline resolved on the BPX50 column. For
prolintane administration samples, the parent drug and metabolites could be effectively
resolved from background matrix peaks. Likewise a 23-drug spike standard in horse urine
blank gave acceptable resolution of the drugs from matrix peaks [03036].
Gas chromatography-microchip atmospheric pressure photoionization-mass

spectrometry
A gas chromatography-microchip atmospheric pressure photoionization-tandem mass

spectrometry (GC-microAPPI-MS/MS) method was developed for the analysis of anabolic
androgenic steroids in urine as their trimethylsilyl derivatives. The method utilizes a heated
nebulizer microchip in atmospheric pressure photoionization mode (microAPPI) with
chlorobenzene as dopant, which provides high ionization efficiency by producing abundant
radical cations with minimal fragmentation. The performance of GC-microAPPI-MS/MS was
evaluated with respect to repeatability, linearity, linear range, and limit of detection (LOD).
The results confirmed the potential of the method for doping control analysis of anabolic
steroids. Repeatability, linearity, and sensitivity (LODs 0.05-0.1ng/mL) were acceptable.
Quantitative performance of the method was tested and compared with that of conventional
GC-electron ionization-MS, and the results were in good agreement [13087].
Gas chromatography-positive chemical ionization triple quadrupole mass

spectrometry
In 2013, the World Anti-Doping Agency (WADA) drastically lowered the minimum required
performance levels (MRPLs) of most doping substances, demanding a substantial increase
in sensitivity of the existing methods. For a number of compounds, conventional electron
impact ionization gas chromatography tandem mass spectrometry (GC-EI-MS/MS) is often
no longer sufficient to reach these MRPLs and new strategies are required. In this study, the
capabilities of positive ion chemical ionization (PICI) GC-MS/MS are investigated for a wide
range of drug related compounds of various classes by injection of silylated reference
standards. Ammonia as PICI reagent gas had superior characteristics for GC-MS/MS
purposes than methane. Compared to GC-EI-MS/MS, PICI (with ammonia as reagent gas)
provided more selective ion transitions and consequently, increased sensitivity by an
average factor of 50. The maximum increase (by factor of 500-1000) was observed in the
analysis of stimulants, namely chlorprenaline, furfenorex and phentermine. In total, improved
743
sensitivity was obtained for 113 out of 120 compounds. A new GC-PICI-MS/MS method has
been developed and evaluated for the detection of a wide variety of exogenous doping
substances and the quantification of endogenous steroids in urine in compliance with the
required MRPLs established by WADA in 2013. The method consists of a hydrolysis and
extraction step, followed by derivatization and subsequent 1microL pulsed splitless injection
on GC-PICI-MS/MS (16 min run). The increased sensitivity allows the set up of a balanced
screening method that meets the requirements for both quantitative and qualitative
compounds: sufficient capacity and resolution in combination with high sensitivity and short
analysis time. This resulted in calibration curves with a wide linear range (e.g. 48-9600
ng/mL for androsterone and etiochanolone) without compromising the requirements for the
qualitative compounds [150128].
Gas chromatography-mass spectrometry
Gas chromatography-mass spectrometry (GC-MS) is the technique that is used most widely
in anti-doping labs. The GC effluent enters the mass spectrometer continuously, and the
mass spectrometer continuously records roughly one mass spectrum (scan) per second.
There are two main modes of MS operation: the full-scan mode and the selected ion
monitoring (SIM) mod. In the full-scan mode, the mass spectrometer records the whole mass
spectrum (from m/z 70 to 400), monitoring hundreds of ions. In the SIM mode, only selected
ions are monitored (e.g. 143, 345, 360); therefore, a longer time is spent recording each ion.
In physics, signal strength (signal-to-noise ratio) increases with the time spent collecting
data. Therefore, on the same instrument SIM is more sensitive than full scan; it can detect
smaller amounts of drug. Other types of MS that are more sensitive include high-resolution
MS, tandem MS, and ion traps. High-resolution MS is designed to measure m/z not only to
the nearest unit or decimal, but out to several more decimals. This makes it possible to
mathematically deduce the molecular formula (how many carbon, hydrogen, oxygen, and
other atoms it contains); the more decimals, the fewer combinations of atoms fit, the
narrower the possibilities. High-resolution MS instruments happen to be inherently more
sensitive. Tandem MS instruments have two mass spectrometers back to back. The first one
can be used to select only one ion, the precursor ion, which can be the molecular ion. The
second mass spectrometer monitors only one (or at most a few) characteristic
fragmentations (transitions to product ions). This is called the multiple reaction monitoring or
selected reaction monitoring mode. (Alternatively, the first mass spectrometer can be used to
select only the molecular ion and the second mass spectrometer can be used to record a full
scan.) Tandem MS is more sensitive because it is blind to interferences. Unlike all of the
above MS types, which let all ions formed continually escape from the ion source, ion traps
trap all ions until they are released, one m/z at a time, to determine their abundance [07046].
One paper described a fast gas chromatographic/mass spectrometric (GC/MS) screening

method for the detection, in urine, of 36 xenobiotics (30 synthetic anabolic steroids, four
narcotics, one diuretic and one stimulant) excreted free or as glucuro-conjugates in urine and
detectable as trimethylsilyl (TMS) derivatives. These drugs (and/or their urinary metabolites)
can be simultaneously extracted by a single liquid/liquid separation step, at alkaline pH, after
enzymatic hydrolysis with beta-glucuronidase and then assayed as TMS derivatives by
GC/MS using electron ionisation (EI) and single ion monitoring (SIM) acquisition mode. The
total time needed for the GC run is less than 8 min. Good reproducibility of the retention
times (CV % <1) and the relative abundances of the diagnostic fragment ions (CV % <10)
was observed for all target analytes. The sensitivity of the method is sufficient to match the
requirements of the World Anti-Doping Agency (WADA) for the accredited laboratories, with
limits of detection (LODs) that are lower than the corresponding WADA minimum required
744
performance limits (MRPLs) for all target compounds [07049].
Gas chromatography-combustion-IRMS (GC-C-IRMS).
In order to detect the misuse of endogenous anabolic steroids, doping control laboratories
require methods that allow differentiation between endogenous steroids and their synthetic
copies. Gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS) is
capable of measuring the carbon isotope ratio of urinary steroids and this allows
differentiation between both. GC-C-IRMS and its application to doping control has evolved a
lot during the last decade and so have the World Anti-Doping Agency (WADA) technical
documents that describe how GC-C-IRMS should be applied. Especially the WADA technical
document of 2014 introduced a number of obligatory quality controls and a fixed
methodology that should be used by all the doping control laboratories. This document
imposed more uniform methods between the laboratories in order to decrease the
interlaboratory standard deviation and acquire similar results for the analysis of the same
urine samples. In this paper, 3 years of drug testing data of our GC-C-IRMS method in
routine doping control practice is described, with an emphasis on the new WADA technical
document and its implementation. Useful data for other doping control laboratories is
presented focussing on general method setup, quality control and data collected from routine
samples. One paper concentrated on how IRMS results shift or remain similar by switching to
the 2014 WADA technical document and gives insight in a straightforward approach to
calculate the measurement uncertainty [150129].
Before application to doping control, it had long been used to detect the fraudulent
substitution of synthetic compounds in place of natural compounds in the food, flavor, and
fragrance industries. The anabolic steroids are extracted from urine and separated by GC.
The separated testosterone enters the pencil-size combustion oven where it is pyrolyzed.
Every carbon atom in the molecule is converted to CO2, and every hydrogen atom is
converted to water (H2O). The water is scrubbed out and only the CO2 enters the IRMS. This
type of MS measures only three m/z: 44 for 12C16O2, and 45 and 46 for variants containing
carbon-13, oxygen-17, or oxygen-18. From the relative abundances, the instrument software
calculates the delta13C (delta) value. It reflects the 13C/12C ratio, but it actually is the
difference between the 13C/12C ratio of the sample and that of an international standard. The
units are ‰ (per mil). By definition, the delta value of the international standard is 0‰.
Examples of values are -24 ‰ for natural testosterone and -29 ‰ for pharmaceutical
testosterone. The values are negative because both compounds contain less carbon-13 than
the international standard: 29 fewer parts per thousand for the pharmaceutical testosterone.
After exogenous testosterone administration, the delta values of urinary testosterone
metabolites become more negative. In contrast, the delta values of testosterone precursors,
or of endogenous steroids not involved in testosterone metabolism, remain unchanged;
therefore, they can be used as endogenous reference compounds. A gap in delta value
between testosterone or its metabolites and an endogenous reference compound indicates
the use of testosterone or of any steroid in its metabolism. If the difference between the delta
values of one metabolite and the endogenous reference compound is three delta units or
more, the WADA requirement for reporting an adverse analytical finding has been met. The
power of this approach is that it can detect the use of not only testosterone itself, but also of
any one of many testosterone precursors and metabolites. The second advantage is that it is
not affected by factors that might influence baseline delta values. For example, diet
influences the carbon-13 content of endogenous steroids – all of them to a similar extent.
Although interpreting vastly different delta values from one individual to the next might be
difficult, a difference in delta values between a testosterone metabolite and an endogenous
reference compound clearly reveals drug use. In short, the approach compensates for
individual variability. The third advantage is that it does not require identifying or even
745
knowing what exact compound was taken. RMS testing has been applied to various
testosterone precursors, testosterone metabolites, and endogenous reference compounds. It
is currently done for samples with T/E greater than 4 or on request by the sports authority
[07011].
An alternative calibration procedure for use when performing carbon isotope ratio
measurements by gas chromatography/combustion/isotope ratio mass spectrometry
(GC/C/IRMS) has been developed. This calibration procedure does not rely on the
corrections in-built in the instrument software, as the carbon isotope ratios of a sample are
calculated from the measured raw peak areas. The method was developed for the
certification of a urine reference material for sports drug testing, as the estimation of
measurement uncertainty is greatly simplified. To ensure that the method is free from bias
arising from the choice of calibration material and instrument, the carbon isotope ratios of
steroids in urine extracts were measured using two different instruments in different
laboratories, and three different reference materials (CU/USADA steroid standards from
Brenna Laboratory, Cornell University; NIST RM8539 mineral oil; methane calibrated against
NIST RM8560 natural gas). The measurements were performed at LGC and the Australian
National Measurement Institute (NMI). It was found that there was no significant difference in
measurement results when different instruments and reference materials were used to
measure the carbon isotope ratio of the major testosterone metabolites androsterone and
etiocholanolone, or the endogenous reference compounds pregnanediol, 11-
ketoetiocholanolone and 11beta-hydroxyandrosterone. The measurement results of this
comparison were used to estimate a measurement uncertainty of delta13C for the certification
of the urine reference material being performed on a single instrument using a single
reference material at NMI [11037].
The confirmation by GC/C/IRMS of the exogenous origin of pseudo-endogenous steroids

from human urine samples requires extracts of adequate purity. A strategy based on HPLC
sample purification prior to the GC/C/IRMS analysis of human urinary endogenous
androgens (i.e. testosterone, androsterone and/or androstenediols), is presented. A method
without any additional derivatization step is proposed, allowing to simplify the urine
pretreatment procedure, leading to extracts free of interferences permitting precise and
accurate IRMS analysis, without the need of correcting the measured delta values for the
contribution of the derivatizing agent. The HPLC extracts were adequately combined to both
reduce the number of GC/C/IRMS runs and to have appropriate endogenous reference
compounds (ERC; i.e. pregnanediol, 11-keto-etiocholanolone) on each GC-IRMS run. The
purity of the extracts was assessed by their parallel analysis by gas chromatography coupled
to mass spectrometry, with GC conditions identical to those of the GC/C/IRMS assay. The
method has been validated according to ISO17025 requirements (within assay precision
below 0.3‰ 13C delta units and between assay precision below 0.6‰ 13C delta units for most
of the compounds investigated) fulfilling the World Anti-Doping Agency requirements [12051].
In gas chromatographic-combustion-isotope ratio mass spectrometry (GC-C-IRMS) doping

control analysis, endogenous androgenic anabolic steroids and their metabolites are
commonly acetylated using acetic anhydride reagent, thus incorporating exogenous carbon
that contributes to the measured isotope ratio. Comparison of the endogenous delta13C of
free, mono-, and di-acetylated steroids requires application of corrections, typically through
straightforward use of the mass balance equation. Variability in kinetic isotope effects (KIE)
due to steroid structures could cause fractionation of endogenous steroid carbon, resulting in
inaccurate results. To test for possible KIE influence on delta13C, acetic anhydride of graded
isotope ratio within the natural abundance range was used under normal derivatization
conditions to test for linearity. In all cases, plots of measured steroid acetate delta 13C versus
acetic anhydride delta13C were linear and slopes were not significantly different. Regression
746
analysis of the delta13C of enriched acetic anhydrides versus delta13C of derivatized steroids
shows that KIE are similar in all cases. It was concluded that delta13C calculated from the
mass balance equation is independent of the delta13C of the acetic anhydride reagent, and
that net KIE under normal derivatization conditions do not bias the final reported steroid
delta13C [12061].
In order to detect the misuse of endogenous anabolic steroids, doping control laboratories
require methods that allow differentiation between endogenous steroids and their synthetic
copies. Gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS) is
capable of measuring the carbon isotope ratio of urinary steroids and this allows
differentiation between both. GC-C-IRMS and its application to doping control has evolved a
lot during the last decade and so have the World Anti-Doping Agency (WADA) technical
documents that describe how GC-C-IRMS should be applied. Especially the WADA technical
document of 2014 introduced a number of obligatory quality controls and a fixed
methodology that should be used by all the doping control laboratories. This document
imposed more uniform methods between the laboratories in order to decrease the
interlaboratory standard deviation and acquire similar results for the analysis of the same
urine samples. In this paper, 3 years of drug testing data of our GC-C-IRMS method in
routine doping control practice is described, with an emphasis on the new WADA technical
document and its implementation. Useful data for other doping control laboratories is
presented focussing on general method setup, quality control and data collected from routine
samples. The paper concentrates on how IRMS results shift or remain similar by switching to
the 2014 WADA technical document and gives insight in a straightforward approach to
calculate the measurement uncertainty [14734].
Distinguishing between endogenous and exogenous steroids

The contamination of commonly used supplements by unknown steroids as well as their
metabolites (parent compounds) become a challenge for the analytical laboratories. Although
the determination of steroids profile is not trivial because of the complex matrix and low
concentration of single compound, one of the most difficult current problem is to distinguish,
during analytical procedure, endogenous androgens such as testosterone,
dehydrotestosterone or dehydroepiandrosterone from their synthetic equivalents. The aim of
this work was to develop and validate an analytical procedure for determination of the steroid
profile in human urine by gas chromatography-combustion-isotope ratio mass spectrometry
(GC/C/IRMS) toward distinguishing between endogenous and exogenous steroids. Beside
the optimization of the experimental parameters for gas chromatography separation and
mass spectrometry, attention was focused on urine sample preparation. Using an optimized
sample preparation protocol it was possible to achieve better chromatographic resolutions
and better sensitivity enabling the determination of 5 steroids, androsterone, etiocholanolone,
testosterone, 5-androstandiol, 11-hydroxyandrdostane, pregnandiol, with the expanded
uncertainty (k=2) below 0.1 percent. The analytical protocol described in this work was
successfully used for the confirmation of positive founding urine by evaluation T/E ratio after
GC/C/IRMS analysis [14735].
The use of performance enhancing drugs in sports is prohibited. For the detection of misuse
of such substances gas chromatography or liquid chromatography coupled to mass
spectrometry are the most frequently used detection techniques. In this work the
development and validation of a fast gas chromatography tandem mass spectrometric
method for the detection of a wide range of doping agents is described. The method can
determine 13 endogenous steroids (the steroid profile), 19-norandrosterone, salbutamol and
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11-nor-delta9-tetrahydrocannabinol.9carboxylic acid in the applicable ranges and to detect
qualitatively over 140 substances in accordance with the minimum required performance
levels of the World Anti-Doping Agency in 1mlLof urine. The classes of substances included
in the method are anabolic steroids, beta2-agonists, stimulants, narcotics, hormone
antagonists and modulators and beta-blockers. Moreover, using a short capillary column and
hydrogen as a carrier gas the run time of the method is less than 8 min [11038].
Gas chromatography-QqQ-MS
A gas chromatography-QqQ-MS method was developed for the detection of over 150
compounds from different classes (steroids, narcotics, stimulants, beta-blockers, beta-2-
agonists and hormone antagonists) in a qualitative way. In the quantitative part, the
traditional steroid profile with the most important endogenous steroids is expanded with six
minor metabolites, which further improves the detection and identification of endogenous
steroid abuse. In addition to these, norandrosterone, salbutamol and the major metabolite of
cannabis are also quantified. Methods developed for anti-doping purposes should be
subjected to the highest level of quality. Here, the addition of a combination of (deuterated)
internal standards allows for an accurate quality control of every single step of the
methodology: hydrolysis efficiency, derivatization efficiency and microbiological degradation
are monitored in every single sample. Additionally, special attention is paid to the
relationships between parameters indicating degradation by micro-organisms and the
reliability of the steroid profile. The impact of the degradation is studied by evaluation of the
quantities and percentages of 5alpha-androstane-3,17-dione and 5beta-androstane-3,17-
dione. The concept of measurement uncertainty was introduced for the evaluation of relative
abundances of mass-to-charge ratios and the obtained ranges were compared with the
World Anti-Doping Agency regulations on tolerance windows for relative ion intensities. The
results indicate that the approaches are similar [12063].
GC/Quadrupole-Orbitrap Mass Spectrometer

Identification of unknown compounds is of critical importance in GC/MS applications
(metabolomics, environmental toxin identification, sports doping, petroleomics, and biofuel
analysis, among many others) and remains a technological challenge. Derivation of
elemental composition is the first step to determining the identity of an unknown compound
by MS, for which high accuracy mass and isotopomer distribution measurements are critical.
Here, we report on the development of a dedicated, applications-grade GC/MS employing an
Orbitrap mass analyzer, the GC/Quadrupole-Orbitrap. Built from the basis of the benchtop
Orbitrap LC/MS, the GC/Quadrupole-Orbitrap maintains the performance characteristics of
the Orbitrap, enables quadrupole-based isolation for sensitive analyte detection, and
includes numerous analysis modalities to facilitate structural elucidation. It was detailed the
design and construction of the instrument, discuss its key figures-of-merit, and demonstrate
its performance for the characterization of unknown compounds and environmental toxins
[14634].
Carbon isotope (CIR)-based analyses
The Achilles’ heel of all carbon isotope ratio (CIR)-based assays however is the necessity of
a significant difference between the CIR of the administered steroid and the employed
endogenous reference compounds (ERCs). As demonstrated earlier various testosterone
formulations of mostly illicit origin exhibit CIRs at natural 13C-values. Here, IRMS analyses
focusing on carbon isotope signatures only might disallow determining the prohibited
administration of a natural steroid. Further to this, the effect of hormones influencing
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testicular activity such as human chorionic gonadotrophin (hCG) on steroid profiles and CIRs
necessitated consideration [12017].
The detection of steroids originating from synthetic precursors against a background of their
chemically identical natural analogues has proven to be a significant challenge for doping
control laboratories accredited by the World Anti-Doping Agency (WADA). The
complementary application of gas chromatography-mass spectrometry (GC-MS) and gas
chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) has been
demonstrated to provide specific detection of endogenous steroid misuse for improved anti-
doping analysis. Markers of synthetically derived steroids are reviewed on the basis of
abnormal urinary excretions and low 13C content. A combinatorial approach is presented for
the interpretation of GC-MS and GC-C-IRMS data in the anti-doping context. This
methodology can allow all relevant information concerning an individual's metabolism to be
assessed in order to make an informed decision with respect to a doping violation [12077].
IRMS (isotope ratio mass spectrometry)
Today, many scientists concerned with forensic and environmental analytics will appreciate
the introduction of isotope ratio mass spectrometry (IRMS) as a most innovative tool.
Significant IRMS-based knowledge about the systematic variation of 13C/12C ratios in nature
was, however, available around 1950. The fundamental depletion of 13C in organic material
was even recognized as early as 1939. This development essentially was brought forward by
Alfred Nier. Any scientist currently concerned with IRMS will immediately be familiar with
Nier’s mass spectrometer design from 1947. In fact, significant development of the ion optics,
source design, etc., has taken place in the meanwhile. But ever since Nier’s inventions,
these instruments feature multiple collectors and the characteristic magnetic sector field.
Even more striking, the fundamental design of isotope ratio mass spectrometers has not
changed since 1940. Nier built his first mass spectrometer in 1936. But on principle, the
concept even dates back to 1918, when AJ Dempster advocated the 180ο design as “A New
Method of Positive Ray Analysis” Five years earlier, Jj Thomson had shown the usefulness
of “Positive Rays as a Method of Chemical Analysis”. So, IRMS is a well proven, rather
traditional methodology. The term “continous-flow isotope ratio mass spectrometry”
(CFIRMS) was presumably introduced in 1988 when Preston and McMillan coupled an
elemental analyzer to an isotope ratio mass spectrometer via a variable leak. Recent
improvements in the methodology mainly concern online coupling and analytical peripherals.
The breakthrough thus achieved for doping control can hardly be overestimated. If still
challenging, 13C/12C analysis of urinary steroids by gas chromatography coupled to IRMS is
now a standard procedure. However, it merely represents one out of a multitude of emerging
applications of stable isotope analysis at natural abundance. The relative nature of d scales
must always be kept in mind. As a most important consequence, divisions are not feasible. It
follows immediately that measurement uncertainties must not be expressed as coefficients of
variation. Due to the design of Nier-type mass spectrometers, the analytical error is largely
independent of the measured isotope ratio anyway. When emphasis is on quantitation of the
respective contributions, the term source apportionment is more appropriate. In doping
control, it is implicitly assumed that there are two distinct sources for urinary steroids:
Endogenous steroids and/ or synthetic steroids. Therefore, low 13C/12C ratios are often
immediately associated with synthetic origin. However, theoretically and practically the
situation is more complicated and this should fundamentlly be considered. Terms and
definitions for possible sources of urinary steroids are not very consistent. For instance,
endogenous steroid is mostly used for a compound synthesized physiologically in an
organism. But sometimes it is also used to classify pharmaceuticals with chemical structures
identical to those of the physiological compounds [12072].
749
Delta 13C and delta13C values of endogenous urinary steroids represent physiological random
variables. Measurement uncertainty and biological scatter likewise contribute to the
variances. The statistical distributions of negative controls are well investigated, but there is
little knowledge about the corresponding distributions of steroid-users. For these reasons
valid discrimination of steroid users from non-users by (13C/12C analysis of endogenous
steroids requires elaborate statistical treatment. Corresponding Bayesian approaches are
presented following an introduction to the rationale. The use of mixture models appears
appropriate. The distribution of routine data has been deconvolved and characterized
accordingly. The mixture components, which presumably represent steroid users and non-
users, exhibit considerable overlap. The validity of a given result depends on both the
analytical uncertainty and the prior probability of doping offenses. Low analytical
uncertainties but high prior probabilities facilitate valid detection of doping offenses. Two
recommendations can be deduced. First, before starting an 13C/12C analysis, any initial
suspicion should be well-substantiated. This precludes use of permissive criteria derived
from the steroid profile. Secondly, knowledge of relevant 13C/12C distributions is required.
This must cover representative numbers of authentic steroid users. Finally, it is desirable that
the conditional probability for steroid administration rather than the measurement uncertainty
is calculated and reported. This quantity possesses superior validity and it is largely
independent of laboratory bias. The findings suggest and facilitate flexible handling of
decision limits [12073].
The detection of steroids originating from synthetic precursors against a background of their
chemically identical natural analogues has proven to be a significant challenge for doping
control laboratories accredited by the World Anti-Doping Agency (WADA). The
complementary application of gas chromatography-mass spectrometry (GC-MS) and gas
chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) has been
demonstrated to provide specific detection of endogenous steroid misuse for improved anti-
doping analysis. Markers of synthetically derived steroids are reviewed on the basis of
abnormal urinary excretions and low 13C content. A combinatorial approach is presented for
the interpretation of GC-MS and GC-C-IRMS data in the anti-doping context. This
methodology can allow all relevant information concerning an individual's metabolism to be
assessed in order to make an informed decision with respect to a doping violation [12074].
Isotope ratio mass spectrometry (IRMS) testing is performed to determine if an atypical

steroid profile is due to administration of an endogenous steroid. Androsterone (Andro) and
etiocholanolone (Etio), and/or the androstanediols (5alpha- and 5beta-androstane-
3alpha,17beta-diol) are typically analyzed by IRMS to determine the 13C/12C ratio. The ratios
of these target compounds are compared to the 13C/12C ratio of an endogenous reference
compound (ERC) such as 5beta-pregnane-3alpha,20alpha-diol (Pdiol). Concentrations of
Andro and Etio are high so 13C/12C ratios can easily be measured in most urine samples.
Despite the potentially improved sensitivity of the androstanediols for detecting the use of
some testosterone formulations, additional processing steps are often required that increase
labour costs and turnaround times. Since this can be problematic when performing large
numbers of IRMS measurements, we established thresholds for Andro and Etio that can be
used to determine the need for additional androstanediol testing. Using these criteria, 105
out of 2639 urine samples exceeded the Andro and/or Etio thresholds, with 52 of these
samples being positive based on Andro and Etio IRMS testing alone. The remaining 53 urine
samples had androstanediol IRMS testing performed and 3 samples were positive based on
the androstanediol results. A similar strategy was used to establish a threshold for Pdiol to
identify athletes with relatively 13C-depleted values so that an alternative ERC can be used to
confirm or establish a true endogenous reference value. Adoption of a similar strategy by
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other laboratories can significantly reduce IRMS sample processing and analysis times,
thereby increasing testing capacity [13095].
Complementary to carbon isotope ratios (CIR), isotope ratio analysis concerning hydrogen
and deuterium (HIR) has received increasing attention in doping controls. Particularly the 2-
dimensional analysis of urinary steroids, i.e. combined evaluation of CIR and HIR, was
considered as a powerful (though time- and cost-intensive) means allowing to lower
reference limits in doping controls and to enable the determination of exogenous steroids
comprising CIR signatures close to endogenous values. In that respect, potentially
confounding factors have to be assessed and the influence of the deuterium content in
drinking water on urinary steroid HIR was measured in a recent study. Despite the drastic
influence on the HIR of the body water, only shifts of approximately 30 ‰ in urinary steroids
were observed. Hence, the HIR analysis proved robust against diet-induced changes,
specifically the ingestion of drinking water with different isotopic signature [13009].
Isoelectric focusing
Isoelectric focusing (IEF) is used to detect recombinant EPO in the urine EPO test.
Historically, the EPO test at the Olympics (2000 to 2006) was done on paired blood and urine
samples collected simultaneously. The blood test is an indirect test because it does not
detect the presence of recombinant EPO. Instead, it measures multiple parameters (eg,
hemoglobin, hematocrit, percentage of reticulocytes) and calculates a score that indicates
whether the individual is on or recently off recombinant EPO. Since 2002, EPO tests done by
United States sports authorities have included only the urine test, a direct test that identifies
recombinant EPO. EPO tests are done on only some of all of the urine samples, upon
request by the sports authority. Endogenous human EPO is a glycoprotein with a known
amino acid sequence and glycosylation pattern. More precisely, it consists of a family of
isoforms (molecules that differ only by their degrees of glycosylation). As a result, the pH at
which each isoform bears as many negative charges as positive charges (isoelectric point or
PI) is different. Recombinant human EPO differs from endogenous human EPO only by its
overall glycosylation pattern (i.e. it consists of a different family of isoforms). The difference in
overall pattern of isoforms allows differentiation between recombinant and endogenous
human EPO. The urine EPO test, also known as the French test or the IEF test, consists of
four steps: sample preparation, IEF, double blotting, and visualization. Sample preparation
concentrates EPO by multiple ultrafiltrations that leave the proteins of desired molecular
weight in the filtration “retentate.” Next, the retentate is deposited on a gel with an embedded
pH gradient, and a current is applied to achieve electrophoretic separation of the isoforms
(IEF). Unknown samples, reference standards, and known positive and negative quality
controls are normally run on each gel. Each sample, standard, or control spreads out in its
own “lane.” Each isoform is charged; therefore, it migrates in the electrical field until it
reaches the distance on the gel at which the pH is equal to its PI. There the isoform is
electrically neutral so it stops migrating. Its position or distance up the gel is key, and the
goal of the remaining steps is to visualize it. The first blotting step transfers all proteins
(erythropoietic and other) to a first membrane. The membrane is incubated with antibodies
specific to erythropoietic proteins. The second blot transfers only these specific antibodies to
the second membrane, thus transferring the isoform pattern, but leaving behind all proteins,
including some that otherwise would obscure the final image. Visualization is based on
chemiluminescence; it involves incubation with a second antibody that binds to the first
antibody and a chemical reaction that emits light. The image (electropherogram) is captured
with a special digital camera. All steps use commonplace molecular biology techniques. The
electropherogram contains one lane per sample, standard, or quality control sample. In each
751
lane, the isoform pattern consists of bands. The pattern (number of bands, positions, relative
intensities) allows identification. In common language, a negative EPO test often is
discussed as if it reflects the absence of EPO, but of course what it means is that there was
no recombinant erythropoietic protein in the urine sample, which normally would (hopefully!)
contain natural, endogenous EPO [07011].
Electrospray ionization
Electrospray ionization (ESI) mass spectra of 15 anti-estrogenic substances, beta2-agonists

and mesocarb were investigated in terms of fragmentation patterns. On the basis of this
product ion information, a simultaneous screening method for anti-estrogenic substances,
beta2-agonists and mesocarb was developed for doping control purposes. After hydrolysis,
liquid-liquid extraction was adopted for the sample preparation. The recoveries for all
compounds were 30 and 96 percent. A single liquid chromatography/tandem mass
spectrometry (LC/MS/MS) analysis could be performed in 13 min for the analysis of 15 anti-
estrogenic substances, beta2-agonists and mesocarb. A quantitative analysis was also
validated. Inaccuracies were below + 12 percent and precisions varied from 0 to 16 percent.
The limit of detection was below 10 ng/mL except formestane (300 ng/mL) and
aminoglutethimide (100 ng/mL). The validated method was applied for the analysis of
excretion samples [07054].
Micellar electrokinetic capillary chromatography and electrospray mass spectrometry
A partial filling micellar electrokinetic capillary chromatography (PF-MEKC) separation of six

anabolic androgenic steroids (androstenedione, metandienone, fluoxymesterone,
methyltestosterone, 17-epimetandienone and testosterone) is introduced. The method
utilises a mixed micellar solution consisting of sodium dodecyl sulphate (SDS) and sodium
taurocholate. The analytes are detected with a photodiode array detector at 247 nm
wavelength. Methyltestosterone is used as internal standard. The detection limits were 39
microg/L for androstenedione, 40 microg/L for testosterone, 45 microg/L for fluoxymesterone,
45-90 microg/L for 17-epimetandienone, 59 microg/L for methyltestosterone and 90 microg/L
for metandienone. Linear correlation between concentration (0.1-5.0 mg/L) and detector
response was obtained with r2 of 0.994 for fluoxymesterone, 0.998 for 17-epimetandienone
and 0.999 for androstenedione, metandienone and testosterone. In addition, ionisation of the
investigated compounds in electrospray mass spectrometry (ESI-MS) was studied in positive
ion mode. The most intense signal (100 %) was the protonated molecular ion [M + H] +,
except for 17-epimetandienone, which gave its strongest signal at m/z corresponding to [M -
H2O + H]+. Finally, separation and identification of fluoxymesterone, androstenedione and
testosterone by PF-MEKC-ESI-MS is described. This is the first use of PF-MEKC and PF-
MEKC-ESI-MS assays for anabolic androgenic steroids [04047].
During the past two decades, chiral capillary electrophoresis (CE) emerged as a promising,
effective and economic approach for the enantioselective determination of drugs and their
metabolites in body fluids, tissues and in vitro preparations. This review discusses the
principles and important aspects of CE-based chiral bioassays, provides a survey of the
assays developed during the past 10 years and presents an overview of the key
achievements encountered in that time period. Applications discussed encompass the
pharmacokinetics of drug enantiomers in vivo and in vitro, the elucidation of the
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stereoselectivity of drug metabolism in vivo and in vitro, and bioanalysis of drug enantiomers
of toxicological, forensic and doping interest. Chiral CE was extensively employed for
research purposes to investigate the stereoselectivity associated with hydroxylation,
dealkylation, carboxylation, sulfoxidation, N-oxidation and ketoreduction of drugs and
metabolites. Enantioselective CE played a pivotal role in many biomedical studies, thereby
providing new insights into the stereoselective metabolism of drugs in different species which
might eventually lead to new strategies for optimization of pharmacotherapy in clinical
practice [10320].
At present the role of capillary electrophoresis in the detection of doping agents in athletes is,
for the most part, nonexistent. More traditional techniques, namely gas and liquid
chromatography with mass spectrometric detection, remain the gold standard of antidoping
tests. This Feature will investigate the in-roads that capillary electrophoresis has made, the
limitations that the technique suffers from, and where the technique may grow into being a
key tool for antidoping analysis [13093].
A new type of diglycidyloxy-calix arene coated fiber made by sol-gel method was initially
prepared for capillary electrophoresis (CE) sample pretreatment. By using headspace solid-
phase microextraction (SPME) combined with a novel back-extraction facility coupled off-line
to capillary zone electrophoresis (CZE), the simultaneous determination of propranolol
enantiomers in human urine was achieved. The clean up effect and preconcentration effect
were realized for the first time without derivatization during the SPME process in terms of
these strong polarity and thermal stable compounds. Ultrasonic back-extraction and field
amplified sample injection (FASI) technologies were employed. Extraction and back-
extraction parameters were optimized. Preconcentration of the sample by calyx arene fiber
based SPME and FASI increased the sensitivity, yielding a limit of detection (LOD) of 0.01
microg/mlLby CZE-diode array detection (DAD). Method repeatability (RSD<6.5 %) and fiber
reusability (>150 extraction procedures) were observed over a linear range (0.05-10
microg/mL) in urine samples. Based on the superior thermal stability, high alkali- and solvent-
resistant ability, marvelous repeatability and long lifetime of the novel fiber, this SPME-FASI-
CZE procedure could meet the demand of minimum required performance limit (MRPL) set
by the World Anti-doping Agency (WADA) for the detection of propranolol in urine samples
[05023].
Capillary electrokinetic chromatography

The separation of three common anabolic steroids (methyltestosterone, methandrostenolone
and testosterone) was performed for the first time by capillary EKC. Different charged CD
derivatives and bile salts were tested as dispersed phases in order to achieve the separation.
A mixture of 10 mmol/L succinylated-beta-CD with 1 mmol/L beta-CD in a 50 mmol/L borate
buffer (pH 9) enabled the separation of the three anabolic steroids in less than 9 min.
Concentration LODs, obtained for these compounds with low absorption of UV light, were
approximately 5 x 10-5 mol/L. The use of online reverse migrating sample stacking with large-
volume injection (the effective length of the capillary) enabled to improve the detection
sensitivity. Sensitivity enhancement factors (SEFs) ranging from 95 (for testosterone) to 149
(for methyltestosterone) were achieved by single stacking preconcentration. Then, the
possibilities of multistep stacking to improve the sensitivity for these analytes were
investigated. SEFs obtained by double stacking preconcentration ranged from 138 to 185,
enabling concentration LODs of 2.79 x 10-7 mol/L (for methyltestosterone), 3.47 x 10-7 mol/L
(for testosterone) and 3.56 x 10-7 mol/L (for methandrostenolone). Although online triple
stacking preconcentration was achieved, its repeatability was very poor and SEFs for the
studied analytes were not calculated [05024].
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Capillary electrophoresis time-of-flight mass spectrometry
Capillary electrophoresis coupled to orthogonal accelerated time-of-flight mass spectrometry

(CE/TOFMS) was used for the analysis of O- and N-glycopeptides of recombinant human
erythropoietin (rhEPO). O(126) and N(83) with a tetraantennary complex type glycan (N(83)-
4Ant) were selected as glycopeptide models to develop an optimum CE/TOFMS
methodology capable of detecting and characterizing the wide variety of glycopeptides
present in the glycoprotein digest. Glycopeptide adsorption in the inner surface of the fused-
silica capillary was prevented after using a capillary conditioning of 1 M HAc between runs.
On the other hand, different acidic conditions in the sheath liquid (SL) and in the background
electrolyte (BGE) were tested with the aim of studying their influence in glycopeptide
fragmentation. Finally, the fragmentor voltage value of the TOF-MS instrument was
optimized to avoid the involuntary fragmentation of the native glycopeptides. Hence, the
established method may be regarded as an excellent starting point to obtain reliable
glycopeptide maps of complex glycoproteins such as rhEPO by CE/TOFMS [11430].
Vacuum MALDI-linear ion trap mass spectrometry
Detection of doping agents in urine frequently requires extensive separation prior to chemical
analyses. Gas or liquid chromatography coupled to mass spectrometry has produced
accurate and sensitive assays, but chromatographic separations require time and,
sometimes, chemical derivatization. To avoid such tedious and lengthy procedures, vacuum
matrix-assisted laser desorption ionization (vMALDI) coupled with the linear ion trap mass
spectrometry (LIT/MS) technique is tested for its applicability as a rapid screening technique.
Commonly used doping agents like nandrolone, boldenone, trenbolone, testosterone, and
betamethasone were chosen as study compounds. Different MALDI matrixes like alpha-
cyano-4-hydroxycinnamic acid (CHCA), dihyroxy benzoic acid (DHB) with and without cetyl
trimethyl ammonium bromide (CTAB), a surfactant, and meso-tetrakis(pentafluorophenyl)
porphyrin (F20TPP) were tested. Among them, F20TPP (MW 974.57 Da) was selected as
the preferred matrix owing to the lack of interfering matrix peaks at the lower mass range
(m/z 100-700). Urine samples spiked with study compounds were processed by solid-phase
extraction (SPE) and consistently detected through a linear range of 0.1-100 ng/mL. The limit
of detection and lower limit of quantification for all five analytes have been determined to be
0.03 and 0.1 ng/mL, respectively, in urine samples. Testosterone-d3 was used as an internal
standard, and the quantitative measurements were achieved by the selective reaction
monitoring (SRM) mode. The method was validated and showed consistency in the results.
Hence, vMALDI-LIT/MS can be used as a rapid screening method to complement the
traditional GC/MS and LC/MS techniques for simultaneous identification, confirmation, and
quantification of doping agents in urine [06039].
Detection of doping agents in urine frequently requires extensive separation prior to chemical
analyses. Gas or liquid chromatography coupled to mass spectrometry has produced
accurate and sensitive assays, but chromatographic separations require time and,
sometimes, chemical derivatization. To avoid such tedious and lengthy procedures, vacuum
matrix-assisted laser desorption ionization (vMALDI) coupled with the linear ion trap mass
spectrometry (LIT/MS) technique is tested for its applicability as a rapid screening technique.
Commonly used doping agents like nandrolone, boldenone, trenbolone, testosterone, and
betamethasone were chosen as study compounds. Different MALDI matrixes like alpha-
cyano-4-hydroxycinnamic acid (CHCA), dihyroxy benzoic acid (DHB) with and without cetyl
trimethyl ammonium bromide (CTAB), a surfactant, and meso-tetrakis(pentafluorophenyl)
porphyrin (F20TPP) were tested. Among them, F20TPP (MW 974.57 Da) was selected as
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the preferred matrix owing to the lack of interfering matrix peaks at the lower mass range
(m/z 100-700). Urine samples spiked with study compounds were processed by solid-phase
extraction (SPE) and consistently detected through a linear range of 0.1-100 ng/mL. The limit
of detection and lower limit of quantification for all five analytes have been determined to be
0.03 and 0.1 ng/mL, respectively, in urine samples. Testosterone-d3 was used as an internal
standard, and the quantitative measurements were achieved by the selective reaction
monitoring (SRM) mode. The method was validated and showed consistency in the results.
Hence, vMALDI-LIT/MS can be used as a rapid screening method to complement the
traditional GC/MS and LC/MS techniques for simultaneous identification, confirmation, and
quantification of doping agents in urine [07055].
Hyphenated mass spectrometric techniques
Hyphenated mass spectrometric techniques, particularly gas chromatography/mass

spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS), are
indispensable tools in clinical and forensic toxicology and in doping control owing to their
high sensitivity and specificity. They are used for screening, library-assisted identification and
quantification of drugs, poisons and their metabolites, prerequisites for competent expertise
in these fields. In addition, they allow the study of metabolism of new drugs or poisons as a
basis for developing screening procedures in biological matrices, most notably in urine, or
toxicological risk assessment. Concepts and procedures using GC/MS and LC/MS
techniques in the areas of analytical toxicology and the role of mass spectral libraries are
presented and discussed in this feature article [06040].
siRNA
Small interfering ribonucleic acid (siRNA) molecules can effect the expression of any gene by
inducing the degradation of mRNA. Therefore, these molecules can be of interest for illicit
performance enhancement in sports by affecting different metabolic pathways. An example
of an efficient performance-enhancing gene knockdown is the myostatin gene that regulates
muscle growth. One study was carried out to provide a tool for the mass spectrometric
detection of modified and unmodified siRNA from plasma samples. The oligonucleotides are
purified by centrifugal filtration and the use of an miRNA purification kit, followed by flow-
injection analysis using an Exactive mass spectrometer to yield the accurate masses of the
sense and antisense strands. Although chromatography and sensitive mass spectrometric
analysis of oligonucleotides are still challenging, a method was developed and validated that
has adequate sensitivity (limit of detection 0.25-1 nmol/mL and performance (precision 11-
21 %, recovery 23-67 %) for typical antisense oligonucleotides currently used in clinical
studies [10442].
Uncovering manipulation of athletic performance via small interfering (si)RNA is an emerging

field in sports drug testing. Due to the potential to principally knock down every target gene in
the organism by means of the RNA interference pathway, this facet of gene doping has
become a realistic scenario. In the present study, two distinct model siRNAs comprising 21
nucleotides were designed as double strands which were perfect counterparts to a sequence
of the respective messenger RNA coding the muscle regulator myostatin of Rattus
norvegicus. Several modified nucleotides were introduced in both the sense and the
antisense strand comprising phosphothioates, 2'-O-methylation, 2'-fluoro-nucleotides, locked
nucleic acids and a cholesterol tag at the 3'-end. The model siRNAs were applied to rats at 1
mg/kg (i.v.) and blood as well as urine samples were collected. After isolation of the RNA by
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means of a RNA purification kit, the target analytes were detected by liquid chromatography -
high resolution/high accuracy mass spectrometry (LC-HRMS). Analytes were detected as
modified nucleotides after alkaline hydrolysis, as intact oligonucleotide strands (top-down)
and by means of denaturing SDS-PAGE analysis. The gel-separated siRNA was further
subjected to in-gel hydrolysis with different RNases and subsequent identification of the
fragments by untargeted LC-HRMS analysis (bottom-up, “experimental RNomics”).
Combining the results of all approaches, the identification of several 3'-truncated urinary
metabolites was accomplished and target analytes were detected up to 24 h after a single
administration. Simultaneously collected blood samples yielded no promising results. The
methods were validated and found fit-for-purpose for doping controls [13106].
Mass spectrometric detection of siRNA
Small interfering ribonucleic acid (siRNA) molecules can effect the expression of any gene by
inducing the degradation of mRNA. Therefore, these molecules can be of interest for illicit
performance enhancement in sports by affecting different metabolic pathways. An example
of an efficient performance-enhancing gene knockdown is the myostatin gene that regulates
muscle growth. One study was carried out to provide a tool for the mass spectrometric
detection of modified and unmodified siRNA from plasma samples. The oligonucleotides are
purified by centrifugal filtration and the use of an miRNA purification kit, followed by flow-
injection analysis using an Exactive mass spectrometer to yield the accurate masses of the
sense and antisense strands. Although chromatography and sensitive mass spectrometric
analysis of oligonucleotides are still challenging, a method was developed and validated that
has adequate sensitivity (limit of detection 0.25-1 nmol/mL) and performance (precision 11-
21 %, recovery 23-67 %) for typical antisense oligonucleotides currently used in clinical
studies [10322].
High-resolution liquid chromatography-time-of-flight mass spectrometry
A unification of doping-control screening procedures of prohibited small molecule substances

– including stimulants, narcotics, steroids, beta2-agonists and diuretics – is highly urgent in
order to free resources for new classes such as banned proteins. Conceptually this may be
achieved by the use of a combination of one gas chromatography-time-of-flight mass
spectrometry method and one liquid chromatography-time-of-flight mass spectrometry
method. In this work a quantitative screening method using high-resolution liquid
chromatography in combination with accurate-mass time-of-flight mass spectrometry was
developed and validated for determination of glucocorticosteroids, beta2-agonists, thiazide
diuretics, and narcotics and stimulants in urine. To enable the simultaneous isolation of all
the compounds of interest and the necessary purification of the resulting extracts, a generic
extraction and hydrolysis procedure was combined with a solid-phase extraction modified for
these groups of compounds. All 56 compounds are determined using positive electrospray
ionisation with the exception of the thiazide diuretics for which the best sensitivity was
obtained by using negative electrospray ionisation. The results show that, with the exception
of clenhexyl, procaterol, and reproterol, all compounds can be detected below the respective
minimum required performance level and the results for linearity, repeatability, within-lab
reproducibility, and accuracy show that the method can be used for quantitative screening. If
qualitative screening is sufficient the instrumental analysis may be limited to positive
ionisation, because all analytes including the thiazides can be detected at the respective
minimum required levels in the positive mode. The results show that the application of
accurate-mass time-of-flight mass spectrometry in combination with generic extraction and
756
purification procedures is suitable for unification and expansion of the window of screening
methods of doping laboratories. Moreover, the full-scan accurate-mass data sets obtained
still allow retrospective examination for emerging doping agents, without re-analyzing the
samples [10036].
Ultra-high-pressure liquid chromatography-quadrupole time-of-flight mass

spectrometry
For doping control, analyses of samples are generally achieved in two steps: a rapid
screening and, in the case of a positive result, a confirmatory analysis. A two-step
methodology based on ultra-high-pressure liquid chromatography coupled to a quadrupole
time-of-flight mass spectrometry (UHPLC-QTOF-MS) was developed to screen and confirm
103 doping agents from various classes (e.g., beta-blockers, stimulants, diuretics, and
narcotics). The screening method was presented in a previous article as part I (i.e. fast
analysis of doping agents in urine by ultra-high-pressure liquid chromatography-quadrupole
time-of-flight mass spectrometry. part I: screening analysis). For the confirmatory method,
basic, neutral and acidic compounds were extracted by a dedicated solid-phase extraction
(SPE) in a 96-well plate format and detected by MS in the tandem mode to obtain precursor
and characteristic product ions. The mass accuracy and the elemental composition of
precursor and product ions were used for compound identification. After validation including
matrix effect determination, the method was considered reliable to confirm suspect results
without ambiguity according to the positivity criteria established by the World Anti-Doping
Agency. Moreover, an isocratic method was developed to separate ephedrine from its isomer
pseudoephedrine and cathine from phenylpropanolamine in a single run, what allowed their
direct quantification in urine [10037].
The urinary steroid profile is constituted by anabolic androgenic steroids, including

testosterone and its relatives, that are extensively metabolized into phase II sulfated or
glucuronidated steroids. The use of liquid chromatography coupled to mass spectrometry
(LC-MS) is an issue for the direct analysis of conjugated steroids, which can be used as
urinary markers of exogenous steroid administration in doping analysis, without hydrolysis of
the conjugated moiety. In this study, a sensitive and selective ultra high-pressure liquid
chromatography coupled to quadrupole time-of-flight mass spectrometer (UHPLC-QTOF-
MS) method was developed to quantify major urinary metabolites simultaneously after
testosterone intake. The sample preparation of the urine (1 mL) was performed by solid-
phase extraction on Oasis HLB sorbent using a 96-well plate format. The conjugated steroids
were analyzed by UHPLC-QTOF-MS(E) with a single-gradient elution of 36 min (including re-
equilibration time) in the negative electrospray ionization mode. MS(E) analysis involved
parallel alternating acquisitions of both low- and high-collision energy functions. The method
was validated and applied to samples collected from a clinical study performed with a group
of healthy human volunteers who had taken testosterone, which were compared with
samples from a placebo group. Quantitative results were also compared to GC-MS and LC-
MS/MS measurements, and the correlations between data were found appropriate. The
acquisition of full mass spectra over the entire mass range with QTOF mass analyzers gives
promise of the opportunity to extend the steroid profile to a higher number of conjugated
steroids [11034].
The potential and applicability of UHPSFC-MS/MS for anti-doping screening in urine

samples were tested for the first time. For this purpose, a group of 110 doping agents with
diverse physicochemical properties was analyzed using two separation techniques, namely
UHPLC-MS/MS and UHPSFC-MS/MS in both ESI+ and ESI- modes. The two approaches
757
were compared in terms of selectivity, sensitivity, linearity and matrix effects. As expected,
very diverse retentions and selectivities were obtained in UHPLC and UHPSFC, proving a
good complementarity of these analytical strategies. In both conditions, acceptable peak
shapes and MS detection capabilities were obtained within 7 min analysis time, enabling the
application of these two methods for screening purposes. Method sensitivity was found
comparable for 46 percent of tested compounds, while higher sensitivity was observed for 21
percent of tested compounds in UHPLC-MS/MS and for 32 percent in UHPSFC-MS/MS. The
latter demonstrated a lower susceptibility to matrix effects, which were mostly observed as
signal suppression. In the case of UHPLC-MS/MS, more serious matrix effects were
observed, leading typically to signal enhancement and the matrix effect was also
concentration dependent, i.e., more significant matrix effects occurred at the lowest
The conditions for the analysis of selected doping substances by UHPSFC-MS/MS were
optimized to ensure suitable peak shapes and maximized MS responses. A representative
mixture of 31 acidic and basic doping agents was analyzed, in both ESI+ and ESI- modes.
The best compromise for all compounds in terms of MS sensitivity and chromatographic
performance was obtained when adding 2 percent water and 10mM ammonium formate in
the CO2/MeOH mobile phase. Beside mobile phase, the nature of the make-up solvent
added for interfacing UHPSFC with MS was also evaluated. Ethanol was found to be the
best candidate as it was able to compensate for the negative effect of 2% water addition in
ESI- mode and provided a suitable MS response for all doping agents. Sensitivity of the
optimized UHPSFC-MS/MS method was finally assessed and compared to the results
obtained in conventional UHPLC-MS/MS. Sensitivity was improved by 5-100-fold in
UHPSFC-MS/MS vs. UHPLC-MS/MS for 56 percent of compounds, while only one
compound (bumetanide) offered a significantly higher MS response (4-fold) under UHPLC-
MS/MS conditions. In the second paper of this series, the optimal conditions for UHPSFC-
MS/MS analysis will be employed to screen >100 doping agents in urine matrix and results
will be compared to those obtained by conventional UHPLC-MS/MS [14738].
Isotachophoresis sample stacking
A simple and effective method of capillary electrophoresis-amperometric detection (CE-AD)

coupled with transient isotachophoresis (tITP) was developed for the trace determination of
doping substances. Compared with the conventional capillary electrophoresis method, the
maximum enhancement factor in terms of peak heights was up to 5500-fold when the tITP
technique was adopted. Under the optimum conditions, detection limit for methylephedrine
(MDP), celiprolol (CEL), sotalol (SOT) and indapamide (IDP) were 4.2 x 10-14, 6.3 x 10-13, 5.8
x 10-14 and 9.5 x 10-13 mol/L, respectively. The proposed method was successfully applied to
determine the contents of SOT and IDP in real urine sample, and the excretion curve of IDP
within 48 h was also investigated. The recoveries of the four doping in urine ranged from 90
to 102 percent [10031].
Polar organic chemical integrative samplers (POCIS)
Polar organic chemical integrative samplers (POCIS) were calibrated in situ for selected illicit
drugs and their metabolites at a sewage treatment works. Eleven out of 13 target compounds
were detected and eight of those exhibited linear uptake kinetics with sampling rates
between 0.035 and 0.150 L/d. Subsequently POCIS were deployed for 2 week periods over
the course of a whole year, in order to examine trends in drug usage. Amphetamine and
758
methamphetamine showed several similar peaks in concentration during the course of the
year as did cocaine and two of its metabolites. Low levels of ecstasy were observed, with a
prominent peak in May and a steady increase toward the end of the year. The antihistamine
Cetirizine showed a clear increase in use during the summer months as expected and back
calculation of the yearly dosage from POCIS accumulations yielded very similar results to
that registered in the Norwegian prescription database. Estimations of cocaine usage using
the parent compound averaged between 0.31 and 2.8 g/d per 1000 inhabitants. POCIS is a
cost-effective technique for the long-term monitoring of drug usage of a defined population
and may overcome the difficulties of representative sampling associated with autosampling
equipment [11431].
Fourier-transform infrared spectroscopy
Doping prevention is mainly directed to providing information on the dangers of doping to

young athletes and to every profession concerned with athletic performance. Unfortunately,
repression is also necessary in the fight against doping. Measurement of performance-
enhancing drugs is complex, partly because of the large number of prohibited substances. A
number of sophisticated analytical techniques are increasingly being used to provide the
maximum detection time window. However, the effectiveness of methods to separate
exogenous from endogenous biological molecules and the cost of antidoping analyses
makes controls invalid or impossible. Moreover, most athletes, because of the metabolic and
psychological stresses caused, legitimately refuse blood testing. It is becoming crucial to
introduce new methods in the form of longitudinal health monitoring, since this is probably
the most effective tool to prevent the use of doping agents when athletes become
overtrained and/or overstressed. This paper describes new methods using Fourier-transform
infrared spectroscopy to analyse serum from 50 microl samples of capillary blood. This
technique has been shown to allow determination of the concentration of a wide range of
biological molecules in a single microsample with clinically useful accuracy, and to provide a
'discriminatory biomolecular profile' to differentiate individuals on the basis of their
physiological status. A specific application of this methodology is to perform longitudinal
health monitoring in athletes, allowing prevention of overtraining. It is proposed to apply such
methods in longitudinal studies for health monitoring and prevention of doping [00026].
Microwave assisted extraction
It was described a fast and efficient method for the liquid/liquid extraction from human urine
of different classes of drugs, included in the list of prohibited substances published every
year by the World Anti-doping Agency, using microwave irradiation. Liquid/liquid extraction
was conducted in a temperature controlled single beam microwave oven equipped with an
extraction unit and closed vessels. The effects of microwave power and time on the
liquid/liquid extraction process were investigated utilizing different organic solvents. The
optimum power was found to be 600 W (generating a temperature of 70 degrees C) with an
incubation time of 30-60 s for the most thermolable constituents such as triamcinolone,
prednisolone, chlorthiazide, chlorthalidone, epi-trembolone and oxandrolone, and 1020W
(generating a temperature of 150 degrees C) with an incubation time of 30-60 s for the other
compounds considered in this study. The effectiveness of this approach was evaluated by
GC-MS (anabolic steroids, beta2-agonists and narcotics) and by LC-MS/MS (diuretics,
glucocorticoids and beta-blockers) analyzing more than 20 different urine samples spiked
with the compounds considered in this study. The results showed that the effect of
microwave irradiation on the liquid/liquid extraction process was very remarkable: the total
759
sample preparation time can be shortened by 9 min compared to the traditional method (30-
60 s instead of 10 min); furthermore, a significant increase in the recovery was recorded for
specific compounds such as terbutaline and several diuretics. In addition to the above the
repeatability of the extraction recoveries, the limits of detection and the matrix interferences
were comparable with the reference methods, presently accredited under the ISO17025,
followed by the World Anti-doping Agency accredited anti-doping laboratory of Rome
[10030].
In one contribution it was tested the possibility to use microwave irradiation for the screening
and confirmation pre-treatment steps of hydroxyethylstarch, with the aim to speed up gas
chromatography-mass spectrometric procedures. Acid hydrolysis and derivatization
processes were conducted in a temperature-controlled single beam microwave oven for
organic synthesis. The kinetics of hydroxyethylstarch chemical hydrolysis and derivatization
were investigated at different microwave power, incubation temperature and incubation time.
The best hydrolysis conditions were found at a microwave power value of 1200W (T 100°C)
with an incubation time of 2 min; whereas the best derivatization conditions were found at a
microwave power value of 1020W (T 100°C) with an incubation time of 5 min. The
effectiveness of this approach was evaluated by gas chromatography-mass spectrometry
analyzing more than 20 different pools of blank urine samples spiked with hydroxyethylstarch
at a concentration of 1mg/mL. The results showed that the effect of microwave irradiation on
the chemical hydrolysis process was very remarkable: the total sample preparation time can
be shortened by 58 min compared to the reference method (2 min instead of 60 min). In
addition to this, the time necessary for the derivatization process can also be drastically
shortened with respect to the reference procedure (5 min instead of 30 min). The
repeatability of the hydrolysis and derivatization recoveries, the limit of detection and the
matrix interferences were comparable to the reference method accredited under the ISO
17025 guidelines and presently followed by the accredited sports anti-doping laboratory of
Rome [10321].
Ultrasound and microwave
A comparison between ultrasonication and microwave irradiation as tools to achieve a rapid

sample treatment for the analysis of banned doping substances in human urine by means of
gas chromatography-mass spectrometry (GC-MS) was performed. The following variables
were studied and optimised: time of treatment, temperature, microwave power and ultrasonic
amplitude. The results were evaluated and compared with those achieved by the routine
method used in the World Anti-Doping Agency (WADA) accredited Antidoping Laboratory of
Rome. Only under the effect of the ultrasonic field was it possible to enhance the enzymatic
hydrolysis reaction rate of conjugated compounds. Similar reaction yield to the routine
method was achieved after 10 min for most compounds. Under microwave irradiation,
denaturation of the enzyme occurs for high microwave power. The use of both ultrasonic or
microwave energy to improve the reaction rate of the derivatisation of the target compounds
with trimethyliodosilane/methyl-N-trimethylsilyltrifluoroacetamide (TMSI/MSTFA/NH(4)I/2-
mercaptoethanol) was also evaluated. To test the use of the two systems in the acceleration
of the reaction with TMSI, a pool of 55 banned substances and/or their metabolites were
used. After 3 min of ultrasonication, 34 of the 55 compounds had recoveries similar to those
obtained with the classic procedure that lasts for 30 min, 18 increased to higher silylation
yields, and for the compounds 13beta,17alpha-diethyl-3alpha,17beta-dihydroxy-5alpha-
gonane (norboletone metabolite 1), metoprolol and metipranolol the same results were
obtained increasing the ultrasonication time to 5 min. Similar results were obtained after
3 min of microwave irradiation at 1,200 W. In this case, 30 of the 55 compounds had
recoveries similar to the classic procedure whilst 18 had higher silylation yields. For the
760
compounds 3alpha-hydroxy-1alpha-methyl-5alpha-androstan-17-one (mesterolone metab-
olite 1), 17alpha-ethyl-5beta-estrane-3alpha,17beta,21-triol (norethandrolone metabolite 1),
epioxandrolone, 4-chloro-6beta,17beta-dihydroxy-17alpha-methyl-1,4-androstadien-3-one
(chlormetandienone metabolite 1), carphedon, esmolol and bambuterol the same results
were obtained after 5 min under microwave irradiation [11045].
Liquid/liquid extraction
It was described a fast and efficient method for the liquid/liquid extraction from human urine
of different classes of drugs, included in the list of prohibited substances published every
year by the World Anti-doping Agency, using microwave irradiation. Liquid/liquid extraction
was conducted in a temperature controlled single beam microwave oven equipped with an
extraction unit and closed vessels. The effects of microwave power and time on the
liquid/liquid extraction process were investigated utilizing different organic solvents. The
optimum power was found to be 600W (generating a temperature of 70 degrees C) with an
incubation time of 30-60s for the most thermolable constituents such as triamcinolone,
prednisolone, chlorthiazide, chlorthalidone, epi-trembolone and oxandrolone, and 1020W
(generating a temperature of 150 degrees C) with an incubation time of 30-60s for the other
compounds considered in this study. The effectiveness of this approach was evaluated by
GC-MS (anabolic steroids, beta2-agonists and narcotics) and by LC-MS/MS (diuretics,
glucocorticoids and beta-blockers) analyzing more than 20 different urine samples spiked
with the compounds considered in this study. The results showed that the effect of
microwave irradiation on the liquid/liquid extraction process was very remarkable: the total
sample preparation time can be shortened by 9min compared to the traditional method (30-
60s instead of 10min); furthermore, a significant increase in the recovery was recorded for
specific compounds such as terbutaline and several diuretics [10033].
"Dilute-and-inject" multi-target screening assay for highly polar doping agents
In the field of LC-MS, reversed phase liquid chromatography is the predominant method of
choice for the separation of prohibited substances from various classes in sports drug
testing. However, highly polar and charged compounds still represent a challenging task in
liquid chromatography due to their difficult chromatographic behavior using reversed phase
materials. A very promising approach for the separation of hydrophilic compounds is
hydrophilic interaction liquid chromatography (HILIC). Despite its great potential and versatile
advantages for the separation of highly polar compounds, HILIC is up to now not very
common in doping analysis, although most manufacturers offer a variety of HILIC columns in
their portfolio. In this study, a novel multi-target approach based on HILIC high
resolution/high accuracy mass spectrometry is presented to screen for various polar
stimulants, stimulant sulfo-conjugates, glycerol, AICAR, ethyl glucuronide, morphine-3-
glucuronide, and myo-inositol trispyrophosphate after direct injection of diluted urine
specimens. The usage of an effective online sample cleanup and a zwitterionic HILIC
analytical column in combination with a new generation Hybrid Quadrupol-Orbitrap® mass
spectrometer enabled the detection of highly polar analytes without any time-consuming
hydrolysis or further purification steps, far below the required detection limits. The
methodology was fully validated for qualitative and quantitative (AICAR, glycerol) purposes
considering the parameters specificity; robustness (rRT < 2.0 %); linearity (R > 0.99); intra-
and inter-day precision at low, medium, and high concentration levels (CV < 20 %); limit of
detection (stimulants and stimulant sulfo-conjugates < 10 ng/mL; norfenefrine;
octopamine < 30 ng/mL; AICAR < 10 ng/mL; glycerol 100 μg/mL; ETG < 100 ng/mL);
761
accuracy (AICAR 103.8-105.5 %, glycerol 85.1-98.3 % at three concentration levels) and ion
suppression/enhancement effects [150133].
Phase I and phase II intact urinary metabolites
In order to improve the detection capabilities of anabolic androgenic steroids (AAS) in sports,
a liquid chromatography-tandem mass spectrometry (LC-MS/MS) screening method for the
simultaneous detection of AAS phase I and phase II intact urinary metabolites (glucuronides
and sulfates) was developed. A total of 36 metabolites (7 unconjugated; 19 glucuronides and
10 sulfates) corresponding to 15 of the most reported AAS were included. Analytes were
extracted from urine using C18 cartridges. LC and MS conditions were studied in-depth to
determine the most sensitive and selective conditions for each analyte. A selected reaction
monitoring method was set up. The optimization of the experimental parameters for 13
metabolites not available as standards was performed using excretion study urines.
Extraction recoveries were above 77 percent for all 23 validated analytes. Intra-day precision
was lower than 21 percent, and LODs were in the range 0.25-4ng/mL for 18 of the 23
analytes. Matrix effect was evaluated using post column infusion and ranged from 92 to
147percent. The method was successfully applied to excretion study urines of different
exogenous AAS. The suitability of the strategy was demonstrated with methyltestosterone
and stanozolol excretion study urines by achieving detection times of 22 and 21 days,
respectively. The method is compliant with the World Antidoping Agency requirements for
most of the studied compounds. It represents a cost-effective approach that improves the
detection capabilities of AAS by increasing the sensitivity for some metabolites and by
including recently described phase II long-term metabolites not detectable using the current
screening strategy [150134].
Two step derivatization
Two-step derivatization procedures were developed for the enhancement of the positive ESI
in LC-MS detection of anabolic androgenic steroids, a class of prohibited substances with
limited ionization efficiency in atmospheric pressure interfaces. The developed procedures
are based on the esterification of hydroxyl groups of anabolic steroids with picolinic acid,
followed by conversion of carbonyl groups to Schiff bases by either Girard's reagent T or 2-
hydrazino pyridin. Ionization efficiency for the model derivatized compounds 19-
norandrosterone (nandrolone main metabolite) and methasterone was higher by almost two
orders of magnitude compared with the respective efficiency of the underivatized
compounds. The obtained derivatives provided a significant improvement in the ESI
sensitivity, compared with those of underivatized molecules in positive LC-ESI-ion trap-MS
full-scan mode [12082].
Adsorption to metallic plasmonic nanoparticles
A comparative study of different plasmonic nanoparticles with different morphologies

(nanospheres and triangular nanoprisms) and metals (Ag and Au) was done in this work and
applied to the ultrasensitive detection of aminoglutethimide (AGI) drug by surface enhanced
Raman spectroscopy (SERS) and plasmon resonance. AGI is an aromatase inhibitor used
as an antitumoral drug with remarkable pharmacological interest and also in illegal sport
doping. The application of very sensitive spectroscopic techniques based on the localization
of an electromagnetic field on plasmonic nanoparticles confirms the previous study of the
762
adsorption of drugs onto a metal surface due to the near field character of these techniques.
The adsorption of AGI on the above substrates was investigated at different pH values and
surface coverages, and the results were analyzed on the basis of AGI/metal affinity,
considering the interaction mechanism, the existence of two binding sites in AGI, and the
influence of the interface on the adsorption in terms of surface charge due to the presence of
other ions linked to the surface. Finally, a comparative quantitative detection of AGI was
performed on both spherical and triangular nanoprism nanoparticles, and a limit of detection
lower than those reported so far was deduced on the latter nanoparticles [12083].
Surface plasmon resonance
In the last 20 years, surface plasmon resonance (SPR) and its advancement with imaging
(SPRi) emerged as a suitable and reliable platform in clinical analysis for label-free,
sensitive, and real-time monitoring of biomolecular interactions. Thus, we report in this
review the state of the art of clinical target detection with SPR-based biosensors in complex
matrices (e.g. serum, saliva, blood, and urine) as well as in standard solution when
innovative approaches or advanced instrumentations were employed for improved detection.
The principles of SPR-based biosensors were summarized first, focusing on the physical
properties of the transducer, on the assays design, on the immobilization chemistry, and on
new trends for implementing system analytical performances (e.g. coupling with
nanoparticles (NPs). Then it was critically reviewed the detection of analytes of interest in
molecular diagnostics, such as hormones (relevant also for anti-doping control) and
biomarkers of interest in inflammatory, cancer, and heart failure diseases. Antibody detection
is reported in relation to immune disorder diagnostics. Subsequently, nucleic acid targets are
considered for revealing genetic diseases (e.g., point mutation and single nucleotides
polymorphism, SNPs) as well as new emerging clinical markers (microRNA) and for
pathogen detection. Finally, examples of pathogen detection by immunosensing were also
analyzed. A parallel comparison with the reference methods was duly made, indicating the
progress brought about by SPR technologies in clinical routine analysis [14036].
Within this communication, consistent evidence of a quantitative biosensing principle for

steroidal residue analysis is presented. One approach uses a simple method for the
quantitative determination of an anabolic agent called stanozolol (Sz). Sz (Mw 328) is widely
used in sports, horse racing and as a growth promoter in animals for human consumption.
Through the use of localised surface plasmons (LSPs), sustained by three-dimensional noble
metal nanostructures, we have developed a highly specific, label-less immunosensor for the
detection of this small organic molecule to low levels (nM range). A main practical advantage
over conventional flat extended film surface plasmon resonance (SPR) systems is the
simplicity of the optical configuration, since there is no need for cumbersome total internal
reflection illumination, thus making integration easier. In addition, the active area of the LSP-
based sensor is smaller, decreasing the minimum detectable number of molecules involved
in the binding event. Assay times are short and the set-up is comprised of relatively cheap
instrumentation. Detection levels found here are comparable with SPR, even at this early
stage of development and with further modifications, we envisage sensing down to pM (10-12)
levels [06042].
Doping analysis relies on the determination of prohibited substances that should not be
present in the body of an athlete or that should be below a threshold value. In the case of
xenobiotics their mere presence is sufficient to establish a doping offence. However, in the
case of human biotics the analytical method faces the difficulty of distinguishing between
endogenous and exogenous origin. For this purpose ingenious strategies have been
763
implemented, often aided by state-of-the-art technological advancements such as mass
spectrometry in all its possible forms. For larger molecules, i.e. protein hormones, the innate
structural complexity, the heterogeneous nature, and the extremely low levels in biological
fluids have rendered the analytical procedures heavily dependent of immunological
approaches. Although approaches these confer specificity and sensitivity to the applications,
most rely on the use of two, or even three, antibody incubations with the consequent
increment in assay variability. Moreover, the requirement for different antibodies that
separately recognise different epitopes in screening and confirmation assays further
contributes to differences encountered in either measurement. The development of analytical
techniques to measure interactions directly, such as atomic force microscopy, quartz crystal
microbalance or surface plasmon resonance, have greatly contributed to the accurate
evaluation of molecular interactions in all fields of biology, and expectations are that this will
only increase. Here, an overview is provided of surface plasmon resonance, and its particular
value in application to the field of doping analysis [11040].
Within this communication, consistent One of a quantitative biosensing principle for steroidal
residue analysis is presented. Our approach uses a simple method for the quantitative
determination of an anabolic agent called stanozolol (Sz). Sz (Mw 328) is widely used in
sports, horse racing and as a growth promoter in animals for human consumption. Through
the use of localised surface plasmons (LSPs), sustained by three-dimensional noble metal
nanostructures, it was developed a highly specific, label-less immunosensor for the detection
of this small organic molecule to low levels (nM range). A main practical advantage over
conventional flat extended film surface plasmon resonance (SPR) systems is the simplicity of
the optical configuration, since there is no need for cumbersome total internal reflection
illumination, thus making integration easier. In addition, the active area of the LSP-based
sensor is smaller, decreasing the minimum detectable number of molecules involved in the
binding event. Assay times are short and the set-up is comprised of relatively cheap
instrumentation. Detection levels found here are comparable with SPR, even at this early
stage of development and with further modifications, we envisage sensing down to pM (10-12)
levels [05025].
Isotope ratio mass spectrometry
Detecting the misuse of endogenously occurring steroids, i.e. steroids such as testosterone
that are produced naturally by humans, is one of the most challenging issues in doping
control analysis. The established thresholds for urinary concentrations or concentration ratios
such as the testosterone/epitestosterone quotient are sometimes inconclusive owing to the
large biological variation in these parameters. For more than 15 years, doping control
laboratories focused on the carbon isotope ratios of endogenous steroids to distinguish
between naturally elevated steroid profile parameters and illicit administration of steroids. A
variety of different methods has been developed throughout the last decade and the number
of different steroids under investigation by isotope ratio mass spectrometry has recently
grown considerably. Besides norandrosterone, boldenone was found to occur endogenously
in rare cases and the misuse of corticosteroids or epitestosterone can now be detected with
the aid of carbon isotope ratios as well. In addition, steroids excreted as sulfoconjugates
were investigated, and the first results regarding hydrogen isotope ratios recently became
available. All of these will were presented in detail within the review together with some
considerations on validation issues and on identification of parameters influencing steroidal
isotope ratios in urine [11041].
764
ETD and CID tandem mass spectrometry
Identification of an unknown substance without any information remains a daunting challenge

despite advances in chemistry and mass spectrometry. However, an unknown cyclic peptide
in a sample with very limited volume seized at a Pennsylvania racetrack has been
successfully identified. The unknown sample was determined by accurate mass
measurements to contain a small unknown peptide as the major component. Collision-
induced dissociation (CID) of the unknown peptide revealed the presence of Lys (not Gln, by
accurate mass), Phe, and Arg residues, and absence of any y-type product ion. The latter,
together with the tryptic digestion results of the unusual deamidation and absence of any
tryptic cleavage, suggests a cyclic structure for the peptide. Electron-transfer dissociation
(ETD) of the unknown peptide indicated the presence of Gln (not Lys, by the unusual
deamidation), Phe, and Arg residues and their connectivity. After all the results were pieced
together, a cyclic tetrapeptide, cyclo[Arg-Lys-N(C(6)H(9))Gln-Phe], is proposed for the
unknown peptide. Observations of different amino acid residues from CID and ETD
experiments for the peptide were interpreted by a fragmentation pathway proposed, as was
preferential CID loss of a Lys residue from the peptide. ETD was used for the first time in
sequencing of a cyclic peptide; product ions resulting from ETD of the peptide identified were
categorized into two types and named pseudo-b and pseudo-z ions that are important for
sequencing of cyclic peptides. The ETD product ions were interpreted by fragmentation
pathways proposed. Additionally, multi-stage CID mass spectrometry cannot provide
complete sequence information for cyclic peptides containing adjacent Arg and Lys residues.
The identified cyclic peptide has not been documented in the literature, its pharmacological
effects are unknown, but it might be a "designer" drug with athletic performance-enhancing
effects [11042].
Protein chips
Sport and doping are a contradiction in terms, however, doping abuse in sports has been a
serious problem for many years. The systematic screening of every athlete for all prohibited
drugs should be an indispensable feature of the Olympic Games. The gas chromatography
mass spectrometry method is reserved as a reference method, but is limited by its low
throughput. The advent of protein chip technology may enable the screening of all athletes
for any illegal use of drugs [06043].
Small molecular analysis
In doping control analysis, the demand for modern analytical strategies with fast turnaround,
high sensitivity, and selectivity in complex matrices is a major concern. The number of
forbidden substances and/or metabolites on the WADA List that should be monitored has
grown continuously over the last several years, reaching approximately 250 entities. In
addition, all these compounds possess very diverse physicochemical properties (e.g.
polarity, molecular weight, and acido-basic properties). Considering these constraints, the
analysis of small molecules remains challenging, and several methods in parallel are
required to cover all the different categories and to ensure the quality of the analytical results.
These methods are generally focused on the direct detection of prohibited substances as
well as their major phase I and phase II metabolites because monitoring of the latter often
improves detection window capabilities in urine. For this purpose, the excretion pattern of
each illicit substance must be carefully examined to ensure the proper selection of the target
compounds for screening purposes, favoring major metabolites or those with long-term
765
urinary excretion profiles [150058].
Sample preparation techniques
When dealing with the bioanalysis of urine or blood by chromatographic methods, sample
preparation is a critical step due to the wide heterogeneity of the analytes and the complexity
of the matrices containing salts, lipids, and proteins. Sample preparation is mandatory to
achieve a sufficient level of sensitivity and selectivity and also to avoid clogging the
chromatographic column and contaminating the mass spectrometer, primarily used as the
detector. Various sample treatments (i.e. dilute and shoot, protein precipitation) or sample
preparation techniques (i.e. solid-phase extraction (SPE), liquid–liquid extraction (LLE), or
supported-liquid extraction (SLE)) can be selected to obtain the best compromise between
good recoveries for most of the analytes and cleanliness of the extract. The choice of the
best sample preparation procedure should be based on the physicochemical properties of
the substances and the employed analytical instrumentation. The sample preparation
procedure for doping control analysis should be as generic as possible because many illicit
substances must be monitored simultaneously during the initial screening procedure. With
this in mind and thanks to the high selectivity and sensitivity offered by the latest generations
of MS detectors, this step can largely be simplified, and today, the initial screening of a high
number of illicit substance classes in urine is often performed after a nonselective dilute-and-
shoot procedure with an appropriate solvent to limit matrix effects. This approach is mostly
used as a screening assay of easily ionizable compounds with a limited metabolism, such as
stimulants, narcotics, diuretics, beta2-agonists, and beta-blockers, for which the required
minimum detection levels in urine are relatively high, in the range of a few tens to hundreds
of ng/mL. This sample pretreatment approach is generic, fast, and inexpensive and does not
require any specific equipment. However, the detectability of analytes is reduced due to the
dilution factor. The dilution factor varies between 1:1 and 1:25, and most of the recent
applications dealing with the determination of sport drugs in urine have been carried out with
a dilution factor of 1:10. Because urine is a complex matrix including, but not limited to,
phospholipids, proteins, salts, urea, and creatinine, as well as a wide range of organic acids
and other inorganic compounds, matrix effects are non-negligible in the case of a simple
dilute-and-shoot procedure and should be evaluated case by case. To compensate for matrix
effects and obtain accurate and reproducible results, the use of an isotope-labeled internal
standard (IL-ISTD) remains the best choice, especially for the quantitative determination of
threshold substances such as carboxy-THC, salbutamol, 19-norandrosterone, morphine, or
ephedrine and its derivatives [150058].
However, in cases for which certain classes of forbidden substances should be monitored at
trace levels in the biological fluid of interest (e.g. anabolic androgenic steroids), a
preconcentration step is mandatory. In this context, the selective LLE procedure has been
historically the most widely used preconcentration technique in doping control analysis
because the sensitivity of LC-MS or GC/MS platforms was limited in the past. Sufficient
elimination of major interferences is obtained with LLE, and the extraction protocol is simple
and cost-efficient and provides clean extracts using solvents such as tert-butyl methyl ether,
diethyl ether, and n-pentane. However, this technique is not adapted to substances with
polar groups in their structure; it requires large volumes of sample and solvent and suffers
from poor recovery and repeatability and lack of automation. In addition, two parallel
extractions, at basic and acidic pH, respectively, are often required to simultaneously extract
acidic and basic substances for screening purposes. Despite this, LLE at basic pH is still
widely used as a routine protocol in antidoping analysis, mainly for compounds such as
endogenous and exogenous steroids as well as glucocorticoids [150058].
With the advent of green chemistry, there is also a trend toward miniaturized sample
766
preparation techniques to reduce the use of organic solvents or substitute organic solvents
for less toxic alternatives. In this context, many liquid-based or solid-based microextraction
procedures have been proposed for a wide range of applications, including clinical and
forensic toxicology. The most conventional liquid-based microextraction methods include
single-drop microextraction (SDME), hollow-fiber liquid-phase microextraction (HF-LPME), or
dispersive liquid–liquid microextraction (DLLME), while most well-known solid-based
microextraction methods are disposable pipette extraction (DPX) or microextraction by
packed-sorbents (MEPS). Until now, these approaches have not yet been applied in routine
laboratories, but it is expected that the interest in these strategies will increase in the future
and that some attempts will certainly be carried out soon in doping control laboratories.
Finally, because blood matrix is known to provide complementary information to urine
analysis, it is necessary to describe the sample preparation procedures used for this matrix.
The simplest procedure is protein precipitation (PP), which is analogous to the dilute-and-
shoot method for urine. Depending on sensitivity and selectivity required, LLE, SLE, or SPE
procedures can also be employed in serum, plasma, and whole blood before GC/MS and
LC-MS analysis, as reported above for urine. Moreover, greater attention has been paid over
the past few years to the use of dried blood spots (DBS) for blood collection in doping
control. Few recent applications for monitoring different categories of small prohibited
compounds, peptides, anabolic steroid esters, and SIRT1 activators have been recently
reported in the literature. DBS consists of collecting a few microliters of whole blood spotted
on absorbent paper and appears to be very promising for doping control blood analysis in
terms of sampling (less invasive than conventional blood collection), easier shipment and
storage, and improved long-term analyte stability. However, although DBS is a promising
sampling technique, there is still some resistance to its widespread application, mainly due to
the limited preconcentration factors achieved and relatively high limits of detection.
Furthermore, in a typical DBS workflow, a small disk of the spotted and dried blood spot is
punched out, and these punches are therefore assumed to contain a fixed blood volume.
However, the viscosity of blood depends on the hematocrit level. Then, if a uniform blood
volume is placed onto a DBS card, the size of the spot formed will decrease as the
hematocrit level increases. This leads to volumetric assay bias associated with the
hematocrit of the blood [150058].
Chromatographic approaches
In doping control analysis, GC-FID and GC/MS quickly became the standard instrumentation
for the detection and quantification of illicit substances. GC is generally coupled to MS
through electron impact (EI) ionization, and this approach has been particularly useful for
toxicological laboratories due to the low interinstrument variability and the possibility of using
existing mass spectral libraries. When using GC, hydrolysis and derivatization steps are
required prior to the analysis of many doping agents to make them sufficiently volatile and
also to improve their sensitivity. These procedures may induce important variability and are
considered expensive and time-consuming because enzymatic hydrolysis can vary from
minutes to hours depending on the incubation temperature, whereas derivatization can be
performed within about 1 h. However, today, GC/MS remains the gold standard method for
the screening and confirmation of anabolic steroids, which is certainly one of the most
challenging classes of doping agents to analyze. Indeed, anabolic steroids are excreted in
urine at very diverse concentrations, and there are also a large number of isomers and
metabolites that are difficult to separate in a satisfactory manner. To improve the resolving
power of GC, comprehensive two-dimensional GC (GCxGC) can be used to tackle the
extreme complexity of samples. In GCxGC, the resolving power is significantly increased by
the use of two orthogonal columns of different polarities and lengths. In addition, GCxGC is
often coupled with high-resolution mass spectrometers possessing fast data acquisition
rates. However, to date, GCxGC-TOF/MS has only been scarcely employed in doping control
767
analysis likely because robust instrumentation only became commercially available very
recently. For quantification purposes, GC/MS(/MS) is also considered fit-for-purpose,
especially regarding the determination of endogenous steroids linked to the testosterone
metabolism in urine, also called the urinary steroid profile integrated in the steroidal module
of the ABP. The technical document describing the sample preparation as well as the
instrument settings imposed by WADA clearly states that GC/MS(/MS) is the preferred
reference method. However, although this technique has been widely applied in the fight
against doping, there is still a lack of standardization between the WADA accredited
laboratories. Not only is the detector (single or triple quadrupole) dissimilar among
laboratories but also the sample preparation (either LLE or SPE and LLE purification steps)
is known to be a potential source of variation for steroid profile quantification. In addition,
many other exogenous or endogenous confounding factors can influence the steroid profile.
Because of all these limitations, the urinary data and individual sequences generated by the
steroidal module of the ABP currently suffer from more inconsistencies compared to the
hematological module for which the preanalytical and analytical conditions are well
established among laboratories [150058].
Another application of GC in doping control analysis is the determination of xenon in urine or

plasma samples. Xenon was listed by the WADA in 2014 as a banned substance
categorized as a hypoxia-inducible factor activator. The presence of xenon in urine can be
successfully assessed using GC/MS/MS with headspace injection down to a detection limit of
approximately 0.5 nmol/mL and up to 40 h postanesthesia. In addition, GC-TOF/MS and
GC/MS/MS have been employed to determine xenon in human plasma or blood, also with
headspace injection. Depending on the type of mass spectrometer, detection limits of 0.5–50
nmol/mL have been achieved, and xenon has been detected up to 30 h after plasma and
blood storage collection. However, further studies are still required to better understand the
detection window for xenon under different gas mixtures and exposure time settings.
Moreover, the sampling and storage of specimens should also be evaluated [150058].
Mass spectrometry detection
Due to its high sensitivity and selectivity and the possibility to confirm the identification of
analytes based on mass spectral information, MS has emerged as the gold standard
detection mode in doping control analysis. Historically, low-resolution instruments have been
preferentially used for small molecules, both for identification and quantitative determination,
while high-resolution analyzers have been mainly dedicated for untargeted applications and
especially the analysis and characterization of different forbidden peptides and proteins. In
routine antidoping laboratories, single quadrupole, triple quadrupole (QqQ), and quadrupole
ion trap (QTrap) are the most widely used low-resolution MS devices because they generally
offer suitable sensitivity, specificity, and dynamic range. For GC-based applications, single
quadrupole MS remains the workhorse in routine laboratories due to its ease of use and the
possibility of employing existing mass spectral libraries for analyte identification. Recently,
triple quadrupole mass analyzers have emerged for the detection of doping agents in
targeted single-reaction monitoring (SRM) mode with GC, thereby significantly improving the
detection performance from complex matrices. Due to the advantages afforded by
GC/MS/MS, limits of detection of routine methods were improved compared to single-stage
MS, especially for critical doping compounds such as steroids and their major urinary
metabolites. However, because GC peaks are extremely narrow (<1 s), ultrafast MS/MS
devices with low SRM dwell times are required. Today, an SRM transition can be monitored
with a dwell time of only 0.5 ms in the latest generation of GC/MS/MS instrumentation
[150058].
Despite the lack of reference libraries, LC-MS is now widely employed for doping control
768
analysis, probably more than GC/MS due to the higher versatility of LC. In LC-based
applications, single quadrupole analyzers are no longer used, and QqQ and QTrap are the
techniques of choice for most of the screening methods. These mass analyzers allow a rapid
polarity switching between positive and negative modes, reducing the number of LC-MS
injections needed for screening purposes by half. Due to the very high sensitivity and
specificity afforded by these instruments, different classes of forbidden compounds (e.g.,
stimulants, diuretics, and beta2-agonists) can be monitored after a simple dilute-and-shoot
procedure from urine samples. However, the electrospray ionization (ESI) source, which is
by far the most commonly employed interface between LC and MS, can suffer from matrix
effects produced by coeluting interferences in the sample. Ion suppression or enhancement
modifies the sensitivity of the method by inducing irreproducibility. As for GC/MS, another
important aspect when using LC-QqQ in doping control analysis is the need for an instrument
able to provide sufficient sensitivity even at low dwell times in SRM mode. With the latest
generation of LC-MS/MS instruments, the dwell time can be reduced down to only 0.8 ms,
allowing the inclusion of a large number of compounds in the screening method. In doping
control analysis, numerous methods based on the detection of multiclass analytes have been
developed with the QqQ analyzer in the past few years, with up to 100–150 banned
substances screened in a single run [150058].
Because the initial screening of doping agents needs to be rapid and simple, there may be
interest in the near future in using ambient mass spectrometry (AMS) techniques for fast,
versatile, and direct analysis of samples in open air, with little or no sample preparation.
Various AMS techniques have been developed over the past few years, such as DESI
(desorption electrospray ionization), DART (direct analysis in real time), and EESI (extractive
electrospray ionization), but none of these is currently routinely used for doping control
analysis of food supplements, pharmaceutical preparations, and/or biological fluids.
However, the potential of DART hyphenated with Orbitrap-MS was evaluated for the fast
identification and quantification of 21 anabolic steroid esters in oily commercial preparations.
Direct analysis in high-resolution scan mode was used to screen for steroid esters based on
the accurate mass measurement. Steroid ester identification was further supported by
collision-induced dissociation (CID) experiments through the generation of two additional
ions. Moreover, the use of labeled internal standards allowed quantitative data to be
recovered on the basis of isotopic dilution. DART-MS was also applied for the rapid
determination of dimethylamylamine (DMAA), which is a stimulant banned by the WADA.
However, current AMS methods suffer from drawbacks, such as poor quantitative
performance, high limits of detection, lack of universality, and lack of convenience for
practical applications, that should be addressed before potential widespread use in a doping
control laboratory [150058].
Finally, another MS-based strategy that becomes increasingly popular is ion mobility
spectrometry-mass spectrometry (IMS-MS). This analytical method separates gaseous
phase ions according to their mobility under an electrical field on a millisecond time scale
using IMS, followed by the detection of ions according to their mass-to-charge ratio on a
microsecond time scale. IMS could thus be considered an ultrafast replacement of
chromatography prior to MS or as an additional dimension when combining liquid
chromatography with IMS-MS. Much work is currently being performed on this technique to
further improve the resolving power when compounds with similar collisional cross sections
need to be separated, and a promising study on the analysis of human insulin and its
analogues was recently published. In the near future, this approach will become valuable for
antidoping control [150058].
Peptide analysis
769
Peptides that stimulate growth hormone (GH) secretion have been particularly investigated in
doping control analysis in the past few years due to their potential misuse as doping agents
in sports. These peptides can be classified into two main groups: (i) growth hormone
releasing hormones (GHRHs) such as sermorelin, tesamorelin, CJC-1288, CJC-1293, and
CJC-1295, all possessing molecular masses between approximately 3 to 5 kDa; and (ii)
growth hormone secretagogues (GHS) and releasing peptides (GHRPs), including, but not
limited to, GHRP-1 to GRHP-6, alexamorelin, hexarelin, and ipamorelin, with smaller
molecular masses of <2 kDa. In addition to these peptides, numerous methods for the
analysis of human and synthetic insulins, with sizes ranging from 5 to 6 kDa as well as
insulin-like growth factors (IGF) of slightly larger sizes, have been developed by doping
control laboratories in recent years. Finally, there is also a range of additional peptides that
need to be monitored, such as desmopressin, LHRH (GnRH) and its agonists (e.g.,
leuprolide, buserelin, and triptorelin), ACTH, and Synacthen and, more recently, several
growth factors such as MGFs, as listed elsewhere. Currently, the key chromatographic
method for analyzing peptidic drugs in sport drug testing is based on the use of RPLC-MS.
When analyzing peptides in RPLC, it is recommended to use 0.1 percent TFA in the mobile
phase to improve peak shapes through ion pairing to neutralize the positive charges at the
surface of the peptides. However, ESI-MS sensitivity is reduced by a factor of approximately
10 compared to the use of 0.1 percent formic acid. If a charge surface hybrid (CSH) C18
stationary phase is employed, the peaks observed in the presence of formic acid remain
highly symmetrical and narrow, while MS sensitivity is not altered. Therefore, the CSH C18
column has been used in a few recent studies dealing with the determination of peptidic
drugs in sports doping and appears as a promising stationary phase [150058].
Several years ago, the trend was to develop dedicated procedures for each class of
peptides, but today, many laboratories want to develop a multiclass/multianalyte initial testing
and confirmatory methods for peptidic substances. The most frequently reported approach
for the analysis of small peptides, such as GHRPs and similar molecules, involves using
mixed-mode weak cation exchange SPE from a few mL of urine samples or a few μL of
protein-depleted serum/plasma specimens, followed by LC-ESI-MS/MS or LC-ESI-
HRMS(/MS). A routine screening for the small GHRPs by LC-MS/MS was successfully
implemented during the winter Olympic Games in 2014, and positive cases were also
recently identified in Montreal and Moscow using a similar methodology. In addition, a
screening assay was developed for the determination of 11 prohibited peptides (9 GHRPs,
desmopressin, and LHRH) containing between 4 and 8 amino acids (<1.5 kDa) by combining
SPE with nano-LC-HRMS. The method was fully validated, and the limits of detection were in
the range of 2–10 pg/mL, which is much better than the most recent WADA
recommendations (MRPL set at 2 ng/mL) for this class of substances. However, one of the
main issues when analyzing small peptide hormones is the short half-life in plasma and their
rapid elimination. Although GHRPs and related substances are extracted and enriched
through SPE alone, peptides >2 kDa (e.g., sermorelin, tesamorelin, CJC-1295, and
Synacthen) should be better isolated from biological matrices using SPE or ultrafiltration
followed by immunoaffinity purification. This allows one to obtain extracts of higher purity and
better detection limits in biological fluids, far below the MRPL recently set by the WADA at 2
ng/mL in urine. In fact, using nano-LC coupled with HRMS detection, detection limits in urine
down to 1–5 pg/mL were reported. This procedure is relatively fast and allows the analysis of
approximately 25 samples per day, which is of utmost importance for fast results reporting.
However, the use of specific antibodies and magnetic beads makes this type of analysis
expensive, limiting its application to targeted specimens based on previously obtained
suspicious results and/or linked to high-risk sport disciplines [150058].
770
Small peptide hormones
Peptide hormones represent an emerging class of potential doping agents. Detection of their
misuse is difficult due to their short half-life in plasma and rapid elimination. Therefore,
investigating their metabolism can improve detectability. Unfortunately, pharmacokinetic
studies with human volunteers are often not allowed because of ethical constraints, and
therefore alternative models are needed. One study was performed in order to evaluate in
vitro models (human liver microsomes and S9 fraction) for the prediction of the metabolism of
peptidic doping agents and to compare them with the established models. The peptides that
were investigated include desmopressin, TB-500, GHRP-2, GHRP-6, hexarelin, LHRH and
leuprolide. Several metabolites were detected for each peptide after incubation with human
liver microsomes, S9 fraction, and serum, which all showed endopeptidase and
exopeptidase activity. In vitro models from different organs (liver vs kidney) were compared,
but no significant differences were recorded. Deamidation was not observed in any of the
models and was therefore evaluated by incubation with alpha-chymotrypsin. In conclusion, in
vitro models are useful tools for forensic and clinical analysts to detect peptidic metabolic
markers in biological fluids [14739].
With the growing availability of mature systems and strategies in biotechnology and the
continuously expanding knowledge of cellular processes and involved biomolecules, human
sports drug testing has become a considerably complex field in the arena of analytical
chemistry. Proving the exogenous origin of peptidic drugs and respective analogs at lowest
concentration levels in biological specimens (commonly blood, serum and urine) of rather
limited volume is required to pursue an action against cheating athletes. Therefore,
approaches employing chromatographic-mass spectrometric, electrophoretic, immunological
and combined test methods have been required and developed. These allow detecting the
misuse of peptidic compounds of lower (such as growth hormone-releasing peptides, ARA-
290, TB-500, AOD-9604, CJC-1295, desmopressin, luteinizing hormone-releasing
hormones, synacthen, etc.), intermediate (e.g. insulins, IGF-1 and analogs, “full-lengt”
mechano growth factor, growth hormone, chorionic gonadotropin, erythropoietin, etc.) and
higher (e.g., stamulumab) molecular mass with desired specificity and sensitivity. A gap
between the technically possible detection and the day-to-day analytical practice, however,
still needs to be closed [14740].
Peptide hormones represent an emerging class of potential doping agents. Detection of their
misuse is difficult due to their short half-life in plasma and rapid elimination. Therefore,
investigating their metabolism can improve detectability. Unfortunately, pharmacokinetic
studies with human volunteers are often not allowed because of ethical constraints, and
therefore alternative models are needed. One study was performed in order to evaluate in
vitro models (human liver microsomes and S9 fraction) for the prediction of the metabolism of
peptidic doping agents and to compare them with the established models. The peptides that
were investigated include desmopressin, TB-500, GHRP-2, GHRP-6, hexarelin, LHRH and
leuprolide. Several metabolites were detected for each peptide after incubation with human
liver microsomes, S9 fraction, and serum, which all showed endopeptidase and
exopeptidase activity. In vitro models from different organs (liver vs kidney) were compared,
but no significant differences were recorded. Deamidation was not observed in any of the
models and was therefore evaluated by incubation with α-chymotrypsin. In conclusion, in
vitro models are useful tools for forensic and clinical analysts to detect peptidic metabolic
markers in biological fluids [150135].
771
Detection of misuse of peptides and proteins as growth promoters is a major issue for sport
and food regulatory agencies. The limitations of current analytical detection strategies for this
class of compounds, in combination with their efficacy in growth-promoting effects, make
peptide and protein drugs highly susceptible to abuse by either athletes or farmers who seek
for products to illicitly enhance muscle growth. Mass spectrometry (MS) for qualitative
analysis of peptides and proteins is well-established, particularly due to tremendous efforts in
the proteomics community. Similarly, due to advancements in targeted proteomic strategies
and the rapid growth of protein-based biopharmaceuticals, MS for quantitative analysis of
peptides and proteins is becoming more widely accepted. These continuous advances in MS
instrumentation and MS-based methodologies offer enormous opportunities for detection and
confirmation of peptides and proteins. Therefore, MS seems to be the method of choice to
improve the qualitative and quantitative analysis of peptide and proteins with growth-
promoting properties. This review aims to address the opportunities of MS for peptide and
protein analysis in veterinary control and sports-doping control with a particular focus on
detection of illicit growth promotion. An overview of potential peptide and protein targets,
including their amino acid sequence characteristics and current MS-based detection
strategies is, therefore, provided. Furthermore, improvements of current and new detection
strategies with state-of-the-art MS instrumentation are discussed for qualitative and
quantitative approaches [150136].
Solid-phase extraction of small biologically active peptides on cartridges
Currently liquid chromatography-mass spectrometry (LC-MS) analysis after solid-phase

extraction (SPE) on weak cation-exchange cartridges is a method of choice for anti-doping
analysis of small bioactive peptides such as growth hormone releasing peptides (GHRPs),
desmoporessin, LHRH, and TB-500 short fragment. Dilution of urine samples with phosphate
buffer for pH adjustment and SPE on weak cation exchange microelution plates was tested
as a means to increase throughput of this analysis. Dilution using 200 mM phosphate buffer
provides good buffering capacity without affecting the peptides recoveries. SPE on
microelution plates was performed on Waters Positive Pressure-96 Processor with
subsequent evaporation of eluates in nitrogen flow. Though the use of smaller sample
volume decreases the pre-concentration factor and increases the limits of detection of 5 out
of 17 detected peptides, the recovery, linearity, and reproducibility of the microelution
extraction were comparable with cartridge SPE. The effectiveness of protocols was
confirmed by analysis of urine samples containing ipamorelin, and GHRP-6 and its
metabolites. SPE after urine sample dilution with buffer can be used for faster sample
preparation. The use of microelution plates decreases consumption of solvents and allows
processing of up to 96 samples simultaneously. Cartridge SPE with manual рН adjustment
remains the best option for confirmation [150137].
Immunoaffinity purification and LC-HRMS/MS for peptide hormones
Bioactive peptides with an approximate molecular mass of 2-12 kDa are of considerable
relevance in sports drug testing. Such peptides have been used to manipulate several
potential performance-enhancing processes in the athlete's body and include for example
growth hormone releasing hormones (sermorelin, CJC-1293, CJC-1295, tesamorelin),
synthetic/animal insulins (lispro, aspart, glulisine, glargine, detemir, degludec, bovine and
porcine insulin), synthetic ACTH (synacthen), synthetic IGF-I (longR(3) -IGF-I) and mechano
growth factors (human MGF, modified human MGF, “full-length” MGF). A combined initial
test method using one analytical procedure is a desirable tool in doping controls and related
disciplines as requests for higher sample throughput with utmost comprehensiveness
772
preferably at reduced costs are constantly issued. An approach modified from an earlier
assay proved fit-for-purpose employing pre-concentration of all target analytes by means of
ultrafiltration, immunoaffinity purification with coated paramagnetic beads, nano-ultra high
performance liquid chromatography (UHPLC) separation, and subsequent detection by
means of high resolution tandem mass spectrometry. The method was shown to be
applicable to blood and urine samples, which represent the most common doping control
specimens. The method was validated considering the parameters specificity, recovery (11-
69%), linearity, imprecision (<25 %), limit of detection (5-100 pg in urine, 0.1-2 ng in plasma),
and ion suppression. The analysis of administration study samples for insulin degludec,
detemir, aspart, and synacthen provided the essential data for the proof-of-principle of the
method [150138].
Affinity-based biosensors
Affinity-based biosensors (ABBs) have started to be considered in sport medicine and doping
control analysis because they are cheap, easy to use and sufficiently selective analytical
devices, characterized by a reversible interaction with the analyte under investigation
allowing the use of the same sensor for multiple analyses. In one review it was described the
main categories of substances reported in the World Anti-Doping Agency Prohibited List and
how ABBs may contribute to their detection. Although several ABBs proposed in the last few
years display limit of detections that are in principle matching the World Anti-Doping Agency
requirements, their application in the framework of 'traditional' antidoping tests seems quite
unlikely, mainly because of the still insufficient selectivity especially in the case of 'pseudo-
endogenous' compounds, and on the lack of complete information regarding potential matrix
effects in real samples and following their routine use. At the same time, ABBs could
contribute to fill a significant information gap concerning complementary evidence that can
be obtained from their use “on the spot”, as well as to preselect a risk population of
individuals to be targeted for a full antidoping test; while in sport medicine they could
contribute to obtaining analytical information of physiological relevance from the
measurement of specific parameters or markers before, during and after physical exercise
[14034].
SIRT1-activating drugs
The NAD(+) depending enzyme SIRT1 regulates the mitochondrial biogenesis, fat and
glucose metabolism through catalyzing the deacetylation of several metabolism-related
protein-substrates. Recently, synthetic activators of SIRT1 referred to as STACs (Sirtuin
activating compounds, e.g. SRT2104) were identified and tested in clinical studies for the
treatment of aging-related diseases such as type 2 diabetes, Alzheimer's and obesity.
Although the mechanism of SIRT1 activation by small molecules has caused considerable
controversy, STACs demonstrated a significant performance enhancement in mice
experiments including an improvement of endurance, muscle strength, and locomotor
behavior. Due to their potential to increase exercise tolerance in healthy individuals, SIRT1
activators are currently being monitored by anti-doping authorities. In the present study, the
in vivo metabolic clearance of three SIRT1 activators was investigated in rats by the
collection of urine, DBS (dried blood spots) and plasma samples following a single oral
administration. The resulting metabolic products were studied by positive electrospray
ionization - (tandem) mass spectrometry and confirmed by the comparison with in vitro
generated metabolites using human and rat liver microsomal preparations. Subsequently, a
screening procedure for five SIRT1 activators and the metabolite M1-SRT1720 in DBS
773
specimens was developed. Liquid-liquid-extraction and liquid chromatography/tandem mass
spectrometry was employed based on diagnostic ion transitions recorded in multiple reaction
monitoring mode and two deuterated internal standards namely d8-SRT1720 and d8-M1-
SRT1720 were utilized. The doping control assay was characterized with regard to
specificity, limit of detection (10-50 ng/mL), recovery (65-83 %) and imprecision (7-20 %) and
ion suppression/enhancement effects (<10 %), demonstrating its fitness-for-purpose for
sports drug testing applications [13101].
The efficiency of Sirtuin1, a major target for the treatment of various metabolic disorders
such as inflammation and type 2 diabetes mellitus, can be modulated via low molecular mass
SIRT1 activators (e.g. resveratrol, SRT1720, and SRT2104).The administration of such
compounds results in increased deacetylation of substrates including p53, FOXO1, and
PGC1alpha, potentially leading to an improved physical performance. Consequently,
proactive and preventive anti-doping measures are required and an assay dedicated to
serum and plasma was desirable. Model substances of emerging SIRT1 drug candidates
were obtained and synthesized and their mass spectrometric behavior following positive or
negative electrospray ionization and collision-induced dissociation was elucidated using low
and high resolution/high accuracy (tandem) mass spectrometry. Subsequently, a screening
and confirmation procedure necessitating 100 microL of plasma was established employing
liquid chromatography/tandem mass spectrometry (LC/MS/MS) based on diagnostic ion
transitions recorded in multiple reaction monitoring mode. Sample preparation consisted of
the addition of two deuterated internal standards (D(8)-SRT1720 and D(4)-resveratrol) to the
plasma specimen and subsequent protein precipitation. Characteristic product ions indicative
of the core structures of the model analytes were characterized and utilized for the
development of a multi-analyte LC/MS/MS detection method applicable to sports drug testing
programs. The doping control assay was validated with regard to specificity, limits of
detection (0.1-1 ng/mL), recoveries (90-98 %), intraday and interday precisions (2-18%), and
ion suppression/enhancement effects. It was concluded that the fragmentation pathways of
SRT1720 and 4 SIRT1 activator models based on a common thiazole-imidazole nucleus as
well as two different complementary activators (SIRT1 activator 3 and CAY10602),
comprising a quinoxaline core, were studied. The resulting information was used to establish
and validate a sports drug testing methodology relevant for an efficient and timely anti-doping
procedure, targeting a new class of emerging therapeutics possessing significant potential
for misuse in elite and amateur sport [13102].
The enzyme SIRT1 is a metabolic key regulator in mitochondrial biogenesis, fat and glucose
metabolism. Its activation through pharmaceutical SIRT1 activators such as SRT2104 results
in an increased deacetylation of substrates representing important targets for the treatment
of metabolic diseases. Moreover, SRT1720 was found to enhance the physical performance
of mice. As SIRT1 activators might therefore be relevant in a doping control context,
metabolism studies of target substances need be conducted in order to develop a detection
assay for SIRT1 activators in urine. In the present study, the in vitro metabolism of five
SIRT1 activators was investigated using human liver microsomes. The mass spectrometric
behavior of the resulting metabolites following positive electrospray ionization and collision-
induced dissociation was elucidated by high-resolution/high-accuracy (tandem) mass
spectrometry, and confirmation of the structure of a major metabolite of SRT1720 was
accomplished by chemical synthesis. Subsequently, a screening procedure for urine samples
was developed employing liquid-liquid-extraction and liquid chromatography/tandem mass
spectrometry based on diagnostic ion transitions recorded in multiple reaction monitoring
mode and the use of d8-SRT1720 as deuterated internal standard. The method was
validated with regard to specificity, sensitivity (limit of detection 0.5 ng/ml), recovery (88-99
%) and imprecision (7-18 %) as well as ion suppression/enhancement effects (<10 %),
demonstrating its fitness-for-purpose for sports drug testing applications [13097].
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Bioassay-guided fractionation
Biological tests can be used to screen samples for large groups of compounds having a
particular effect, but it is often difficult to identify a specific compound when a positive effect
is observed. The identification of an unknown compound is a challenge for analytical
chemistry in environmental analysis, food analysis, as well as in clinical and forensic
toxicology. In this study bioassay-guided fractionation, ultra high performance liquid
chromatography combined with time-of-flight mass spectrometry (UHPLC/TOFMS) and
accurate mass database searching was tested to detect and identify unknown androgens.
Herbal mixtures and sport supplements were tested using an androgen bioassay and
modifications in sample preparations were carried out in order to activate inactive pro-
androgens, androgen esters and conjugated androgens to enable their detection in the
bioassay. Two of the four herbal mixtures tested positive and bioassay-guided fractionation
followed by UHPLC/TOFMS of positive fractions resulted in the identification of
nortestosterone phenylpropionate, testosterone cyclohexanecarboxylate and
methyltestosterone. Three of the four sport supplements reacted toxic in the bioassay or
gave inconclusive results and were further investigated using UHPLC/TOFMS in combination
with data processing software and an accurate mass database having approximately 40,000
entries. This accurate mass database was derived from the PubChem database on the
internet and coupled to the TOFMS software. This resulted in the tentative identification of
several androgens, including methylboldenone, testosterone and the androgen esters
methyltestosterone propionate or testosterone isobutyrate, testosterone buciclate and
methylenetestosterone acetate. The study showed that bioassay-guided fractionation in
combination with UHPLC/TOFMS analysis is a useful procedure to detect, isolate and
identify unknown androgens in suspected samples. As an alternative, the use of data
processing software in combination with an accurate mass database and coupled on-line
with the TOFMS instrument software enabled the identification of androgens and androgen
esters in the chromatogram even without bioassay-guided fractionation [10038].
Unknown fusion proteins
Even without clinical approval, many performance-enhancing drugs are available on the
black market and can therefore be easily obtained by cheating athletes. The misuse of these
preparations can be associated with unforeseeable health risks – either due to a poor quality
of the drugs or as a result of an insufficient clinical assessment. Moreover, confiscated black
market products have frequently been shown to contain ingredients other than those
declared on the label as well as additional by-products or compounds with a modified
molecular structure. This communication describes the identification of an unknown fusion
protein observed in several unlabelled black market products obtained from independent
sources. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and liquid
chromatography-tandem mass spectrometry (LC-MS/MS) analysis of the confiscated
preparations indicated the presence of an 18-kDa fusion protein consisting of the bacterial
redox protein thioredoxin-1 (Trx, 12 kDa) and a 6-kDa peptide of unassigned composition.
Trx has no relevance as performance enhancing agent but is routinely used as solubility tag
for recombinant protein production. Further evaluation of the acquired MS/MS data revealed
both an additional His tag and a thrombin cleavage site between the tags and the presumed
bioactive peptide. However, thrombin cleavage of the fusion protein and LC-MS/MS analysis
of the resulting peptide fragment finally suggested that the unknown protein is only the
product of an empty expression vector without the DNA insert of interest. These findings are
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a further alarming example for the high level of risk that athletes take when misusing drugs
obtained from the black market [14635].
In vitro studies
In one study, the use of equine liver/lung microsomes and S9 tissue fractions were used to
study the metabolism of the androgenic/anabolic steroid stanozolol as an example of the
potential of in vitro technologies in sports drug surveillance. In vitro incubates were analysed
qualitatively alongside urine samples originating from in vivo stanozolol administrations using
LC-MS on a high-resolution accurate mass Thermo Orbitrap Discovery instrument, by LC-
MS/MS on an Applied Biosystems Sciex 5500 Q Trap and by GC-MS/MS on an Agilent
7000A. Using high-resolution accurate mass full scan analysis on the Orbitrap, equine liver
microsome and S9 in vitro fractions were found to generate all the major phase-1 metabolites
observed following in vivo administrations. Additionally, analysis of the liver microsomal
incubates using a shallower HPLC gradient combined with various MS/MS functions on the
5500 Q trap allowed the identification of a number of phase 1 metabolites previously
unreported in the equine or any other species. Comparison between liver and lung S9
metabolism showed that the liver was the major site of metabolic activity in the equine.
Furthermore, using chemical enzyme inhibitors that are known to be selective for particular
isoforms in other species suggested that an enzyme related to CYP2C8 may be responsible
the production of 16-hydroxy-stanozolol metabolites in the equine. In summary, the in vitro
and in vivo phase 1 metabolism results reported herein compare well and demonstrate the
potential of in vitro studies to compliment the existing in vivo paradigm and to benefit animal
welfare through a reduction and refinement of animal experimentation [10039].
Mammalian reporter gene bioassays
Anabolic androgenic steroids (AAS) share the activation of the androgen receptor (AR) as
common mechanism of action. The mammalian androgen responsive reporter gene assay
(AR CALUX bioassay), measuring compounds interacting with the AR can be used for the
analysis of AAS without the necessity of knowing their chemical structure beforehand,
whereas current chemical-analytical approaches may have difficulty in detecting compounds
with unknown structures, such as designer steroids. One study demonstrated that AAS
prohibited in sports and potential designer AAS can be detected with this AR reporter gene
assay, but that also additional steroid activities of AAS could be found using additional
mammalian bioassays for other types of steroid hormones. Mixtures of AAS were found to
behave additively in the AR reporter gene assay showing that it is possible to use this
method for complex mixtures as are found in doping control samples, including mixtures that
are a result of multi drug use. To test if mammalian reporter gene assays could be used for
the detection of AAS in urine samples, background steroidal activities were measured. AAS-
spiked urine samples, mimicking doping positive samples, showed significantly higher
androgenic activities than unspiked samples. GC-MS analysis of endogenous androgens and
AR reporter gene assay analysis of urine samples showed how a combined chemical-
analytical and bioassay approach can be used to identify samples containing AAS. The
results indicate that the AR reporter gene assay, in addition to chemical-analytical methods,
can be a valuable tool for the analysis of AAS for doping control purposes [10040].
Transcriptome analysis
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Evolving challenges require evolving responses. The use of illicit performance enhancing
drugs by athletes permeates the reality and the perception of elite sports. New drugs with
ergogenic or masking potential are quickly adopted, driven by a desire to win and the
necessity of avoiding detection. To counter this trend, anti-doping authorities are continually
refining existing assays and developing new testing strategies. In the post-genome era,
genetic- and molecular-based tests are being evaluated as potential approaches to detect
new and sophisticated forms of doping. Transcriptome analysis, in which a tissue's
complement of mRNA transcripts is characterized, is one such method. The quantity and
composition of a tissue's transcriptome is highly reflective of milieu and metabolic activity.
There is much interest in transcriptional profiling in medical diagnostics and, as
transcriptional information can be obtained from a variety of easily accessed tissues, similar
approaches could be used in doping control. One article briefly reviewed current
understanding of the transcriptome, common methods of global analysis of gene expression
and non-invasive sample sources. While the focus of the article is on anti-doping, the
principles and methodology described could be applied to any research in which non-
invasive, yet biologically informative sampling is desired [10041].
Over the course of the past decade, technical progress has enabled scientists to investigate
genome-wide RNA expression using microarray platforms. This transcriptomic approach
represents a promising tool for the discovery of basic gene expression patterns and for
identification of cellular signalling pathways under various conditions. Since doping
substances have been shown to influence mRNA expression, it has been suggested that
these changes can be detected by screening the blood transcriptome. In one review, it was
critically discuss the potential but also the pitfalls of this application as a tool in doping
research. Transcriptomic approaches were considered to potentially provide researchers with
a unique gene expression signature or with a specific biomarker for various physiological and
pathophysiological conditions. Since transcriptomic approaches are considerably prone to
biological and technical confounding factors that act on study subjects or samples, very strict
guidelines for the use of transcriptomics in human study subjects have been developed.
Typical field conditions associated with doping controls limit the feasibility of following these
strict guidelines as there are too many variables counteracting a standardized procedure.
After almost a decade of research using transcriptomic tools, it still remains a matter of future
technological progress to identify the ultimate biomarker using technologies and/or
methodologies that are sufficiently robust against typical biological and technical bias and
that are valid in a court of law [11564].
Compound-specific isotope analysis (CSIA)
Compound-specific isotope analysis (CSIA) by gas chromatography combustion isotope ratio

mass spectrometry (GCC-IRMS) is a powerful technique for the sourcing of substances,
such as determination of the geographic or chemical origin of drugs and food adulteration,
and it is especially invaluable as a confirmatory tool for detection of the use of synthetic
steroids in competitive sport. We review here principles and practices for data processing
and calibration of GCC-IRMS data with consideration to anti-doping analyses, with a focus
on carbon isotopic analysis (13C/12 C). After a brief review of peak definition, the isotopologue
signal reduction methods of summation, curve-fitting, and linear regression were described
and reviewed. Considerations for the anti-doping analyst are reviewed [12075].
Dual-color bioluminescent bioreporter
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Bioassays represent promising complementary techniques to conventional analytical
approaches used in doping analysis to detect illicit drugs like anabolic-androgenic steroids
(AAS). The fact that all AAS share a common mechanism of action via the human androgen
receptor (hAR) enables the use of bioassays, relying on the activation of hAR as antidoping
screening tools. Previously, it was developed a dual-color bioreporter based on yeast cells
engineered to express hAR and androgen response elements driving the expression of the
bioluminescent (BL) reporter protein Photinus pyralis luciferase. A second reporter protein,
the red-emitting luciferase PpyRE8, was introduced in the bioreporter as internal viability
control. Here, we report the first forensic application of a straightforward, accurate, and cost-
effective bioassay, relying on spectral resolution of the two BL signals, in 96-microwell
format. The bioreporter responds to dihydrotestosterone as reference androgen in a
concentration-dependent manner from 0.08 to 1,000 nM with intra- and inter-assay variation
coefficients of 11.4 and 13.1 percent, respectively. It was also demonstrated the suitability of
this dual-color bioreporter to assess (anti)-androgenic activity of pure AAS, mixtures of AAS,
and other illicit drugs provided by the Scientific Police. Significant anti-androgenic activity
was observed in samples labeled as marijuana and hashish, containing delta(9)-
tetrahydrocannabinol as major constituent [13100].
Two-dimensional gas chromatography with heart-cutting
The accuracy and precision of gas chromatography combustion isotope ratio mass
spectrometry (GC-C-IRMS) measurements are highly dependent on analyte purity. Reliable
analysis of urinary steroids for doping control therefore requires extensive and time-
consuming sample preparation (i.e. liquid chromatography fraction collection) prior to GC-C-
IRMS analysis. The use of two-dimensional GC (GC-GC) with heart-cutting (Deans Switch)
as a possible approach to reduce the sample purification required for IRMS analysis is
described herein. The system uses a low thermal mass oven (LTM) incorporated into an
existing GC-C-IRMS system. GC-GC allowed the use of a cyanopropyl/phenyl column in the
first dimension to optimize the separation of underivatized steroids, while a phenyl-
methylpolysiloxane column in the second dimension focuses the selectively cut analytes into
narrower peaks for more sensitive and reliable MS analysis. In addition, to confirm analyte
identity, eluent from the second GC was split, with 20 percent entering a scanning MS, and
80 percent flowing to the IRMS. As a proof concept, the developed method was then used to
analyze a single spot urine (5 ml) from an individual receiving T therapy (2 × 50 mg sachets of
Testogel®). The T delta value (-27.8 ‰, [T] = 38 ng/ml) was clearly distinct from 11-
ketoetiocholanolone (-22.5 ‰) (used as an endogenous reference compound (ERC)),
indicating T as being of exogenous origin. The simultaneous analysis by the scanning MS
yielded a full scan mass spectrum of the same chromatographic peak, thus confirming the
peak to be T [12078].
RNA sequencing
The abuse of anabolic substances in animal husbandry is forbidden within the EU and well
controlled by detecting substance residues in different matrices. The application of newly
designed drugs or substance cocktails represents big problems. Therefore developing
sensitive test methods is important. The analysis of physiological changes caused by the use
of anabolic agents on the molecular level, for example, by quantifying gene expression
response, is a new approach to develop such screening methods. A novel technology for
holistic gene expression analysis is RNA sequencing. In one study, the potential of this high-
throughput method for the identification of biomarkers was evaluated. The effect of
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trenbolone acetate plus estradiol on gene expression in liver from Nguni heifers was
analyzed with RNA sequencing. The expression of 40 selected candidate genes was verified
via RT-qPCR, whereby 20 of these genes were significantly regulated. To extract the
intended information from these regulated genes, biostatistical tools for pattern recognition
were applied and resulted in a clear separation of the treatment groups. Those candidate
genes could be verified in boars and in calves treated with anabolic substances. These
results show the potential of RNA sequencing to screen for biomarker candidates to detect
the abuse of anabolics. The verification of these biomarkers in boars and calves leads to the
assumption that gene expression biomarkers are independent of breed or even species and
that biomarkers, identified in farm animals could also act as potential biomarker candidates
to detect the abuse of anabolic substances in human sports [12079].
DNA typing
A clear positive case for anabolic steroids doping was confounded by alleged urine
tampering during doping control procedures. Review of the chain of custody showed no
flaws, but nevertheless the athlete was adamant that the urine sample should be analyzed
for DNA in order to support her contention that she was not the donor of the sample. The
results obtained showed that the urine sample that scored positive for steroids contained
nuclear DNA that could not be matched to the DNA obtained from the athlete's blood. On the
other hand, the same urine sample contained mitochondrial DNA whose nucleotide
sequences spanning the hyper variable regions HV1 and HV2 proved to be identical to those
determined in mitochondrial DNA amplified from the athlete's blood. The occurrence of an
extraneous genotype is compatible with exogenous nuclear DNA admixture to the athlete's
urine. Alternatively, taking in consideration the mitochondrial DNA, we could not exclude that
a sibling or a maternal relative of the athlete could have acted as a donor of the urine utilized
for doping control and DNA analysis. Both situations point to possible tampering of the urine
by the athlete. Adjudication at CAS maintained previous national and international federation
decision that there was no proof of a chain of custody flaw to justify the athlete's allegation of
urine substitution after collection [05065].
Urinary steroid sulfate metabolites
The need for laboratories accredited by the World Anti-Doping Agency (WADA) to develop
methods of analysis for steroids excreted primarily as their sulfate conjugates has faced
significant analytical challenges. One of the issues relates to the extraction of these
metabolites from urine in a relatively pure state. The use of (-)-N,N-dimethylephedrinium
bromide as an ion pairing reagent was optimised to produce a method that is selective for the
extraction of steroid sulfates prior to GC-MS or LC-MS analysis, with minimal contributions
from the urine matrix. The recovery of androsterone from its sulfate conjugate was
determined to be 67 percent with a relative quantitative uncertainty of + 14 percent [05064].
The urine marker test
Urine sample collection for doping control tests is a key component of the World Anti-Doping
Agency's fight against doping in sport. However, a substantial number of athletes experience
difficulty when having to urinate under supervision. Furthermore, it cannot always be ensured
that athletes are actually delivering their own urine. A method that can be used to alleviate
the negative impact of a supervised urination procedure and which can also identify urine as
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coming from a specific athlete is the urine marker test. Monodisperse low molecular weight
polyethylene glycols (PEGs) are given orally prior to urination. Urine samples can be traced
to the donor by analysis of the PEGs previously given. The objective of one study was to
investigate the use of the urine marker during urine doping control testing. Two studies
investigated athletes' acceptance of this new method via two questionnaires (n=253).
Furthermore, a third study (n=91) investigated whether ingestion of the marker can identify
the urine as coming from a specific person and whether the marker interferes with the
detection of prohibited substances. The results indicate that this new method finds wide
acceptance both from athletes who have only heard about the procedure and those who
have actually tested the new method. Furthermore, the marker, which can identify urine as
coming from a specific person, does not interfere with the detection of prohibited substances
[150139].
Hydrogen isotope ratio of urinary steroids
The hydrogen isotope ratio (HIR) of body water and, therefore, of all endogenously
synthesized compounds in humans, is mainly affected by the HIR of ingested drinking water.
As a consequence, the entire organism and all of its synthesized substrates will reflect
alterations in the isotope ratio of drinking water, which depends on the duration of exposure.
To investigate the effect of this change on endogenous urinary steroids relevant to doping-
control analysis the hydrogen isotope composition of potable water was suddenly enriched
from -50 to 200 ‰ and maintained at this level for two weeks for two individuals. The steroids
under investigation were 5beta-pregnane-3alpha,20alpha-diol, 5alpha-androst-16-en-3alpha-
ol, 3alpha-hydroxy-5alpha-androstan-17-one (Andro), 3alpha-hydroxy-5beta-androstan-17-
one (Etio), 5alpha-androstane-3alpha,17beta-diol, and 5beta-androstane-3alpha,17beta-diol
(excreted as glucuronides) and Etio, Andro and 3beta-hydroxyandrost-5-en-17-one (excreted
as sulfates). The HIR of body water was estimated by determination of the HIR of total native
urine, to trace the induced changes. The hydrogen in steroids is partly derived from the total
amount of body water and cholesterol-enrichment could be calculated by use of these data.
Although the sum of changes in the isotopic composition of body water was 150 ‰, shifts of
approximately 30 ‰ were observed for urinary steroids. Parallel enrichment in their HIR was
observed for most of the steroids, and none of the differences between the HIR of individual
steroids was elevated beyond recently established thresholds. This finding is important to
sports drug testing because it supports the intended use of this novel and complementary
methodology even in cases where athletes have drunk water of different HIR, a plausible
and, presumably, inevitable scenario while traveling [12080].
Dried blood spots (DBS)
The growing interest in measuring drugs and drug candidates from whole blood and dried
blood spots (DBS) in general and particularly in doping controls continus. The combined
advantages of blood testing and DBS sampling, transport, and storage have contributed to a
renaissance of the importance of blood testing in human doping controls, certainly supported
by significant improvements in analytical instrumentation. Requiring minimal-invasive
sampling devices and minimizing the possibility of sample manipulation, frequent and cost-
efficient sample collections are enabled that allow the detection of numerous intact drugs and
drug candidates prohibited according to WADA regulations. Further, enhanced analyte
stabilities are reported, which is a central aspect of modern doping controls. It remains to be
clarified however which legitimacy applies in case of presumptive analytical findings as the
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established A- and B-sample procedure is not in place for DBS testing today.
Notwithstanding, proof-of-concept studies with DBS have been conducted, demonstrating
their value to anti-doping organizations [14715].
Dried blood spot (DBS) sampling, a technique for whole blood sampling on a piece of filter
paper, has more than 50-years tradition, particularly in the diagnostic analysis of metabolic
disorders in neonatal screening. Due to the minimal invasiveness, straightforwardness,
robustness against manipulation and fastness DBS sampling recommends itself as an
advantageous technique in doping control analysis. The present approach highlights the
development of a screening assay for the analysis of eight anabolic steroid esters
(nandrolone phenylpropionate, trenbolone enanthate, testosterone acetate, testosterone
cypionate, testosterone isocaproate, testosterone phenylpropionate, testosterone decanoate
and testosterone undecanoate) and nandrolone in DBS. The detection of the intact esters
allows an unequivocal proof of the administration of conjugates of exogenous testosterone
and its derivatives. Precise, specific and linear conditions were obtained by means of liquid
chromatography high resolution/high accuracy mass spectrometry. Sensitivity in the low ppb
range was accomplished by the preparation of the methyloxime derivatives of the target
compounds. Labeled internal standards (d3-nandrolone, d3-nandrolone caproate and d3-
nandrolone undecanoate) were applied to compensate for the broad range in chain length of
the esters. The assay presented here outlines the application of DBS for the analysis of
anabolic steroid esters in doping controls for the first time providing great potential to simplify
the proof of exogenous administration of testosterone [14632].
Although not (yet) a frequent doping control specimen, blood samples are advantageous
over urine specimens in a doping control context in at least two ways they commonly contain
the intact drug rather than metabolites, which represents a workaround when new or entirely
unknown (designer) compounds are misused and metabolism studies are not (or not
publicly) available, and they provide information on drug concentrations at the time of
sampling, which is of utmost importance concerning those drugs prohibited in-competition
only. As a consequence, the option to expand doping controls from urine and (less
frequently) plasma or serum to whole blood shortly before or after competition was evaluated
and assays for the analysis of minimal-invasively collected dried blood spots (DBS) were
reported in 2011 and 2012. DBS, created from a volume of 25 ml, were excised from blood
collection cards and consecutively extracted into methanol/ tert.-butyl-methyl ether and
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acetone. After prosessing the blood spots, the combined targeted qualitative and quantitative
analysis was possible and data for non-target substances for retrospective evaluation or
homology searches based on conserved and common molecular structures were recorded
[12017].
In one study, a new type of mass spectrometer combining a quadrupole mass filter, a higher
collision dissociation (HCD) cell and an Orbitrap detector, was evaluated for the analysis of
dried blood spots (DBS) in doping controls. DBS analysis is characterized by the necessity to
detect prohibited compounds in sub-nanogram-per-milliliter levels with high identification
capacity. After extraction of DBS with an organic solvent and liquid chromatographic
separation (using a regular C18-RP-analytical UHPLC-column) of target analytes, mass
spectrometry is performed with a high-resolution full scan in positive and negative mode by
means of electrospray ionisation. Single-product ion mass spectra are acquired using the
data-dependent analysis mode (employing an inclusion list) for previously selected
precursors of known prohibited compounds with fixed retention time ranges. Besides, a
sensitive screening in a targeted approach, non-targeted analysis for retrospective data
evaluation is thus possible. The chosen experimental design enables the determination of
various drugs from different classes with one generic sample preparation which is shown for
26 selected model compounds delta9-tetrahydrocannabinol (THC), tetrahydrocannabinol-9-
carboxylic acid (THC-COOH), methylhexaneamine, methylphenidate, cocaine, nikethamide,
3,4-methylenedioxyamphetamine, N-methyl-3,4-methylenedioxyamphetamine, strychnine,
mesocarb, salbutamol, formoterol, clenbuterol, metandienone, stanozolol, bisoprolol,
propranolol, metoprolol, anastrazole, clomiphene, exemestane, dexamethasone,
budesonide, selective androgen receptor modulator (SARM) S4 (andarine), SARM S1,
hydrochlorothiazide). Generally, only qualitative result interpretation was focussed upon, but
for target analytes with deuterium-labelled internal standards (salbutamol, clenbuterol,
cocaine, dexamethasone, THC-COOH and THC) quantitative analysis was also possible.
Especially the most challenging analytes, THC and its carboxy metabolite, were detected in
DBS at relevant concentrations (<0.5 ng/mL) using targeted HCD experiments. The method
was validated for the parameters: specificity, linearity (0-20 ng/mL), precision (<25 %),
recovery (mean 60 %), limit of detection/quantification, ion suppression, stability and
accuracy (80-120 %). Six isotope-labelled analogues used as internal standards facilitate a
quantitative result interpretation which is of utmost importance especially for in-competition
drug sports testing [12081].
Whole blood sample collection on cellulose paper has a more than 30-year-long tradition,
especially in neonatal screening. The sampling is minimally invasive, fast, discreet, and
robust against manipulation. The present approach highlights the potential to determine
doping agents in dried blood spots (DBS) after extraction and subsequent analysis by liquid
chromatography-mass spectrometry (LC-MS). The assay is focused on selected model
compounds of which the circulating target concentration is of particular interest. Here, pre- or
post-competition testing with DBS allows probing for the conditions (i.e. presence or absence
of relevant drugs) in the athlete's circulation during competition, which complements earlier
approaches towards the identification of urinary indicators for the temporal application of
substances prohibited in-competition only. Precise (< 20 %), linear, and robust conditions
with limits of detection in low ng/ml range were accomplished by means of LC coupled to
high resolution/high accuracy mass spectrometry for the selected model compounds
benzoylecgonine, cocaine, pseudoephedrine, amphetamine, salbutamol, and JWH-018.
Deuterium-labelled internal standards were used to yield reliable quantitative results. In
addition, the non-targeted screening approach (positive/negative switching combined with
tandem mass spectrometry (MS/MS) experiments) enables the retrospective qualitative data
evaluation for a comprehensive selection of known and unknown substances as exemplarily
shown by the extraction of 20 target compounds (corticosteroids, aromatase inhibitors,
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anabolic steroids, beta-blockers, etc.) at 20 ng/mL. The simple and fast nature of the assay
allows for an easy implementation into existing procedures and will potentially enhance the
effectiveness of testing by reducing costs and effort of pre-analysis workload. The
automation of laboratory processes has continuously been improved, and the simplification
and acceleration of pre-analytical steps such as sample collection, transfer, and storage are
desirable benefits in most medico-analytical arenas. The collection of whole blood samples,
dried on a piece of paper, offers various advantages over conventional venopuncture-based
blood sampling concerning time consumption, workload and costs without compromising the
required quality. The use of dried blood spots (DBS) was reported as early as the 1960s
when this technique was applied to the sample collection for testing for phenylketonuria in
newborns by a simple heel prick. In comparison to conventional venous cannula sampling,
the collection of a drop of blood (usually 10-30 microL) from a heel, finger, or ear prick is
considerably less invasive. This minimizes the risk of infections especially for sensitive
patients such as infants on the one hand, and enables the more frequent collection of
samples in pharmacokinetic studies with small laboratory animals (mice, rats, etc) on the
other hand. The stability of the cellulose-fixed target analytes is generally described to be
superior to plasma, serum, or urine storage conditions due to inactivation of enzymatic
degradation processes. All assays follow the common strategy to perform a more or less
simple extraction, whereby the extraction conditions (organic/aqueous solvent ratio) strongly
depend on the chemical properties of the target analytes. In the present study, an assay for
selected prohibited threshold substances by means of liquid chromatographic separation
coupled to high resolution/high mass accuracy mass spectrometry (LC-MS) is reported.
Samples are manually punched from DBS cards, fortified with labelled internal standards,
and extracted with an organic solvent prior to the LC-MS measurement. Target substances
implemented in the method are cocaine, benzoylecgonine, salbutamol, ephedrine/pseudo-
ephedrine, amphetamine and JWH-018, and validation for qualitative and quantitative
purposes has been performed [11565].
A new hyphenated mass spectrometry
Occasionally, doping analysis has been recognized as a competitive challenge between

cheating sportsmen and the analytical capabilities of testing laboratories. Both have made
immense progress during the last decades, but obviously the athletes have the questionable
benefit of frequently being able to switch to new, unknown and untested compounds to
enhance their performance. Thus, as analytical counteraction and for effective drug testing, a
complementary approach to classical targeted methods is required in order to implement a
comprehensive screening procedure for known and unknown xenobiotics. One study
provided a new analytical strategy to circumvent the targeted character of classical doping
controls without losing the required sensitivity and specificity. Using 50 microL of plasma
only, the method potentially identifies illicit drugs in low ng/mL concentrations. Plasma
provides the biological fluid with the circulating, unmodified xenobiotics; thus the identification
of unknown compounds is facilitated. After a simple protein precipitation, liquid
chromatographic separation and subsequent detection by means of high resolution/high
accuracy orbitrap mass spectrometry, the procedure enables the determination of numerous
compounds from different classes prohibited by the World Anti-Doping Agency (WADA). A
new hyphenated mass spectrometry technology was employed without precursor ion
selection for higher collision energy dissociation (HCD) fragmentation experiments. Thus the
mass spectra contained all the desired information to identify unknown substances
retrospectively. The method was validated for 32 selected model compounds for qualitative
purposes considering the parameters specificity, selectivity, limit of detection (<0.1-10
ng/mL), precision (9-28 %), robustness, linearity, ion suppression and recovery (80-112 %).
783
In addition to the identification of unknown compounds, the plasma samples were
simultaneously screened for known prohibited targets [10042].
mRNA transcripts
MicroRNAs (miRNAs) are small non-coding RNAs that regulate a variety of biological
processes. Cell-free miRNAs detected in blood plasma are used as specific and sensitive
markers of physiological processes and some diseases. Circulating miRNAs are highly
stable in body fluids, for example plasma. Therefore, profiles of circulating miRNAs have
been investigated for potential use as novel, non-invasive anti-doping biomarkers. This
review describes the biological mechanisms underlying the variation of circulating miRNAs,
revealing that they have great potential as a new class of biomarker for detection of doping
substances. The latest developments in extraction and profiling technology, and the technical
design of experiments useful for anti-doping, are also discussed. Longitudinal measurements
of circulating miRNAs in the context of the athlete biological passport are proposed as an
efficient strategy for the use of these new markers. It was also emphasizesd potential
challenges for the translation of circulating miRNAs from research into practical anti-doping
applications [13105].
Evolving challenges require evolving responses. The use of illicit performance enhancing
drugs by athletes permeates the reality and the perception of elite sports. New drugs with
ergogenic or masking potential are quickly adopted, driven by a desire to win and the
necessity of avoiding detection. To counter this trend, anti-doping authorities are continually
refining existing assays and developing new testing strategies. In the post-genome era,
genetic- and molecular-based tests are being evaluated as potential approaches to detect
new and sophisticated forms of doping. Transcriptome analysis, in which a tissue's
complement of mRNA transcripts is characterized, is one such method. The quantity and
composition of a tissue's transcriptome is highly reflective of milieu and metabolic activity.
There is much interest in transcriptional profiling in medical diagnostics and, as
transcriptional information can be obtained from a variety of easily accessed tissues, similar
approaches could be used in doping control. One article briefly reviewed current
understanding of the transcriptome, common methods of global analysis of gene expression
and non-invasive sample sources. While the focus of the article was on anti-doping, the
principles and methodology described could be applied to any research in which non-
invasive, yet biologically informative sampling is desired [09040].
Deuterium/hydrogen ratio
The hydrogen isotope ratio (HIR) of body water and, therefore, of all endogenously
synthesized compounds in humans, is mainly affected by the HIR of ingested drinking water.
As a consequence, the entire organism and all of its synthesized substrates will reflect
alterations in the isotope ratio of drinking water, which depends on the duration of exposure.
To investigate the effect of this change on endogenous urinary steroids relevant to doping-
control analysis the hydrogen isotope composition of potable water was suddenly enriched
from -50 to 200 permille and maintained at this level for two weeks for two individuals. The
steroids under investigation were 5beta-pregnane-3alpha,20alpha-diol, 5alpha-androst-16-
en-3alpha-ol, 3alpha-hydroxy-5alpha-androstan-17-one (ANDRO), 3alpha-hydroxy-5beta-
androstan-17-one (ETIO), 5alpha-androstane-3alpha,17beta-diol, and 5beta-androstane-
3alpha,17beta-diol (excreted as glucuronides) and ETIO, ANDRO and 3beta-
hydroxyandrost-5-en-17-one (excreted as sulfates). The HIR of body water was estimated by
determination of the HIR of total native urine, to trace the induced changes. The hydrogen in
784
steroids is partly derived from the total amount of body water and cholesterol-enrichment
could be calculated by use of these data. Although the sum of changes in the isotopic
composition of body water was 150 permille, shifts of approximately 30 permille were
observed for urinary steroids. Parallel enrichment in their HIR was observed for most of the
steroids, and none of the differences between the HIR of individual steroids was elevated
beyond recently established thresholds. This finding is important to sports drug testing
because it supports the intended use of this novel and complementary methodology even in
cases where athletes have drunk water of different HIR, a plausible and, presumably,
inevitable scenario while traveling [13099].
The development and application of a combined gas chromatography/thermal

conversion/isotope ratio mass spectrometry (GC/TC/IRMS) method for D/H ratio
determination of endogenous urinary steroids are presented. The key element in sample
preparation was the consecutive cleanup with high-performance liquid chromatography of
initially native and subsequently acetylated steroids. This strategy enabled sufficient cleanup
off all target analytes for determination of their respective D/H values. Ten steroids (11beta-
hydroxyandrosterone, 5alpha-androst-16-en-3alpha-ol, pregnanediol, androsterone,
etiocholanolone, testosterone, epitesto-sterone, 5alpha-androstan-3alpha,17beta-diol, 5beta-
androstan-3alpha,17beta-diol and dehydroepiandrosterone) were measured from a single
urine specimen. Depending on the biological background, the determination limit for all
steroids ranged from 10 to 15 ng/mL for a 20 mL specimen. The method was validated by
application of linear mixing models on each steroid and covered repeatability and
reproducibility. The specificity of the procedure was ensured by gas chromatography/mass
spectrometry (GC/MS) analysis of the sample using equivalent chromatographic conditions
to those employed in the GC/TC/IRMS measurement. Within the sample preparation, no
isotopic fractionation was observed, and no amount-dependent shift of the D/H ratios during
the measurement was noticed. Possible memory effects occurring during IRMS
measurements were corrected by applying a simple rule of proportion. In order to determine
the naturally occurring D/H ratios of all implemented steroids, a population of 18 male
subjects was analyzed. Relevant mean delta values among selected steroids were
calculated which allowed us to study the metabolic pathways and production sites of all the
implemented steroids with additional consideration of the corresponding 13C/12C ratios
[09041].
Quantitative structure-retention relationships
Quantitative structure-retention relationship (QSRR) is a technique capable of improving the

identification of analytes by predicting their retention time on a liquid chromatography column
(LC) and/or their properties. This approach is particularly useful when LC is coupled with a
high-resolution mass spectrometry (HRMS) platform. The main aim of the present study was
to develop and describe appropriate QSRR models that provide usable predictive capability,
allowing false positive identification to be removed during the interpretation of metabolomics
data, while additionally increasing confidence of experimental results in doping control area.
For this purpose, a dataset consisting of 146 drugs, metabolites and banned compounds
from World Anti-Doping Agency (WADA) lists, was used. A QSRR study was carried out
separately on high quality retention data determined by reversed-phase (RP-LC-HRMS) and
hydrophilic interaction chromatography (HILIC-LC-HRMS) systems, employing a single
protocol for each system. Multiple linear regression (MLR) was applied to construct the linear
QSRR models based on a variety of theoretical molecular descriptors. The regression
equations included a set of three descriptors for each model: ALogP, BELe6, R2p and
ALogP(2), FDI, BLTA96, were used in the analysis of reversed-phase and HILIC column
models, respectively. Statistically significant QSRR models indicate a strong correlation
785
between retention time and the molecular descriptors. An evaluation of the best correlation
models, performed by validation of each model using three tests (leave-one-out, leave-many-
out, external tests), demonstrated the reliability of the models. One paper provided a
practical and effective method for analytical chemists working with LC/HRMS platforms to
improve predictive confidence of studies that seek to identify small molecules [13089].
Solid phase extraction (SPE) procedure
The development of a generic analytical method remains difficult when a high number of
compounds has to be simultaneously considered. One study proposed an innovative strategy
for the development of a solid phase extraction (SPE) procedure before liquid
chromatography-mass spectrometry analysis of 34 diuretics and beta-blockers in urine
samples. These compounds have been selected since they are often encountered in anti-
doping control. The principle is based on the selection of representative analytes during SPE
protocol optimization, allowing a drastic reduction of generated data and development time.
To select the representative compounds, all substances were classified based on their SPE
behavior with a generic method and groups were formed with the help of a chemometric tool,
namely hierarchical cluster analysis (HCA). One representative analyte per group was
selected and used for subsequent SPE method development. Once the SPE method was
developed, compounds were analyzed by LC-MS and matrix effects were evaluated to
determine the influence of the matrix on the SPE process and MS signal alteration due to
endogenous compounds. As a result, matrix effects evaluation must be performed on all
analytes; representative compounds previously selected for SPE development were unable
to predict matrix effects [09042].
17beta-19-nortestosterone (17beta-NT) has been illegally used in antifatigue functional foods

to promote muscle growth and improve endurance. A rapid and sensitive solid-phase
extraction-enzyme-linked immunosorbent assay (SPE-ELISA) method was developed and
successfully applied to analyze the levels of 17beta-NT in antifatigue functional foods. A
polyclonal antibody against 17beta-NT was produced from rabbits immunized with the
17beta-NT-BSA conjugate, and a competitive direct enzyme-linked immunosorbent assay
was developed for the rapid detection of 17beta-NT. The concentration causing 50 percent
inhibition (IC50) and the limit of detection (LOD) were found to be 0.08 and 0.0055 ng/mL,
respectively; this was better than methods previously reported that had a LOD of 2.4 ng/mL.
18
C cartridges were investigated for use in removing the effects of matrix in foods, and the
sample purification protocol was optimized. Using the developed SPE-ELISA method,
recoveries of functional food samples were obtained in the range of 71 percent to 92 percent.
Moreover, rwo kinds of antifatigue functional foods were analyzed using the established
ELISA and HPLC methods. The correlation coefficient of the results obtained using the 2
methods was greater than 0.98. Thus, the preliminary evaluation of the SPE-ELISA method
proved that it is a specific, sensitive, and precise tool that can be used for the practical
detection of 17beta-NT in various antifatigue functional food samples [09043].
Solid-phase microextraction
A fully automated, high-throughput method based on thin-film solid-phase microextraction

(SPME) and liquid chromatography-mass spectrometry was developed for simultaneous
quantitative analysis of 110 doping compounds, selected from ten classes and varying in
physical and chemical properties. Among four tested extraction phases, C18 blades were
chosen, as they provided optimum recoveries and the lowest carryover effect. The SPME
786
method was optimized in terms of extraction pH, ionic strength of the sample, washing
solution, extraction and desorption times for analysis of urine samples. Chromatographic
separation was obtained in reversed-phase model; for detection, two mass spectrometers
were used: triple quadrupole and full scan orbitrap. These combinations allowed for selective
analysis of targeted compounds, as well as a retrospective study for known and unknown
compounds. The developed method was validated according to the Food and Drug
Administration (FDA) criteria, taking into account Minimum Required Performance Level
(MRPL) values required by the World Anti-Doping Agency (WADA). In addition to analysis of
free substances, it was also shown that the proposed method is able to extract the
glucuronated forms of the compounds. The developed assay offers fast and reliable analysis
of various prohibited substances, an attractive alternative to the standard methods that are
currently used in anti-doping laboratories [13090].
Yeast analysis
The classical analytical method for detection of anabolic steroid abuse is gas
chromatography followed by mass spectrometry (GC/MS). However, even molecules with a
chemical structure typical for this class of substances, are sometimes not identified in routine
screening by GC/MS when their precise chemical structure is still unknown. A supplementary
approach to identify anabolic steroid abuse could be a structure-independent identification of
anabolic steroids based on their biological activity. To test the suitability of such a system, it
was analyzed the yeast androgen receptor (AR) reporter gene system to identify anabolic
steroids in human urine samples. Analysis of different anabolic steroids dissolved in buffer
demonstrated that the yeast reporter gene system is able to detect a variety of different
anabolic steroids and their metabolites with high specificity, including the so-called ”designer
steroid” tetrahydrogestrinone. In contrast, other non-androgenic steroids, like
glucocordicoids, progestins, mineralocordicoids and estrogens had a low potency to
stimulate transactivation. To test whether the system would also allow the detection of
androgens in urine, experiments with spiked urine samples were performed. The androgen
reporter gene in yeast responds very sensitive to 5alpha-dihydrotesto-sterone (DHT), even at
high urine concentrations. To examine whether the test system would also be able to detect
anabolic steroids in the urine of anabolic steroid abusers, anonymous urine samples
previously characterized by GCMS were analyzed with the reporter gene assay. Even when
the concentration of the anabolic metabolites was comparatively low in some positive
samples it was possible to identify the majority of positive samples by their biological activity.
In conclusion, the results demonstrated that the yeast reporter gene system detects anabolic
steroids and corresponding metabolites with high sensitivity even in urine of anabolic steroid
abusing athletes. But most importantly, a biological test system does not require knowledge
of the chemical structure of androgenic substances and therefore suitable to detect
previously unidentified substances, especially those of the class of so-called designer
steroids [08189].
Anabolic-androgenic steroids are frequently misused compounds in sports, and they belong
to the controlled substances according to the requirements of the World Anti-Doping Agency.
The classical techniques of steroid detection are mass spectrometry coupled to gas or liquid
chromatography. Biological methods that base on the ability of substances to bind the steroid
receptor are not applied in routine doping control procedures so far, but they appear to be
useful for characterization of steroid androgenic potential. In this study we used the yeast
androgen receptor reporter system (YAS), which in the past has already successfully been
787
applied to both various androgenic substances and also urine samples. Giving attention to
the androgenic potential of steroidal dietary supplements, we exemplified the analysis using
both mass spectrometry techniques and the YAS-based assay on the product "Syntrax
Tetrabol" which was a confiscated dietary supplement and marketed as a steroid precursor.
Identification, structure and the kinetic behavior of its excreted metabolites were analyzed by
NMR, GC-MS and LC-MS/MS. The androgenic potential of the parent compound as well as
its metabolites in urine was evaluated with the help of the YAS. The application of urine
samples with a previous deconjugation and the inclusion of urine density values were carried
out and led to increased responses on the YAS. Further, the possibility of a complementary
application of structure-based instrumental analysis and biological detection of androgenicity
with the help of the YAS seems to be desirable and is discussed [12084].
Yeast and mammalian cell-based androgen bioassays
Androgenic steroids marketed online as nutraceuticals are a growing concern in sport

doping. The inability of conventional mass spectrometry (MS)-based techniques to detect
structurally novel androgens has led to the development of in vitro androgen bioassays to
identify such designer androgens by their bioactivity. The objective of this study was to
determine the androgenic bioactivity of novel steroidal compounds isolated from
nutraceuticals using both yeast and mammalian cell-based androgen bioassays. We
developed two new in vitro androgen bioassays by stably transfecting HEK293 and HuH7
cells with the human androgen receptor (hAR) expression plasmid together with a novel
reporter gene vector (enhancer/ARE/SEAP). The yeast β-galactosidase androgen bioassay
was used for comparison. Our new bioassay featuring the enhancer/ARE/SEAP construct (-
S) displayed simpler assay format and higher specificity with lower sensitivity compared with
the commonly used mouse mammary tumour virus (MMTV)-luciferase. The relative
potencies (RP), defined as EC50 of testosterone of steroid, of nutraceutical extracts in the
yeast, HEK293-S, and HuH7-S, were 34, 333, and 80,000 for Hemapolin; 208, 250, and 80
for Furazadrol; 0.38, 10, and 106 for Oxyguno; 2.7, 0.28, and 15 for Trena; and 4.5, 0.1, and
0.4 for Formadrol, respectively. The wide discrepancies in rank RP of these compounds was
reconciled into a consistent potency ranking when the cells were treated with meclofenamic
acid, a nonselective inhibitor of steroid metabolizing enzymes. These findings indicate that
steroids extracted from nutraceuticals can be converted in vitro into more or less potent
androgens in mammalian but not in yeast cells. It was conclude that the putative androgenic
bioactivity of a new compound may depend on the bioassay cellular format and that
mammalian cell bioassays may have an added benefit in screening for proandrogens but
sacrifice specificity for sensitivity in quantitation [11043].
Laser desorption
New data on sample preparation and matrix selection for the fast screening of androgenic
anabolic steroids (AAS) by matrix-assisted laser desorption/ionisation time-of-flight mass
spectrometry (MALDI-TOF-MS) was presented. The rapid screening of 15 steroids included
in the WADA prohibited list using MALDI was evaluated. Nine organic and two inorganic
matrices were assessed in order to determine the best matrix for steroid identification in
terms of ionisation yield and interference by characteristic matrix ions. The best results were
achieved for the organic matrices 2-(4-hydroxyphenylazo)benzoic acid (HABA) and trans-3-
indoleacrylic acid (IAA). Good signals for all the steroids studied were obtained for
concentrations as low as 0.010 and 0.050 microg/mL on the MALDI sample plate for the
HABA and IAA matrices, respectively. For these two matrices, the sensitivity achieved by
788
MALDI is comparable with the sensitivity achieved by gas chromatography/mass
spectrometry (GC/MS), which is the conventional technique used for AAS detection.
Furthermore, the accuracy and precision obtained with MALDI are very good, since an
internal mass calibration is performed with the matrix ions. For the inorganic matrices, laser
fluences higher than those used with organic matrices are required to obtain good MALDI
signals. When inorganic matrices were used in combination with glycerol as a dispersing
agent, an important reduction of the background noise was observed. Urine samples spiked
with the study compounds were processed by solid-phase extraction (SPE) and the
screening was consistently positive [09045].
Non-target metabolomics
The molecular diversity of the human urinary metabolome is very well reflected by the
existing databases displaying thousands of metabolites classified in as much as 70 different
structural classes. The majority of these metabolites are small molecular weight compounds
having molecular weights between 100 and 800 Dalton (Da) and small peptide fragments
with very different physico-chemical properties (solubility, polarity, proton affinity, etc).
Despite this astonishing chemo-diversity, for quite some time, mainly targeted studies, often
restricted to a particular chemical family or to compounds having similar properties have
been applied into doping control studies. Moreover, the metabolites and the changes were
often regarded in an univariate manner and the correlations between them were often
disregarded. Nowadays, the progresses made in analytical fields like sample preparation,
chemical analysis and data processing offer a wider view of the metabolome and greatly
contribute to our understanding of the biochemical transformations. Among these, two are
believed to have played a key role: the introduction of high (Time of Flight mass
spectrometry, TOF) and ultra-high resolution techniques (Fourier Transform Ion Cyclotron
Resonance mass spectrometry, FT-ICR/MS) and the development of algorithms capable of
handling the thousands of signals generated by such analytical platforms. Indeed, techniques
such as FT-ICR/MS are becoming more and more available and their advantages can be
now fully exploited. Thus, molecular formulae generation based on exact masses and
relevant database search are now possible due to the high resolution and accuracy of this
type of technique. Of equal importance, the recent advances in the pre-processing,
mathematical modeling and the statistical analysis lead to more comprehensive biological
interpretation of the metabolomics data. Up to present, this strategy has been applied in a
variety of fields, including drug discovery, nutrition, toxicology, clinical trials and more
recently chemical submission. It is of particular interest in areas like doping control as new
approaches are needed to fight the ongoing development of performance-enhancing
methods. It was therefore detected differences in metabolite levels between doped athletes,
clean athletes, and volunteers (non athletes). This outcome is obtained by comparing results
of measurements from two analytical platforms: UHPLC-QTOF/MS and FT-ICR/MS. Twenty-
seven urine samples tested positive for glucocorticoids or beta-2-agonists and twenty
samples coming from volunteers and clean athletes were analyzed with the two different
mass spectrometry approaches using both positive and negative electrospray ionization
modes. Urine is a highly complex matrix containing thousands of metabolites having different
chemical properties and a high dynamic range. It was used multivariate analysis techniques
to unravel this huge data set. Thus, the several groups created were studied by Principal
Components Analysis (PCA) and Partial Least Square regression (PLS-DA and OPLS) in the
search of discriminating m/z values. The selected variables were annotated and placed on
pathway by using MassTRIX [13107].
Composite metabonomic data sets
789
It was introduce a statistical approach for integrating data from several analytical platforms. It
was illustrate this approach using 1H-13C heteronuclear multiple bond connectivity nuclear
magnetic resonance spectroscopy (1H-13C HMBC NMR) and pyrolysis metastable atom
bombardment time-of-flight mass spectrometry (Py-MAB-TOF-MS) to perform metabolic
fingerprinting on cattle treated with anabolic steroids. Multiple factor analysis (MFA)
integrates complementary aspects from NMR and MS data into a unique metabolic signature
describing the biomarkers related to the dose-response. This work also indicates that, from a
practical point of view, metabonomics and other "-omics" biotechnologies can benefit
significantly from a generalized multi-platform integrative approach using multiple factor
analysis [05027].
FCMIA
There are a range of applications that require the measurement of multiple drugs such as
urine analysis, drug determination in water, and screening for drug contamination on
surfaces. Some of the procedures used such as enzyme-linked immunosorbent assay
(ELISA) are simple but can only determine one drug at a time, and others such as GC-MS or
LC-MS are complex, time-consuming, and expensive. In one study, fluorescence covalent
microbead immunosorbent assay (FCMIA) was investigated as a simple method for the
measurement of multiple drugs simultaneously in three matrices: diluted urine, water, and on
surfaces. Five different drugs of abuse or their metabolites (methamphetamine, caffeine,
benzoylecgonine (a metabolite of cocaine), tetrahydrocannabinol (THC), the active ingredient
in marijuana, and oxycodone) were studied over the range 0-15 ng/ml. There was no
measureable cross-reactivity among the drugs at the concentrations studied. Urine dilutions
from 1/50 to 1/2.5 were studied and dilutions less than 1/20 had a significant effect on the
methamphetamine assay but limited effects on the benzoylecgonine and oxycodone assays
and almost no effect on the THC assay. For assays performed in 1/20 urine dilution, water,
and diluted surface sampling buffer, least detectable doses (LDD) were 1 ng/ml or less for
the drugs. Surfaces spiked with drugs were sampled with swabs wetted with surface
sampling buffer and recoveries were linear over the range 0-100 ng/100 cm2 surface loading
for all drugs. FCMIA has potential to be used for the measurement of multiple drugs in the
matrices studied [10443].
Lack of influence of NSAIDs
When focusing on steroid glucuronides as diagnostic parameters in doping controls, the

influence of dietary components on relevant enzymes (i.e. UDP-glucuronosyltransferases)
involved in the conjugation reactions in vivo must be considered. A variety of reports
demonstrating glucuronide inhibiting properties of pharmaceuticals (e.g. non-steroidal anti-
inflammatory drugs, NSAIDs) and ingredients of green tea or red wine were published, the
majority of which however was done in vitro. It was therefore investigated the influence of
NSAIDs (ibuprofen and diclofenac) on the renal elimination of TG and epiTG in a controlled,
randomized cross-over study with 23 male volunteers with two (n=8), one (n=7), or no (n=8)
allele of the UGT2B17 gene, thus representing the above mentioned ins/ins, ins/del, and
del/del genotype, respectively. Both the baseline T/epiT ratios as well as the T/epiT values
following an intramuscular injection of 500 mg of testosterone enanthate were not
significantly influenced by repeated maximum daily doses of the NSAIDs, which suggests
that the commonly employed steroid profile approach is not compromised by NSAID
790
In hair
In France during a famous bicycle race, the newspapers documented the degree in which
doping seemed to be supervised in some teams by managers and doctors. Use of anabolic
steroids and other substances was officially banned in the mid-seventies by sports
authorities. This policy has been enforced through urine testing before competition. It is well
known, however, that a latency period is all that is necessary to defeat these tests.
Nevertheless, hair analysis could be a promising tool when testing for periods that are not
accessible to urinalysis any more. It was developed different sensitive methods for testing
hair for amphetamines, anabolic steroids and their esters and corticosteroids. For
amphetamines, 50 mg of hair were digested with 1 M NaOH, extracted with ethyl acetate,
derivatized with TFA and analyzed by gas chromatography positive chemical-ionization mass
spectrometry. For corticosteroids, 50 mg of powdered hair were treated with methanol in an
ultrasonic bath and subsequently purified using a C18 solid phase extraction column.
Analysis was realized by high performance liquid chromatography coupled to electrospray-
ionization tandem mass spectrometry. For anabolic steroids and their esters, 100 mg of
powdered hair were treated with methanol in an ultrasonic bath for extraction of esters, then
alkaline digested with 1 M NaOH for an optimum recovery of other drugs. The two liquid
preparations were subsequently extracted with ethyl acetate, pooled, then finally highly
purified using a twin solid phase extraction on aminopropyl and silica cartridges. Residue
was derivatized with MSTFA prior to injection. Analysis was conducted by gas
chromatography coupled to a triple quadrupole mass spectrometer. Thirty cyclists were
sampled and tested both in hair and in urine. Amphetamine was detected 10 times in hair
(out of 19 analyses) compared to 6 times in urine (out of 30 analyses). Corticosteroids were
detected 5 times in hair (methylprednisolone 1 case, triamcinolone acetonide 3 cases and
hydrocortisone acetate 1 case) in hair (out of 12 analyses) compared to 12 times
(triamcinolone acetonide 10 cases and betamethasone 2 cases) in urine (out of 30
analyses). Anabolic steroids were detected twice (nandrolone 1 case, and testosterone
undecanoate 1 case) in hair (out of 25 analyses) compared to none in urine (out of 30
analyses) [00027].
Lists of banned classes of doping agents are released by the International Olympic
Committee, adopted by other sports authorities and updated regularly, including the
substance classes stimulants, narcotics, diuretics, anabolic agents, peptide hormones, beta-
blockers etc. There are different classes of restriction: anabolic and masking agents
(anabolic steroids, diuretics etc.) are always banned for athletes regardless of their topical
activity (training or competition) several substances are permitted with certain restrictions
(caffeine below a cut-off value, or inhalation of some beta 2 agonists) beta-blockers are
prohibited in competitions of certain sports disciplines the majority of the substances
(stimulants, narcotics etc.) is prohibited during competitions, so that they do not have to be
analysed in out-of-competition samples. A differentiation between training and competition
period is impossible by means of hair analysis due to the uncertainty of (especially short-
term) kinetic considerations related to hair growth. Therefore, the analytical identification of
doping relevant substances in hair is not always a sufficient criterion for a doping offence and
the identification of stimulants, beta-blockers etc. in hair would be entirely irrelevant. The
most interesting target substances are certainly the anabolic agents, because their desired
action (enhanced muscle strength) lasts longer than the excretion, leading to sophisticated
procedures to circumvent positive analytical results in competition control. Besides the
analysis of out-of-competition control samples, the long term detection of steroids in hair
could provide complementary information. An analytical approach to the identification of
791
exogenous steroids in hair requires consideration of the presence of many other steroids in
the hair matrix interfering the analysis at trace levels, and of a limited chemical stability. The
analysis of endogenous steroids in hair appears to be even more complicated, because the
possibility of many biotransformation reactions from (into) other precursors (metabolites) has
to be taken into account. Precursor substances of anabolic steroids (especially esters as
application forms) are very promising analytical targets of hair analysis, because they can
only be detected after an exogenous intake. The quantitative evaluation of active parent
compounds like testosterone (which is actively involved in physiological processes of hair
growth) in hair is still controversial. Clinical applications under reproducible conditions can be
useful, but the biovariability of these parameters will probably prevent the definition of
acceptable cut-off levels as a criterion of abuse [00028].
When positive drug results are reported, a common interpretive question posed is whether or
not it is possible to put a quantitative finding into context. A standard answer to this inquiry is
that a positive hair testing result can be interpreted as meaning that the donor has chronically
or repetitively used the drug identified in the hair, but that chronic or repetitive are not defined
in the same way for all individuals. The Society of Hair Testing published on June 16, 1999, a
consensus opinion on the use of hair in doping situations. However, although accepted in
most courts of justice, hair analysis is not yet recognised by the International Olympic
Committee. To be considered as a valid specimen for doping control, some issues still need
to be addressed. The scientific community has demonstrated significant concern over the
proper role that hair drug testing should serve in toxicological applications. Among the
unanswered questions, five are of critical importance: (1) What is the minimal amount of drug
detectable in hair after administration? (2) What is the relationship between the amount of
the drug used and the concentration of the drug or its metabolites in hair? (3) What is the
influence of hair color? (4) Is there any racial bias in hair testing? (5) What is the influence of
cosmetic treatments? The present report documents scientific findings on these questions,
with particular attention to the applications of hair in doping control [00029].
The actual antidoping control rules applied in sports (as established by the International
Olympic Committee and the International Sport Federations) state that a positive case is
chemically established by the unequivocal detection of a forbidden parent molecule and/or
any of its metabolite(s) in urine, no matter the amounts which were administered and when
the drug was taken. Screening is accomplished most of the time by using GC-MS
procedures. These have been optimized to detect most if not all of the forbidden compounds
which are put on a list. Recently, attempts have been made on scalp hair to demonstrate the
value of this matrix as a possible means for differentiating between therapeutic use and
doping abuse. In particular, GC-mass selective detector and GC-high resolution MS were
successfully applied to treated animals and body-builders for anabolic agents (steroids and
beta-2-agonists) at high sensitivity detection (low ng/g level). Naturally occurring molecules,
like testosterone and its metabolites, could also be differentiated from their synthetic
counterparts. Positive cases are more often challenged in courts and retrospectivity in time of
the drug(s) intake is becoming an important issue for evaluating the responsibility of the
person. This is can be based on hair analyses if the drugs have been taken at regular
intervals. Stimulants and narcotics are often used in sports like drug of abuse in the ordinary
social contexts. On the other hand, anabolic agents, when taken to improve the physical
performances, follow complex regimens with the mixing of various formulas and dosages.
Scalp hair references ranges for these as well as for endogenous substances still wait to be
established statistically for competing, well-trained athletes. The incorporation rate into blond
or gray hair is poorer than that of dark colored hair raising the question of individuals’ equality
against the controls, a very important matter of concern for the sport's governing bodies. The
frequency of hair cutting and short hair cuts necessary to gain speed in specific sports like
swimming are other critical factors. On the other hands, irregular hair growth, associated with
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the washout effect through multiple washing and staining processes over expanded time
intervals can cause concentrating or diluting effects. So far, a minority of prohibited
substances could be detected in scalp hair with the sensitivity and specificity required in the
context of the sport's activities. From the above, clear limitations of the usefulness of hair
analysis in doping control analysis are obvious until a lot more data relevant to this particular
field have been collected [00030].
Hair analysis is a complex problem. Hair is a solid tissue which biology and physiology have
not been understood in all details yet. It is an annex of skin, originated from the hair follicle in
which the germination centre is formed by matrix cells. The matrix cells give rise to the
different layers of the hair shaft, including cuticle, cortex and medulla. In the root, cells are in
active proliferation, whereas within the hair shaft above the skin the metabolism is negligible.
The most important components of hair are fibrous proteins (keratins), melanins and lipids.
Hair follicles are located 3-4 mm below the surface of the skin and are surrounded by rich
blood capillary system. Three glands are associated with the hair follicle – the apocrine,
sebaceous and sweat ones. The secretions of the first two glands bathe the hair shaft in the
follicle and the one of the sweat gland bathe it above the surface of the skin. Hair grows in
cycles: the anagen (active growing stage), the catagen (transition stage) and telogen (resting
stage). The individual length of hair depends on the mutual duration of these stages and on
the growth rate. Average values for the anagen stage in human are 4-8 years, the catagen a
few weeks, and the telogen stage 4-6 months. The scalp hair growth rate is reported to be in
the range 0.6-1.4 cm per month in general. There are significant differences both in the
proportions anagen/telogen hair and both in the growth rate between hair from various
anatomical part of the body. Both parameters are dependent on race, gender, age, and
health stage. On the scalp of an adult, the approximately 85 percent of the hair is in the
growing phase (anagen) and the remaining 15 percent is in a resting phase (telogen). The
consequence of the cyclic hair growth is the nonhomogenity of the hair bunch at the
horizontal level, at a certain distance from the skin [05026].
Because of its peculiar characteristics, hair analysis provides a way of obtaining information
that cannot be acquired by other commonly used forensic medical analyses, such as blood
or urine analysis. In the keratin matrix many xenobiotics are incorporated permanently, in
contrast to the situation with blood or urine where they are generally only detectable for a few
hours or days. Therefore hair analysis should be the method of choice in the clinical and
forensic toxicology field when the assessment of repeated or chronic exposure to a drug is
required, e.g. in the case of criminal responsibility, revocation/restoration of a driving licence
or in workplace testing. Some factors that can affect the concentrations of drugs in hair, such
as passive contamination, age, ethnicity and cosmetic treatment, must be considered.
Analytical methodology is also very important: GC/MS/MS has proved to be a highly
sensitive and specific technique for the detection of very low concentrations of such drugs in
hair. In this study five cases of the application of hair analyses using this technique for the
determination of abused drugs (opiates, cocaine, amphetamine, anabolic steroids) are
described [05067].
Using hair as a medium to analyze drug use has been receiving increased attention during
years because of less embarrassing circumstances of collection and because hair does not
decompose like other body fluids or tissues. Hair testing offers also a wider detection window
after drug exposure than urine testing. The heavy metals were the first toxic substances
which could be analyzed in hair matrix by means of atomic absorption spectroscopy to
document the exposure in the past time. Later on, gradual development of analytical
technologies, offering methods with sufficient sensitivity, enabled hair analysis also for
organic substances. In 1979, it was extracted opiates from hair of heroin user and analyzed
the final buffer extract with Abuscreen RIA. The concentration along the hair shaft differed
793
and corresponded with the time of drug intake. After tenths years of scientific research the
analytical part itself has reached a general standard. Preliminary immunochemical tests for
selected drug groups may be accepted as the first step. GC-MS is a method of choice in
general practice and various tandem mass spectrometry methods (GC-MS-MS or LC-MS-
MS) are used for targeted analyses of low dosed compounds (e. g. fentanyl, buprenorphine,
flunitrazepam), or for detection of some important specific metabolites present in trace
concentrations in hair (e. g. carboxy metabolite of delta-9-tetrahydrocannabinol), or detection
single doses in previous time. Over more than 20 years hair analysis for drugs has been
gaining increasing attention and recognition in various toxicological fields as preemployment
and employment screening, forensic sciences, doping control of banned substances, clinical
diagnostics in health problems. Hair analysis for drugs can expand the toxicological
examination of conventional materials and thus contribute with additional important
information to the complex evaluation of a certain case. Hair is a unique material for the
retrospective investigation of chronic drug consumption, intentional or unintentional chronic
poisoning in criminal cases, gestational drug exposure or environmental exposure to
pollutants and adulterants and with specific ultrasensitive procedures allow to demonstrate
even a previous single dose administration in a very low amount. Assuming the ideal hair
steady and uniform growth, segmental hair analysis can provide the information about the
time course of the substance use or exposure. However, the physiological background of
hair growth, mechanisms of drug incorporation are not simple, not yet understood in full
details and need not be evaluated exactly in all cases. The hair sampling, storage, sample
preparation, analytical performance themselves are also very important for final results.
Different laboratory attitudes can produce different results. The full information on
circumstances of the case examined must be taken into account during interpretation. The
pitfalls in hair analysis should be known and avoided to assure the responsible and correct
interpretation of laboratory results adequate to an individual case [05026].
The precise mechanisms involved in the incorporation of drugs into hair have not been
clarified completely and more research is still necessary. The ideal model assumes that
drugs or chemicals enter hair by passive diffusion from blood capillaries into the growing
cells at the base of hair follicle. However, experimental data indicate that drugs may enter
hair in different locations and in different times from different sources by various
mechanisms. The drugs can be transported from blood and also from deep skin
compartments not only into hair growing cells but with some time delay also into
keratogenous zone during hair shaft formation. The other mechanisms are diffusion from
sweat or sebum secretions. A contamination from external environment need not be
excluded on the hair surface exiting the skin. This multicompartment model has been
demonstrated in details. The three key factors which influence the drug incorporation into
hair are melanin content in a hair, lipophilicity and basicity of a drug substance. The
physicochemical properties of drugs, lipophilicity and basicity related to molecular structure
clearly affect the drug incorporation into hair and on the other side, hair structure and its
colour plays a very important role too. The pH of melanocytes is between 3 and 5 and
significant melanin affinity for basic drugs has been demonstrated in several experimental
studies both with animals and humans or in vitro. It was confirmed that drug concentration in
pigmented hair was much higher than in blond or grey hair after the same dosage. The
second important factor is the polarity of a drug or its metabolite. It has been many times
documented that more polar metabolites benzoylecgonine, morphine or amphetamine enter
the hair in a lesser extent than their more lipophilic precursors cocaine or 6-
monoacetylmorphine or methamphetamine [05026].
The acidity of basicity of a drug substance is the third important factor. The matrix of hair is
more acidic than blood pH 7.4, therefore the resulting pH gradient is more convenient for
transfer of bases than for neutral molecules or acids. For example the acidic carboxy
794
metabolite of delta-9-tetrahydrocannabinol enters the hair only in tiny traces. All aspects of
bioavailability and disposition of drugs in hair must be taken into account in evaluation of a
real case. The genetic context, the age, gender and health conditions have been discussed
with the capability of drug deposition in the inner space of hair. The retention and stability of
drugs in hair is considered to be good, nevertheless it can be affected by cosmetic
treatments as bleaching or dyeing and permanent wave application. Cocaine was less
affected than morphine or 6-monoacetylmorphine. The extent of concentration decline of a
drug is dependent on the value prior to cosmetic treatment and also on individual hair matrix.
The long term effects of wheather (sunshine, rain, wind) may cause the damage of hair shaft
with impacts to changes of concentration in hair. The findings indicate that not only the
stability of a particular drug is important but also the influence of UV light and water on the
hair pigment. In case of long hair, above all the structure of distal part could be damaged and
its analysis should be avoided. Continuous advances in analytical technologies have been
resulting in lowering detection limits of analytical methods, improving their accuracy and thus
allowing better scientific understanding and interpretation of test data. This will influence their
acceptance as useful and objective tool of evidence or important information for subsequent
measures with impacts to an individual. So far the target drugs in the analysis are mostly
typical drugs of abuse. With modern laboratory facilities, lower and lower quantities will be
detectable in hair and thus some other harmful substances will be of analytical interest.The
weak points of nowadays hair analysis are well known and should be considered [05026]:
- it is difficult to prepare reference hair standards containing accurate concentration of

drugs which are necessary for calibration
- the question of efficiency of drug extraction from solid matrix is very important and
this parameter need to be evaluated for each type of drug in every laboratory. The
standardization of decontamination and extraction procedures is also desirable
- minimal performance standards should be kept in different laboratories to assure
interlaboratory comparability of test results. A sufficient LOD values, comparable cut-
off values will support correct identification of drugs and metabolites in hair
At present state of knowledge, the data of hair analysis are rather of semiquantitative value
and in segmental analysis the data have mutual relative character. The interpretation on time
and dose from results of segmental analysis may not be possible in full exact details and
must be careful considering many aspects of the individual case. Frequency of drug
consumption need not to be quite known and lag time between consumption time and drug
appearance in hair above the skin may be variable from a person to a person. Variability in
hair rate growth, multiple mechanisms of drug incorporation, role of hair pigmentation,
stability and retention of drugs in hair under cosmetic treatment – all these factors and
related phenomena are to be taken into account during interpretation. There are still
remaining unresolved questions in hair analysis and more scientific understanding and
further research are necessary in this developing technology. Hair analysis may be useful in
any situation in which the history of past rather than recent drug intake is taken into account
[05026].
Alternative matrices are steadily gaining recognition as biological samples for toxicological
analyses. Hair presents many advantages over traditional matrices, such as urine and blood,
since it provides retrospective information regarding drug exposure, can distinguish between
chronic and acute or recent drug use by segmental analysis, is easy to obtain, and has
considerable stability for long periods of time. For this reason, it has been employed in a
wide variety of contexts, namely to evaluate workplace drug exposure, drug-facilitated sexual
assault, pre-natal drug exposure, anti-doping control, pharmacological monitoring and
alcohol abuse. In this article, issues concerning hair structure, collection, storage and
analysis are reviewed. The mechanisms of drug incorporation into hair are briefly discussed.
795
Analytical techniques for simultaneous drug quantification in hair are addressed. Finally,
representative examples of drug quantification using hair are summarized, emphasizing its
potentialities and limitations as an alternative biological matrix for toxicological analyses
[13108].
The detection of a single drug exposure in hair (doping offence, drug-facilitated crime) is
based on the presence of the compound of interest in the segment corresponding to the
period of the alleged event. However, in some cases, the drug is detected in consecutive
segments. As a consequence, interpretation of the results is a challenge that deserves
particular attention. Literature evaluation and data obtained from the 20-year experience in
drug testing in hair of the author are used as the basis to establish a theory to validate the
concept of single exposure in authentic forensic cases where the drug is detected in 2 or 3
segments. The gained experience recommends to wait for 4-5 weeks after the alleged event
and then to collect strands of hair. Assuming normal hair growth rate (1 cm/mo), it is
advisable to cut the strand into 3 segments of 2 cm to document eventual exposure.
Administration of a single dose would be confirmed by the presence of the drug in the
proximal 2 cm segment (root), whereas not detected in the 2 other segments. However, in
the daily experience of the author, it was noticed that sometimes (about 1 case from 10
examinations), the drug can be detected in 2 or 3 consecutive segments. Such a disposition
was even observed in volunteer experiments in the literature. As it was also described for
cocaine in early 1996, there is considerable variability in the area over which incorporated
drug can be distributed in the hair shaft and in the rate of axial distribution of drug along the
hair shaft. This can explain why a small amount of drug, as compared with the concentration
in the proximal segment, can be measured in the second segment, as a result of an irregular
movement. Another explanation for broadening the band of positive hair from a single dose is
that drugs and metabolites are incorporated into hair during formation of the hair shaft via
diffusion from sweat and other secretions. The presence of confounding interferences in the
hair matrix or changes in the hair structure due to cosmetic treatments might mislead the
final result of hair analysis. To qualify for a single exposure in hair, the author proposes to
consider that the highest drug concentration must be detected in the segment corresponding
to the period of the alleged event (calculated with a hair growth rate at 1 cm/mo) and that the
measured concentration be at least 3 times higher than those measured in the previous or
the following segments. This must only be done using scalp hair after cutting the hair directly
close to the scalp [13109].
In recent years hair has become a fundamental biological specimen, alternative to the usual
samples blood and urine, for drug testing in the fields of forensic toxicology, clinical
toxicology and clinical chemistry. Moreover, hair-testing is now extensively used in workplace
testing, as well as, on legal cases, historical research etc. This article reviews methodological
and practical issues related to the application of hair as a biological indicator of drug
use/abuse or of chronic exposure to environmental toxicants. Hair structure and the
mechanisms of drug incorporation into it are commented. The usual preparation and
extraction methods as well as the analytical techniques of hair samples are presented and
commented on. The outcomes of hair analysis have been reviewed for the following
categories: drugs of abuse (opiates, cocaine and related, amphetamines, cannabinoids),
benzodiazepines, prescribed drugs, pesticides and organic pollutants, doping agents and
other drugs or substances. Finally, the specific purpose of the hair testing is discussed along
with the interpretation of hair analysis results regarding the limitations of the applied
procedures [06044].
The monitoring of anabolic steroid residues in hair is undoubtedly one of the most efficient
strategies to demonstrate the long-term administration of these molecules in meat production
animals. A multi-residue sample preparation procedure was developed and validated for 28
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steroids. A 100 mg hair sample was grinded into powder and extracted at 50 degrees C with
methanol. After acidic hydrolysis and extraction with ethyl acetate, phenolsteroids, such as
estrogens, resorcyclic acid lactones and stilbens in one hand, are separated from androgens
and progestagens in the other hand. Solid phase extractions were performed before applying
a specific derivatisation for each compound sub-group. Detection and identification were
achieved using gas chromatography-tandem mass spectrometry with acquisition in the
selected reaction monitoring mode after electron ionisation. The method was validated
according to the 2002/657/EC guideline. Decision limits (CCalpha) for main steroids were in
the 0.1-10 microg/kg range [06045].
Given the limitations of self-reports on drug use, testing for drugs of abuse is important for
most clinical and forensic toxicological situations, both for assessing the reality of the
intoxication and for evaluation of the level of drug impairment. It is generally accepted that
chemical testing of biological fluids is the most objective means of diagnosis of drug use. The
presence of a drug analyte in a biological specimen can be used to document exposure. The
standard in drug testing is the immunoassay screen, followed by the gas chromatographic-
mass spectrometric confirmation conducted on a urine sample. In recent years, remarkable
advances in sensitive analytical techniques have enabled the analysis of drugs in
unconventional biological specimens such as hair. The advantages of this sample over
traditional media, like urine and blood, are obvious: collection is noninvasive, relatively easy
to perform, and in forensic situations it may be achieved under close supervision of law
enforcement officers to prevent adulteration or substitution. The window of drug detection is
dramatically extended to weeks, months or even years when testing hair. It seems that the
value of alternative specimen analysis for the identification of drug users is steadily gaining
recognition. This can be seen from its growing use in preemployment screening, in forensic
sciences, in clinical applications and for doping control. Hair analysis may be a useful adjunct
to conventional drug testing in urine. Methods for evading urinalysis do not affect hair
analysis. The aim of one review was to document toxicological applications of hair analysis in
drug detection [06046].
The abuse of esters of natural androgenic steroids in cattle fattening and sports is hard to
control via routine urine testing. The esters are rapidly hydrolysed in vivo into substances
which are also endogenously present in urine. In veterinary control strange findings of
17beta-testosterone and 17alpha-testosterone in urine are often ignored because of the lack
of statistically sound reference data of naturally occurring levels. An interesting alternative for
inconclusive urine analyses in veterinary control can be provided by the analysis of the
administered steroids themselves, i.e. the analysis of intact steroid esters in hair.
Unfortunately, the analysis of intact steroid esters is complicated not only by the vulnerability
of the esters which precludes alkaline hydrolysis of the hair, but also by the wide polarity
range of short and long-chain esters yielding very poor recoveries for either the one or the
other. In one study, a multi-steroid esters LC/MS/MS screening method is presented for trace
analysis of the synthetic intact esters of 17beta-testosterone and the undecylenate ester of
17beta-boldenone in bovine hair. The method, requiring only 200 mg of pulverised hair,
features a mild digestion procedure using tris(2-carboxyethyl)phosphine hydrochloride
(TCEP) and the use of four deuterium-labelled steroid esters as internal standards covering
the wide polarity range of the analytes. In spiked hair samples for most of the analytes the
limit of detection and the accuracy using isotope dilution were 2-5 ng/g and 97-105 percent,
respectively. The applicability was demonstrated using hair samples from a controlled
experiment in which six bovines were injected intramuscularly with two different doses of two
commercial mixtures of testosterone esters, and with two different doses of boldenone
undecylenate. Depending on the dose all administered testosterone- and boldenone esters
were found to be incorporated in bovine hair following a single intramuscular injection, except
testosterone propionate which dose might have been too low [06047].
797
Sensitive, specific, and reproducible methods for the quantitative determination of eight
anabolic steroids in guinea pig hair have been developed using LC/MS/MS and GC/MS/MS.
Methyltestosterone, stanozolol, methandienone, nandrolone, trenbolone, boldenone,
methenolone and DHEA were administered intraperitoneally in guinea pigs. After the first
injection, black hair segments were collected on shaved areas of skin. The analysis of these
segments revealed the distribution of anabolic steroids in the guinea pig hair. The major
components in hair are the parent anabolic steroids. The time courses of the concentrations
of the steroids in hair (except methenolone, which does not deposit in hair) demonstrated
that the peak concentrations were reached on days 2-4, except stanozolol, which peaked on
day 10 after administration. The concentrations in hair appeared to be related to the
physicochemical properties of the drug compound and to the dosage. These studies on the
distribution of drugs in the hair shaft and on the time course of their concentration changes
provide information relevant to the optimal time and method of collecting hair samples. Such
studies also provide basic data that will be useful in the application of hair analysis in the
control of doping and in the interpretation of results [09044].
Doping control of anabolic substances is normally carried out with urine samples taken from
athletes and horses. Investigation of alternative specimens, e.g. hair samples, is restricted to
special cases, but can also be worthwhile, in addition to urine analysis. Moreover, hair
material is preferred in cases of limited availability or complicated collection of urine samples,
e.g. from horses. In this work, possible ways of interpretation of analytical results in hair
samples are discussed and illustrated by practical experiences. The results demonstrate the
applicability of hair analysis to detect anabolic steroids and also to obtain further information
about previous abuse. Moreover, the process of incorporation of steroids into hairs is
described and the consequences on interpretation are discussed, e.g. on the retrospective
estimation of the application date. The chosen examples deal with the detection of the
anabolic agent testosterone propionate. Hair samples of an application study, as well as a
control sample taken from a racing horse, were referred to. Hair material was investigated by
a screening procedure including testosterone, nandrolone and several esters (testosterone
propionate, phenylpropionate, decanoate, undecanoate, cypionate; nandrolone decanoate,
dodecanoate and phenylpropionate; limits of detection (LODs) between 0.1 and 5.0 pg/mg).
Confirmation of testosterone propionate (LOD 0.1 pg/mg) was carried out by an optimised
sample preparation. Trimethylsilyl (TMS) and tert-butyl dimethylsilyl derivatives were
detected by gas chromatography-high-resolution mass spectrometry (GC-HRMS) and gas
chromatography-tandem mass spectrometry (GC-MS/MS) [08190].
Beside the traditionally used body-fluids, defining the abuse-material by the use of hair
samples is more and more widespread in the forensic toxicological practice. Using the hair
allows the rectrospective examination of the abuse-material, and due to the sensitive
measuring technics, even one-time use can be proven. A further possibility is the segment-
analysis which allows investigation of the abuse-history retroactive for months depending on
the length of the hair. The quantitative parameters of the abuse can not always be estimated
precisely since the details of the build-up in the hair are complicated and are not clear even
today. Furthermore, the sampling, sample preparation and the measuring method will all
influence the results. One paper reviewsedthe opiates, cocain, amfetamin derivatives,
cannabinoids, alcohol-consumption markers and the frequently found drugs in the forensic
toxicology as determined by using hair samples [10430].
Hair testing is a convenient, tamper resistant and non-invasive technique for the analysis of
many controlled drugs and drugs of abuse as compared to blood tests and urinalysis. Hair
testing can be used to screen for the parent drug and for metabolites and could be used to
complement urinalysis. In addition to urine tests, for the past three decades hair analysis has
798
been employed to detect chronic social drug use and in the fight against doping in sport.
Longer term histories of drug use can be detected as hair grows by approximately 1 cm per
month. Hair analysis provides a wider window of detection thus hair samples of a few
centimetre lengths will provide more accurate information of drug use than blood and urine
and it has good potential for out of competition doping. Most controlled drugs and drugs of
abuse are integrated into the hair matrix in a number of ways: (i) an endogenous-exogenous
pathway; transfer or absorption of drug molecules into hair duct in the form of sweat and
sebum from transdermal secretion, or (ii) by an endogenous pathway; the drug molecules
diffuse into growing hair from circulatory system using passive transport. Dosage,
bioavailability, physiochemical properties and pharmacokinetics also affect the process of
drug incorporation into the hair matrix. The concentration of drug detected in hair is also
influenced by an individual’s metabolic pathway, cosmetic treatments and hair pigmentation.
Hair also favours the incorporation of undissociated basic drugs due to carrying no net
charge due to an isoelectric pH of about 6. Furthermore, due to the less polar nature of the
parent drugs, its incorporation into the keratin matrix is favoured in contrast to their
metabolites. While in urine it is the other way around and relatively low concentrations of
parent drugs are excreted in urine as compared to their metabolites. Doping in sport both in
and out of competition is a persistent problem and therefore it is highly desirable to have a
high throughput multi-analyte mass spectrometry method for the detection of controlled drugs
and drugs of abuse in hair. Gas chromatography-mass spectrometry (GC-MS), requires
additional derivatisation and lacks the required sensitivity to simultaneously detect large
numbers of drugs in hair samples. Liquid chromatography-mass spectrometry (LC-MS/MS)
when used in multiple reaction monitoring (MRM) mode has been a powerful tool for
detecting and confirming the presence of drugs in complex biological matrices. Recently,
researchers have reported the hybrid triple-quadrupole mass spectrometer (QTrap) to screen
and confirm 300, 100 and 88 drugs in human blood samples, respectively. While others
reported a 3200 Q Trap(R) LC-MS/MS system for analysis of 700 and 301 drugs in serum
and urine samples. A set of over 500 negative-ion MS-MS spectra was collected from three
libraries applied in screening and systematic toxicological analysis is also reported.
Moreover, the mass spectra characteristics of more than 2,500 illegal drugs and metabolites
have also been measured in urine and plasma using hybrid quadrupole time-of-flight mass
spectrometry (LC-QTOF-MS). All of the above techniques are limited to human blood or
urine samples. Furthermore, the LOD for most of these drugs were not suitable for doping
control purpose as the methods of detections were not sensitive enough. However, there is
an analytical method of metabolomic approach used for hair analysis using time of flight
detector (TOF) and high resolution mass spectrometry (HRMS), which is limited to metabolite
analysis and not the actual compounds and hence not widely used. An UPLC-TOF-MS
method for simultaneous screening and quantification of 52 drugs in hair was also developed
and validated but the analysis of drugs were limited and only 15 autopsy hair samples were
tested using this method [14707].
Considerable efforts are being extended to develop more effective methods to detect drugs
in forensic science for applications such as preventing doping in sport. The aim of one study
was to develop a sensitive and accurate method for analytes of forensic and toxicological
nature in human hair at sub-pg levels. The hair test covers a range of different classes of
drugs and metabolites of forensic and toxicological nature including selected anabolic
steroids, cocaine, amphetamines, cannabinoids, opiates, bronchodilators, phencyclidine and
ketamine. For extraction purposes, the hair samples were decontaminated using
dichloromethane, ground and treated with 1 M sodium hydroxide and neutralised with
hydrochloric acid and phosphate buffer and the homogenate was later extracted with hexane
using liquid-liquid extraction (LLE). Following extraction from hair samples, drug-screening
employed liquid chromatography coupled to tandem mass spectrometric (LC-MS/MS)
analysis using dynamic multiple reaction monitoring (DYN-MRM) method using proprietary
799
software. The screening method (for > 200 drugs/metabolites) was calibrated with a tailored
drug mixture and was validated for 20 selected drugs for this study. Using standard additions
to hair sample extracts, validation was in line with FDA guidance. A Zorbax Eclipse plus C18
(2.1 mm internal diameter × 100 mm length × 1.8 μm particle size) column was used for
analysis. Total instrument run time was 8 minutes with no noted matrix interferences. The
LOD of compounds ranged between 0.05-0.5 pg/mg of hair. 233 human hair samples were
screened using this new method and samples were confirmed positive for 20 different drugs,
mainly steroids and drugs of abuse. This was the first report of the application of this
proprietary system to investigate the presence of drugs in human hair samples. The method
is selective, sensitive and robust for the screening and confirmation of multiple drugs in a
single analysis and has potential as a very useful tool for the analysis of large array of
controlled substances and drugs of abuse [14707].
Hair color as a potential biasing factor in hair analysis
It was reviewed eight different data sets in this paper for the purposes of assessing the
possibility that reported color of hair can produce a systematic bias in the interpretation of
hair assays. We review studies or data sets that include heroin and its metabolites, cocaine
and its metabolites, MDMA and its analogs, and amphetamine and methamphetamine. The
studies have utilized a variety of different degrees of color categorization, ranging from the
simple dichotomy of brown and black, to a high of 12 categories. The mean number of
categories reported approaches 6 (mean = 5.875). There are a total of 2791 data points in
this analysis. We utilize two major statistical techniques for assessing significance; one-way
analysis of variance, and Tukey's Honestly Significant Difference procedure. In
circumstances were only dichotomous contrasts are possible, one-way analysis of variance
is used. In contrasts involving three or more categorical groups, Tukey's procedure is used.
In circumstances where the homogeneity of group variances is not sustained by the Levene
statistic, we use the Tamahane procedure, allowing an assessment that assumes unequal
variances. The analysis of this data fails to discern a significant color effect. We speculate
that it may be that variance is large in many domains affecting analyte recovery from hair. In
large groups these variations tend to regress towards a typical or mean value. Thus the data
here show that while there are group or aggregate differences in these 'typical' values, they
are not great when considered in relation to the within-group variations which exist for those
values. It is the view that color may play a role in the accumulation of drugs in hair, however
it is likely to account for only a very small part of the complex process of drug accumulation
[00031].
Trace elements
The primary aim of one study is to estimate the effect of different physical activity levels on
hair trace element content in male and female students. A total of 113 students (59 women
and 54 men) at a university took part in the current investigation. According to the level of the
physical activity, all students were divided into three groups: high, medium, and low physical
activity. Essential and toxic metal content (microg/g) in hair samples was assessed by
inductively coupled plasma mass spectrometry using NexION 300D + NWR213 (Perkin-
Elmer, USA). The obtained data show that hair iodine, zinc, arsenic, nickel, and tin levels are
not related to physical activity in male and female students. At the same time, increased
physical activity is associated with decreased hair copper, vanadium, bismuth, and mercury
content in comparison to the low physical activity groups. Students with higher physical
activity are also characterized by significantly higher hair cobalt, iron, manganese, selenium,
cadmium, lithium, and lead concentrations. Finally, statistical analysis has revealed maximal
gender differences in hair trace element content in the high physical activity groups, whereas
800
in the low activity groups, the hair metal concentrations were nearly similar in females and
males [14742].
Children
Hair testing was used to investigate the prevalence of unsuspected exposure to drugs of
abuse in a group of children presenting to an urban paediatric emergency department
without suggestive signs or symptoms. Hair samples were obtained from 114 children
between 24 months and 10 years of age attending the emergency room of Hospital del Mar
in Barcelona, Spain. Hair samples from the accompanying parent were also collected. The
samples were analyzed for the presence of opiates, cocaine, amphetamines, and
cannabinoids by ultra-performance liquid chromatography-tandem mass spectrometry.
Parental sociodemographics and possible drug of abuse history were recorded. Hair samples
from twenty-three children (20 %) were positive for cocaine (concentration range 0.15-3.81
ng/mg hair), those of thirteen children (11 %) to cannabinoids (D9-THC concentration range
0.05-0.54 ng/mg hair), with four samples positive to codeine (0.1-0.25 ng/mg hair), one
positive for 2.09 ng methadone per mg hair and one to 6-MAM (0.42 ng/mg hair) and
morphine (0. 15 ng/mg hair). In 70 and 69 percent of the positive cocaine and cannabinoids
cases respectively, drugs was also found in the hair of accompanying parent. Parental
sociodemographics were not associated with children exposure to drugs of abuse. However,
the behavioural patterns with potential harmful effects for the child's health (e.g. tobacco
smoking, cannabis, benzodiazepines and/or antidepressants use) were significantly higher in
the parents of exposed children. In the light of the obtained results (28 % overall children
exposure to drugs of abuse) and in agreement with 2009 unsuspected 23 percent cocaine
exposure in pre-school children from the same hospital, it was supported general hair
screening to disclose exposure to drugs of abuse in children from risky environments to
provide the basis for specific social and health interventions [14037].
Hair and saliva
It is generally accepted that chemical testing of biologic fluids is the most objective means of
diagnosis of drug use. The presence of a drug analyte in a biologic specimen can be used to
document exposure. The standard for drug testing in toxicology is an immunoassay screen
conducted on a urine sample, followed by confirmation by gas chromatography with mass
spectrometric detection. In recent years, remarkable advances in sensitive analytic
techniques have enabled the analysis of drugs in unconventional biologic specimens such as
saliva or hair. The aim of one review was to document the current status of drugs of abuse
testing in saliva and some doping agents in hair. The influence on drug concentration of the
procedure of saliva sampling is described. Screening procedures along with specific methods
are reviewed for the determination of amphetamines, cannabis, cocaine, and opiates in
saliva. Before an extensive review on the detection of anabolics, corticosteroids, and beta-
adrenergic stimulants in hair, the place of this specimen in doping control is discussed, with a
focus on the potential problems of this new technology [02021].
Alternative specimens (e.g. hair and saliva) are well established in forensic toxicology and
provide significant benefits as noninvasive, inexpensive alternatives to blood with access to
improved long-term retrospection. Based on these experiences, the question of potential
applications and limitations of alternative specimens in doping control arose. Compounds
prohibited at all times (e.g. clenbuterol, beta2 agonists, estrogen-receptor modulators) may
be successfully tested and clearly interpreted in alternative specimens. In contrast,
prohibition of certain compounds in sport are limited to time ranges (e.g., stimulants are only
prohibited in-competition), dosages or administration routes (e.g. systemic uptake of
801
glucocorticosteroids). This cannot be properly differentiated by semiquantitative tests (e.g.
hair analyses), but may be distinguished in saliva. Similarly, proof of external administration
of endogenous steroids (e.g. testosterone) only seems to be achievable by quantitative
analysis of saliva. Moreover, the retrospective monitoring of the relevance of social drugs or
upcoming (unapproved) substances represents promising applications of hair tests in doping
control [12086].
The influence on drug incorporation of melanin affinity, lipophilicity, and membrane

permeability is of paramount importance. Despite their high lipophilicity, some drugs have
quite low incorporation rate into hair, suggesting that the higher incorporation rates of basic
drugs (cocaine, amphetamines.) than neutral (steroids, benzodiazepines, cannabinoids…) or
acidic ones are strongly related to the penetrating ability of the drug to break through the
membrane based on the pH gradient between blood and the acidic hair matrix. When using
hair analysis as a matrix during investigative analysis, e.g. workplace drug testing, doping,
driving under the influence, drug-facilitated crime, the question of importance is to know
whether the analytical procedure was sensitive enough to identify traces of drugs; this is
particularly important when the urine sample(s) of the subject was positive and the hair
sample(s) was negative. It has been accepted in the forensic community that a negative hair
result cannot exclude the administration of a particular drug, or one of its precursors and the
negative findings should not overrule a positive urine result. Nevertheless, the negative hair
findings can, on occasion, cast doubt on the positive urine analysis, resulting in substantial
legal debate and various consequences for the subject. The concept of minimal detectable
dosage in hair is of interest to document the negative findings, but limited data is currently
available in the scientific literature. Such data includes cocaine, codeine, ketamine, some
benzodiazepines and some unusual compounds. Until laboratories will have sensitive
enough methodologies to detect a single use of drug, care should be taken to compare urine
and hair findings [12087].
In saliva
On-site sample preparation is an analytical approach based on direct sampling from the
system under investigation. It has the advantage of combining sampling and sample
preparation into a single step, thus generally is fast, minimizes the potential sources of error
and eliminates the risks for analytes instability. For such analysis solid phase microextraction
in thin film geometry (TF-SPME) can provide robust and convenient in vivo sampling, offering
in the same time faster analysis and higher extraction recovery (i.e. better sensitivity) due to
large surface to volume ratio. In this study, TF-SPME in coated blade and membrane formats
with a single extraction phase were used for in vivo and ex vivo saliva extraction and
separation by LC and GC, respectively. Due to applicability for wide range of polarity of
analytes as well as thermal and solvent stability during the desorption, hydrophilic lipophilic
balanced particles (HLB) were chosen as extraction phase and used for fast (5 min) in vivo
and ex vivo sampling. The results of metabolomic profiling of the saliva are indicating that
even 5min in vivo sampling using TF-SPME followed by GC and LC analyses provides
complementary coverage of wide range of analytes with different physical and chemical
properties. To demonstrate the applicability of the method for doping analyses, the SPME-
LC-MS/MS method was validated for simultaneous quantification of 49 prohibited substances
with limit of quantification (LOQ) ranging between 0.004 and 0.98 ng/mL. Moreover, the
method was also validated and successfully applied for determination of endogenous
steroids in saliva where the concentrations of the analytes are substantially low. The
developed assay offers fast and reliable multiresidue analysis of saliva as an attractive
alternative to the standard analysis methods [14743].
802
Finger nails
In an attempt to obtain alternative doping control matrices, the utility of fingernails as a

source of keratinaceous samples (comparable to hair) have been evaluated concerning
testosterone, testosterone propionate, and stanozolol. Although the study demonstrated the
incorporation of steroidal agents at the proximal nail fold and nail bed, the approach failed to
provide the required sensitivity and most likely also the viability in an authentic doping control
setting [12016].
In sweat
Sweat is a biofluid with present scant use as clinical sample. This review tries to demonstrate
the advantages of sweat over other biofluids such as blood or urine for routine clinical
analyses and the potential when related to metabolomics. With this aim, critical discussion of
sweat samplers and equipment for analysis of target compounds in this sample is made.
Well established routine analyses in sweat as is that to diagnose cystic fibrosis, and the
advantages and disadvantages of sweat versus urine or blood for doping control have also
been discussed. Methods for analytes such as essential metals and xenometals, ethanol and
electrolytes in sweat in fact constitute target metabolomics approaches or belong to any
metabolomics subdiscipline such as metallomics, ionomics or xenometabolomics. The higher
development of biomarkers based on genomics or proteomics as omics older than
metabolomics is discussed and also the potential role of metabolomics in systems biology
taking into account its emergent implementation. Normalization of the volume of sampled
sweat constitutes a present unsolved shortcoming that deserves investigation. Foreseeable
trends in this area are outlined [14039].
Sweat is an alternative biological matrix useful to detect drugs of abuse intake. It is produced
by eccrine and apocrine glands originating in the skin dermis and terminating in secretory
canals that flow into the skin surface and hair follicles. Since many years it has been
demonstrated that endogenous and exogenous chemicals are secreted in this biological
sample hence its collection and analysis could show the past intake of xenobiotics. From the
seventies the excretion of drugs of abuse has been investigated in human skin excretion;
later in nineties forensic scientists began to experiment some techniques to trap sweat for
analyses. Even if the use of skin excretions for drug testing has been restricted mainly by
difficulties in sample recovery, the marketing of systems for the sample collection has
allowed successful sweat testing for several drugs of abuse. In the recent years sweat
testing developed a noninvasive monitoring of drug exposure in various contexts as criminal
justice, employment and outpatient clinical settings. This paper provides an overview of
literature data about sweat drug testing procedures for various xenobiotics especially cocaine
metabolites, opiates, cannabis and amphetamines. Issues related to collection, analysis and
interpretation of skin excretions as well as its advantages and disadvantages are discussed.
Moreover the chance to apply the technique to some particular situation such as workplace
drug testing, drivers, doping or prenatal diagnosis, the comparison between sweat and other
non conventional matrices are also reviewed. According to literature data the analysis of
sweat may be usefully alternative for verifying drug history and for monitoring compliance
[13103].
Sweat is a biofluid with present scant use as clinical sample. One review tried to demonstrate
the advantages of sweat over other biofluids such as blood or urine for routine clinical
803
analyses and the potential when related to metabolomics. With this aim, critical discussion of
sweat samplers and equipment for analysis of target compounds in this sample is made.
Well established routine analyses in sweat as is that to diagnose cystic fibrosis, and the
advantages and disadvantages of sweat versus urine or blood for doping control have also
been discussed. Methods for analytes such as essential metals and xenometals, ethanol and
electrolytes in sweat in fact constitute target metabolomics approaches or belong to any
metabolomics subdiscipline such as metallomics, ionomics or xenometabolomics. The higher
development of biomarkers based on genomics or proteomics as omics older than
metabolomics is discussed and also the potential role of metabolomics in systems biology
taking into account its emergent implementation. Normalization of the volume of sampled
sweat constitutes a present unsolved shortcoming that deserves investigation [13104].
Sweat is an alternative biological matrix useful to detect drugs of abuse intake. It is produced
by eccrine and apocrine glands originating in the skin dermis and terminating in secretory
canals that flow into the skin surface and hair follicles. Since many years it has been
demonstrated that endogenous and exogenous chemicals are secreted in this biological
sample hence its collection and analysis could show the past intake of xenobiotics. From
the seventies the excretion of drugs of abuse has been investigated in human skin excretion;
later in nineties forensic scientists began to experiment some techniques to trap sweat
for analyses. Even if the use of skin excretions for drug testing has been restricted mainly by
difficulties in sample recovery, the marketing of systems for the sample collection have
allowed successful sweat testing for several drugs of abuse. In the recent years sweat
testing developed a noninvasive monitoring of drug exposure in various context as criminal
justice, employment and outpatient clinical settings. This paper provides an overview of
literature data about sweat drug testing procedures for various xenobiotics especially cocaine
metabolites, opiates, cannabis and amphetamines. Issues related to collection, analysis and
interpretation of skin excretions as well as its advantages and disadvantages are discussed.
Moreover the chance to apply the technique to some particular situation such as workplace
drug testing, drivers, doping or prenatal diagnosis, the comparison between sweat and other
non conventional matrices are also reviewed. According to literature data the analysis of
sweat may be usefully alternative for verifying drug history and for monitoring compliance
[12085].
The option of using sweat for doping control purposes is interesting, suggesting this matrix
as a viable means for detecting drugs of basic pH (e.g. stimulants and narcotics) due to their
accumulation in perspired liquid. However, although declared as a “preferred sample for
doping control” by the authors, the substantial limitations (control of sample volume,
restricted analyte coverage, limited knowledge on drug distribution, etc.) and few advantages
(non-invasiveness/intrusiveness) do not seem to promote this matrix in the focus of sports
drug testing whilst its utility in clinical settings, for example in the diagnosis of cystic fibrosis,
is undisputed [14715].
In oral fluid
Currently, urine and blood are the only matrices authorized for antidoping testing by the
World Anti-Doping Agency (WADA). Although the usefulness of urine and blood is proven,
issues remain for monitoring some drug classes and for drugs prohibited only in competition.
The alternative matrix oral fluid (OF) may offer solutions to some of these issues. OF
collection is easy, noninvasive, and sex neutral and is directly observed, limiting potential
adulteration, a major problem for urine testing. OF is used to monitor drug intake in
workplace, clinical toxicology, criminal justice, and driving under the influence of drugs
804
programs and potentially could complement urine and blood for antidoping testing in sports.
Onereview outlines the state of knowledge and the advantages and limitations of OF testing
for each of the WADA drug classes and the research needed to advance OF testing as a
viable alternative for antidoping testing. Doping agents are either prohibited at all times or
prohibited in competition only. Few OF data from controlled drug administration studies are
available for substances banned at all times, whereas for some agents prohibited only in
competition, sufficient data may be available to suggest appropriate analytes and cutoffs
(analytical threshold concentrations) to identify recent drug use. Additional research is
needed to characterize the disposition of many banned substances into OF; OF collection
methods and doping agent stability in OF also require investigation to allow the accurate
interpretation of OF tests for antidoping monitoring [14038].
Currently, urine and blood are the only matrices authorized for antidoping testing by the
World Anti-Doping Agency (WADA). Although the usefulness of urine and blood is proven,
issues remain for monitoring some drug classes and for drugs prohibited only in competition.
The alternative matrix oral fluid (OF) may offer solutions to some of these issues. OF
collection is easy, noninvasive, and sex neutral and is directly observed, limiting potential
adulteration, a major problem for urine testing. OF is used to monitor drug intake in
workplace, clinical toxicology, criminal justice, and driving under the influence of drugs
programs and potentially could complement urine and blood for antidoping testing in
sports.Content:This review outlines the present state of knowledge and the advantages and
limitations of OF testing for each of the WADA drug classes and the research needed to
advance OF testing as a viable alternative for antidoping testing.Summary:Doping agents are
either prohibited at all times or prohibited in competition only. Few OF data from controlled
drug administration studies are available for substances banned at all times, whereas for
some agents prohibited only in competition, sufficient data may be available to suggest
appropriate analytes and cutoffs (analytical threshold concentrations) to identify recent drug
use. Additional research is needed to characterize the disposition of many banned
substances into OF; OF collection methods and doping agent stability in OF also require
investigation to allow the accurate interpretation of OF tests for antidoping monitoring
[13112].
Designer drugs
The number and diversity of potentially performance-enhancing substances is continuously

growing, fueled by new pharmaceutical developments but also by the inventiveness and, at
the same time, unscrupulousness of black-market (designer) drug producers and providers.
In terms of sports drug testing, this situation necessitates reactive as well as proactive
research and expansion of the analytical armamentarium to ensure timely, adequate, and
comprehensive doping controls. This review summarizes literature published over the past 5
years on new drug entities, discontinued therapeutics, and 'tailored' compounds classified as
doping agents according to the regulations of the World Anti-Doping Agency, with particular
attention to analytical strategies enabling their detection in human blood or urine. Among
these compounds, low- and high-molecular mass substances of peptidic (e.g. modified
insulin-like growth factor-1, TB-500, hematide/peginesatide, growth hormone releasing
peptides, AOD-9604, etc.) and non-peptidic (selective androgen receptor modulators,
hypoxia-inducible factor stabilizers, siRNA, S-107 and ARM036/aladorian, etc.) as well as
inorganic (cobalt) nature are considered and discussed in terms of specific requirements
originating from physicochemical properties, concentration levels, metabolism, and their
amenability for chromatographic-mass spectrometric or alternative detection methods
[14242].
805
Virtual screening
Parallel ligand- and structure-based virtual screenings of 269 steroids with anabolic activity
evaluated in vivo were performed. The quantitative structure-activity relationship (QSAR)
model expressed by selected descriptors as the octanol-water partition coefficient, the molar
volume and the quantum mechanical calculated charge values on atoms C1, C2, C5, C9,
C10, C14 and C17 of the steroid skeleton, expresses structural features of anabolic steroids
(AS) contributing to the transport and steroid-receptor interaction. On the other hand,
computational simulations of a candidate ligand binding to a receptor study (a "docking"
procedure) predict the association of these AS with the human androgen receptor (AR).
Fourteen compounds were identified as lead; the most potent was the 7alpha-methylestr-4-
en-3, 17-dione. It was concluded that a good anabolic activity requires hydrogen bonding
interactions between both Arg752 and Gln711 residues in the cycles A with O3 atom of the
steroid and either Asn705 and Thr877 residues in the cycles D of steroid with O17 atom
[13110].
Artificial networks
The computational generation of gradient retention time data for retrospective detection of
suspected sports doping species in postanalysis human urine sample data is presented
herein. Retention data for a selection of 86 compounds included in the London 2012 Olympic
and Paralympic Games drug testing schedule were used to train, verify, and test a range of
computational models for this purpose. Spiked urine samples were analyzed using solid
phase extraction followed by ultrahigh-pressure gradient liquid chromatography coupled to
electrospray ionization high-resolution mass spectrometry. Most analyte retention times
varied ≤0.2 min over the relatively short runtime of 10 min. Predicted retention times were
within 0.5 min of experimental values for 12 out of 15 blind test compounds (largest error:
0.97 min). Minimizing the variance in predictive ability across replicate networks of identical
architecture is presented for the first time along with a quantitative discussion of the
contribution of each selected molecular descriptor toward the overall predicted value. The
performance of neural computing predictions for isobaric compound retention time is also
discussed. One work presented the application of neural networks to the prediction of
gradient retention time in archived high-resolution urine analysis sample data for the first time
in the field of anti-doping [13111].
Appearance and Performance Enhancing Drug Use Schedule (APEDUS)
Appearance-and-performance enhancing drug (APED) use is a form of drug use that

includes use of a wide range of substances such as anabolic-androgenic steroids (AASs)
and associated behaviors including intense exercise and dietary control. To date, there are
no reliable or valid measures of the core features of APED use. One study described the
development and psychometric evaluation of the Appearance and Performance Enhancing
Drug Use Schedule (APEDUS) which is a semi-structured interview designed to assess the
spectrum of drug use and related features of APED use. Eighty-five current APED using men
and women (having used an illicit APED in the past year and planning to use an illicit APED
in the future) completed the APEDUS and measures of convergent and divergent validity.
Inter-rater agreement, scale reliability, one-week test-retest reliability, convergent and
divergent validity, and construct validity were evaluated for each of the APEDUS scales. The
806
APEDUS is a modular interview with 10 sections designed to assess the core drug and non-
drug phenomena associated with APED use. All scales and individual items demonstrated
high inter-rater agreement and reliability. Individual scales significantly correlated with
convergent measures (DSM-IV diagnoses, aggression, impulsivity, eating disorder
pathology) and were uncorrelated with a measure of social desirability. APEDUS subscale
scores were also accurate measures of AAS dependence. The APEDUS is a reliable and
valid measure of APED phenomena and an accurate measure of the core pathology
associated with APED use. Issues with assessing APED use are considered and future
research is considered [11567].
807
ANABOLIC ANDROGENIC SUBSTANCES
Overview
Since its discovery in 1935, numerous derivatives of testosterone have been synthesized,
with the goals of prolonging its biological activity in vivo, producing orally active androgens,
and developing products, commonly referred to as anabolic-androgenic steroids (AAS), that
are more anabolic and less androgenic than the parent molecule. One article reviewed the
structure, biotransformation, and mechanism of action of testosterone and some of the most
commonly used AAS. Clinical applications of the AAS are discussed, and guidelines and
therapeutic maneuvers for minimizing their side effects are outlined. Literature for inclusion in
this review was identified using the libraries of the University of Wisconsin Medical School
and School of Pharmacy, the author's files, and searches of MEDLINE, Science Citation
Index, Biological Abstracts, and Chemical Abstracts. The myotrophic action of testosterone
and its derivatives and their stimulatory effects on the brain have led to widespread use of
AAS by athletes and "recreational" drug users. Consequently, all AAS were classified as
class III controlled substances in 1991. Nonetheless, AAS have shown benefit in a variety of
human disorders, including HIV-related muscle wasting and other catabolic conditions such
as chronic obstructive pulmonary disease, severe burn injuries, and alcoholic hepatitis.
Because of their diverse biological actions, AAS have been used to treat a variety of other
conditions, including bone marrow failure syndromes, constitutional growth retardation in
children, and hereditary angioedema. AAS therapy is associated with various side effects
that are generally dose related; therefore, illicit use of megadoses of AAS for the purpose of
bodybuilding and enhancement of athletic performance can lead to serious and irreversible
organ damage. The most common side effects of AAS are some degree of masculinization in
women and children, behavioral changes (e.g. aggression), hepatotoxicity, and alteration of
blood lipid levels and coagulation factors. To minimize or avoid serious toxicities with AAS
therapy, close medical supervision and periodic monitoring are important, with dose
adjustment as appropriate to achieve the minimum effective dose. Given the biological
effects and potential adverse effects of AAS, administration of these agents should be
avoided in pregnant women, women with breast cancer or hypercalcemia, men with
carcinoma of the prostate or breast, and patients with nephrotic syndromes or significant liver
dysfunction [01025].
Humans are basically competitive. For centuries, athletes have used various substances to
enhance performance, increase strength, and prolong endurance. In the early 1940s,
research indicating that testosterone improved a sense of well-being, appearance, and
sexual performance led to the use of anabolic steroid hormones by a select few athletes.
Today, even among high school students, the use of androgenic steroid hormones is
prevalent, with 1 to 2 percent of adolescent girls and 4 to 6 percent of adolescent boys
having used an anabolic steroid at least once. An estimated 1 million people in the United
States are current of former users of anabolic-androgenic steroid hormones, with men having
a higher prevalence of use than women. Androgenic steroid use has been associated with
the use of other illicit drugs, cigarette smoking, and alcohol use. Nevertheless, anabolic-
androgenic steroid hormones appear to have legitimate uses in certain patients. In HIV-
infected, hypogonadal men, anabolic steroid hormones optimize muscle strength and muscle
mass when combined with resistance exercise. Although a large number of people have
used these drugs for many years, no studies of the long-term health effects have been done.
However, when taken in supraphysiologic doses, these drugs are known to cause a wide
range of acute adverse effects. When used in less then supraphysiologic doses in eugonadal
or hypogonadal HIV-infected patients, these drugs reverse HIV-related hypogonadism,
808
muscle wasting, and perhaps lipodystrophy. Provided that the oral preparations are not used
and patients are closely monitored, anabolic-androgenic steroid hormones offer HIV-infected
patients a better quality of life and an improved sense of well-being [01026].
Anabolic steroids are synthetic derivatives of testosterone modified to enhance the anabolic
rather than the androgenic actions of the hormone. The anabolic effects are considered to be
those promoting protein synthesis, muscle growth and crythopoiesis. There are numerous
side-effects to anabolic steroids, including hypertension and atherosclerosis, blood clotting,
jaundice, hepatic carcinoma, tendon damage, psychiatric and behavioural effects and, in
males, reduced fertility and gynaccomastia. Anabolic steroids were added to the International
Olympic Committee's list of banned substances in 1975. The majority of “evidence”
concerning the efficacy of anabolic steroids as performance enhancing agents is anecdotal.
In the main, experimental investigations have been poorly designed scientifically, clinically
and statistically. The percentage of positive test results from IOC accredited laboratories has
remained consistently low. However, athletes take their steroids during training and out-of-
competition testing is not conducted in all countries, although international co-operation is
now under consideration. Despite the lack of conclusive evidence, steroids users will
continue to hold the view that their effects are efficacious and they are therefore unlikely to
be persuaded to curtail their use [00032].
The term "anabolic steroids" refers to testosterone derivatives that are used either clinically
or by athletes for their anabolic properties. However, scientists have questioned the anabolic
effects of testosterone and its derivatives in normal men for decades. Most scientists
concluded that anabolic steroids do not increase muscle size or strength in people with
normal gonadal function and have discounted positive results as unduly influenced by
positive expectations of athletes, inferior experimental design, or poor data analysis. There
has been a tremendous disconnect between the conviction of athletes that these drugs are
effective and the conviction of scientists that they aren't. In part, this disconnect results from
the completely different dose regimens used by scientists to document the correction of
deficiency states and by athletes striving to optimize athletic performance. Recently, careful
scientific study of suprapharmacologic doses in clinical settings – including aging, human
immunodeficiency virus, and other disease states – supports the efficacy of these regimens.
However, the mechanism by which these doses act remains unclear. "Anabolism" is defined
as any state in which nitrogen is differentially retained in lean body mass, either through
stimulation of protein synthesis and/or decreased breakdown of protein anywhere in the
body. Testosterone, the main gonadal steroid in males, has marked anabolic effects in
addition to its effects on reproduction that are easily observed in developing boys and when
hypogonadal men receive testosterone as replacement therapy. However, its efficacy in
normal men, as during its use in athletes or in clinical situations in which men are eugonadal,
has been debated. A growing literature suggests that use of suprapharmacologic doses can,
indeed, be anabolic in certain situations; however, the clear identification of these situations
and the mechanism by which anabolic effects occur are unclear. Furthermore, the
pharmacology of "anabolism" is in its infancy: no drugs currently available are "purely"
anabolic but all possess androgenic properties as well [02022].
The most easily available drugs for doping are the anabolic steroids (AS), derivatives of
testosterone, that are used by athletes for their anabolic properties. The mechanisms of
action include the stimulation of protein synthesis, antagonism of the catabolic effects of
glucocorticoids, increase the capacity for more intensive training and through central nervous
system effects increase motivation and decrease fatigue. However, they also possess
virilizing effects that are more evident in children and women. Commonly used androgens
are the orally administered mesterolone and testosterone undecanoate and the parenterally
administered testosterone enanthate. The most widely used AS are metenolone enanthate
809
and nadrolone decanoate AS are often combined with stimulants, supplements and/or
growth factors. Combinations of at least two drugs have been frequent with constant
increase of daily dose. There have been long-standing discussions, mainly due to different
dose regimens used to correct deficient states as compared to those used by athletes to
enhance athletic performance, regarding the efficacy of the AS. Testosterone, the main
gonadal steroid in males, has clear anabolic effects in developing boys or when used as
replacement therapy. Its efficacy in eugonal men has been debated. However, data suggest
that the use of suprapharmacologic doses can indeed be anabolic [03002].
Ever since the ancient Olympic Games, athletes have long sought the ability to enhance their
performance in sports and continue to do so in the modern era of elite competition. Over the
past few decades, athletes have attempted to enhance their performance with the use of
exogenous hormones including androgens, erythropoietin and growth hormone. Androgens
are the most effective form of sports doping and are the most common type of performance
enhancing substances detected in screening tests. It was not until the 1972 Munich Olympic
Games that the International Olympic Committee (IOC) introduced screening tests for
exogenous androgens. Since then, the World Anti-Doping Agency (WADA) enforces the
banning of androgens through urine screening to detect even trace amounts of an extensive
list of banned and prohibited substances which athletes are screened for prior, and during,
competition. This list relies on screening urine samples with highly specific and sensitive
techniques such as gas chromatography-mass spectrometry (GC-MS). For some athletes,
the prospect of success outweighs the health and unethical concerns of sports doping. In an
attempt to bypass screening methods, designer androgens have been created that have
different chemical structures to known androgens and, therefore, cannot be easily detected
by GC-MS. However, because designer androgens have biological activity they activate the
androgen receptor (AR), and therefore can be detected by androgen bioassays. Therefore,
androgen bioassays may prove to be a suitable tool for routine screening of nutraceutical or
biological samples suspected to contain an androgen [13084].
Androgenic-anabolic steroids (AAS) is an official definition for all male sex steroid hormones,
their synthetic derivatives and their active metabolites. AAS are drugs with specific
therapeutic indications, yet they are popularly known because of their worldwide non-
therapeutic use in a large number of healthy individuals. Doping with AAS has become so
widespread in athletics that it affects the outcome of sports contests. Furthermore, AAS non-
therapeutic use is increasing particularly among adolescents and females, becoming one of
the main causes of iatrogenic diseases due to drug abuse. All physicians must be aware
about the large diffusion and side effects related to AAS non-therapeutic use, in order to
discover clinical signs of AAS abuse and/or to start adequate preventive and/or therapeutic
actions [05029].
Androgens are the class of sex steroids responsible for male sexual characteristics, including
increased muscle mass and decreased fat mass. Illicit use of androgen doping can be an
attractive option for those looking to enhance sporting performance and/or physical
appearance. The use of in vitro bioassays to detect androgens, especially designer or
proandrogens, is becoming increasingly important in combating androgen doping associated
with nutritional supplements. The nutritional sports supplement market has grown rapidly
throughout the past decade. Many of these supplements contain androgens, designer
androgens or proandrogens. Many designer or proandrogens cannot be detected by the
standard highly-sensitive screening methods such as gas chromatography-mass
spectrometry because their chemical structure is unknown. However, in vitro androgen
bioassays can detect designer and proandrogens as these assays are not reliant on knowing
the chemical structure but instead are based on androgen receptor activation. For these
810
reasons, it may be advantageous to use routine androgen bioassay screening of
nutraceutical samples to help curb the increasing problem of androgen doping [13084].
Anabolic steroids are widely used for doping, in professional and non-professional sports.
The mechanism of action may differ somewhat depending on the specific molecule due to
structural differences that influence the specificity of binding with steroid receptors. When
used by athletes in training, they can improve performance to levels that cannot be attained
by almost any combination of sophisticated nonchemical support by modern sport science.
The severity of the undesired effects of anabolic steroids depends on a variety of factors,
from the type and combination of them, the dose and duration of administration, as well as
the gender of the person taking the drug. Younger individuals and women show greater
effects caused by anabolic steroids in terms of performance, but are also at greater risk of
side effects [09047].
The anabolic-androgenic steroids (AAS) have been used by elite athletes since the 1950s,
but they did not become widespread drugs of abuse in the general population until the
1980s. Thus, knowledge of the medical and behavioral effects of illicit AAS use is still
evolving. Surveys suggest that many millions of boys and men, primarily in Western
countries, have abused AAS to enhance athletic performance or personal appearance. AAS
use among girls and women is much less common. Taken in supraphysiologic doses, AAS
show various long-term adverse medical effects, especially cardiovascular toxicity.
Behavioral effects of AAS include hypomanic or manic symptoms, sometimes accompanied
by aggression or violence, which usually occur while taking AAS, and depressive symptoms
occurring during AAS withdrawal. However, these symptoms are idiosyncratic and afflict only
a minority of illicit users; the mechanism of these idiosyncratic responses remains unclear –
the personality of the victim before taking the drugs semms to be of importance. AAS users
may also ingest a range of other illicit drugs, including both "body image" drugs to enhance
physical appearance or performance, and classical drugs of abuse. In particular, AAS users
appear particularly prone to opioid use. There may well be a biological basis for this
association, since both human and animal data suggest that AAS and opioids may share
similar brain mechanisms. Finally, AAS may cause a dependence syndrome in a substantial
minority of users. AAS dependence may pose a growing public health problem in future
years but remains little studied [09048].
Anabolic Androgenic Steroids (AASs) are chemical and pharmacological derivatives of the
male hormone testosterone which are widely used for increasing burst and sprinting activities
in sports. Although AASs are thought to be transversal to the plurality of sports disciplines,
they are principally misused by bodybuilders, weightlifters, shot, hammer, discus or javelin
throwers, rugby and American football players as well as by swimmers and runners. AAS
exert a kaleidoscope of effects on human biology, principally through the 5-alpha-reductase-
mediated conversion into dihydrotestosterone, the aromatase-mediated conversion into
female sex hormones, a competitive antagonism to the glucocorticoid receptors, the potential
stimulation of erythropoietin secretion as well as psychoactive effects on the brain. The
influence of AASs on physical performance is still undefined, since the large numbers of
studies published so far have described discordant and often contradictory outcomes.
Nevertheless, animal and human investigations support the hypothesis that the
administration of AASs might increase lean body mass, muscle mass, and maximal voluntary
strength especially in men, so that they would represent an appealing form of doping for
increasing power capacity, sustaining intensive training periods and, last but not least, as a
cosmetic muscle makeover [11061].
The World Anti-Doping Agency regulations governing anti-doping in elite sports found in
2005 anabolic compounds as the most frequently detected doping agents, accounting for
811
about 43 percent of positive results in 2005. Among these testosterone, nandrolone and
stanozolol were predominant [08011].
With increasing availability of designer androgens, significant efforts are needed by

antidoping authorities to develop sensitive methods to detect their use. The PubMed and
Google Scholar search engines were used to identify publications addressing various forms
of doping, methods employed in their detection, and adverse effects associated with their
use.. It appears that the use of AAS continues to be associated with premature mortality
(especially cardiovascular) in addition to suppressed spermatogenesis, gynecomastia, and
virilization. The attention that androgen abuse has received lately should be used as an
opportunity to educate both athletes and the general population regarding their adverse
effects. The development of sensitive detection techniques may help discourage (at least to
some extent) the abuse of these compounds. Investigations are needed to identify ways to
hasten the recovery of the gonadal axis in anabolic androgenic steroids users and to
determine the mechanism of cardiac damage by these compounds [10043].
Anabolic steroids regulate the building blocks for the adolescent growth spurt and body
composition. Even the body uses the combination of testosterone, hGH and IGF-I to
subserve the adolescent growth spurt and the "partitioning" of food energy into the various
compartments of body composition. As noted previously the early attainment of pubertal
development confers some advantage on adolescent athletes compared to their "on-time"
peers, although this is likely less important later in puberty given that sport-specific skills
become more important as the majority of athletes will have entered and progressed through
pubertal development [10001].
An anabolic steroid is a sex hormone that promotes the development and maintenance of the
male sex characteristics. Testosterone is the principal secreted androgen in men. Androgens
have both masculinizing (development of male secondary sex characteristics, including hair
growth) and anabolic effects (increase in skeletal muscle mass and strength). For decades,
pharmaceutical companies have attempted to develop androgens that have preferential
anabolic activity and reduced or no androgenic activity; these compounds have been referred
to as anabolic steroids. There is feedback control of androgen synthesis and secretion
involving the hypothalamus (GnRH) and the pituitary. During adolescence there are
remarkable alterations in both secretion and feedback sensitivity that underpin male pubertal
development. Testosterone is strongly bound to sex-hormone binding globulin (SHBG),
loosely bound to albumin, and a small proportion circulates as the free hormone. The
biological activity is thought to reside in the free and loosely bound (albumin-bound) fractions
[10001].
Androgens mediate their action through their binding to the androgen receptor (AR) which is
mainly expressed in androgen target tissues, such as the prostate, skeletal muscle, liver and
central nervous system. There is a wide spectrum of testosterone and synthetic AAS
structure modifications related to the intended enhancement in anabolic activity [08124].
Anabolic androgenic steroids are the most abused class of prohibited substances, with
testosterone accounting for many positive cases. Testosterone abuse is problematic because
synthetic testosterone is indistinguishable from endogenous testosterone by routine
screening methods such as gas chromatography–mass spectrometry. In the 1980s, it was
discovered that testosterone use alters the ratio of testosterone glucuronide to
epitestosterone glucuronide (T/E ratio) in urine. Epitestosterone is a naturally occurring
biologically inactive epimer of testosterone that remains relatively constant in urine. A
population-based T/E ratio cutoff of 6.0 was initially used to indicate synthetic testosterone
use; the T/E ratio cutoff was lowered to 4.0 in 2005. Based on data from several laboratories,
812
the average T/E ratio ranges from 0.9 to 1.6 for healthy male adolescents and men. At the
UCLA Olympic Analytical Laboratory, it was found that the average T/E ratio during a 31-
month period was 1.1 (median 0.9 %). Approximately 99.6 percent of urine samples have a
T/E ratio <4.0, and 99.8 percent have a T/E ratio <6.0 [08124].
The T/E ratio is typically used as a screening test, and urine samples with a ratio >4.0 are
submitted for confirmation testing by gas chromatography/combustion/isotope ratio mass
spectrometry (GC/C/IRMS). GC/C/IRMS has excellent specificity and can measure very
small differences in the 13C to 12C ratio of testosterone and steroid metabolites. The 13C
content of natural (endogenously produced) testosterone is influenced by plant and animal
sources consumed in food. In contrast, synthetic (pharmaceutical) testosterone is produced
from plant precursors (stigmasterol) containing less 13C. This results in smaller 13C/12C ratios
for synthetic testosterone, compared to natural testosterone. Although GC/C/IRMS is used to
confirm testosterone use, the technique has low analytical sensitivity and is labor-intensive
(owing to extensive sample cleanup) and costly to perform, and thus cannot be used as a
screening test [08124].
An androgen (anabolic-androgenic steroid) is a derivative of the major sex steroid in men

and most mammals, testosterone (T). Others might answer in the more generic sense: any
compound that is an agonist (or partial agonist) at the androgen receptor (AR). There are
seemingly enough forms of T activity available to satisfy the needs of the medical population
with oral, buccal, cutaneous patches (really, drug delivery devices) and gels, injectables of
various durations (days to months), and implantable forms with a duration of action of 6
months or more. It seems that performance enhancement for human (and at least equine)
athletes provide the backbone for the design of new anabolic steroids to circumvent the
sophisticated testing procedures that surround major national and international competitions
[12113].
Anabolic steroids are synthetic derivatives of testosterone, modified to enhance its anabolic
actions (promotion of protein synthesis and muscle growth). They have numerous side
effects, and are on the International Olympic Committee's list of banned substances. Gas
chromatography-mass spectrometry allows identification and characterisation of steroids and
their metabolites in the urine but may not distinguish between pharmaceutical and natural
testosterone. Indirect methods to detect doping include determination of the
testosterone/epitestosterone glucuronide ratio with suitable cut-off values. Direct evidence
may be obtained with a method based on the determination of the carbon isotope ratio of the
urinary steroids. One paper aimed to give an overview of the use of anabolic-androgenic
steroids in sport and methods used in anti-doping laboratories for their detection in urine,
with special emphasis on doping with testosterone. It was made a review of the recent
literature of anabolic steroid testing, athletic use, and adverse effects of anabolic-androgenic
steroids.Procedures used for detection of doping with endogenous steroids are outlined. The
World Anti-Doping Agency provided a guide in August 2004 to ensure that laboratories can
report, in a uniform way, the presence of abnormal profiles of urinary steroids resulting from
the administration of testosterone or its precursors, androstenediol, androstenedione,
dehydroepiandrosterone or a testosterone metabolite, dihydrotestosterone, or a masking
agent, epitestosterone. Technology developed for detection of testosterone in urine samples
appears suitable when the substance has been administered intramuscularly. Oral
administration leads to rapid pharmacokinetics, so urine samples need to be collected in the
initial hours after intake. Thus there is a need to find specific biomarkers in urine or plasma to
enable detection of long term oral administration of testosterone [06048].
Androgens remain the most effective and widely abused ergogenic drugs in sport. The use of
particularly anabolic androgenic steroids has changed from being a problem restricted to
813
sports to one of public-health concern. In a review the prevalence of misuse, the evidence
that some drugs improve performance in sport, their side-effects, and the long-term
consequences of AAS misuse for society at large were discussed. There is substantial
under-reporting of the side-effects of AAS to health authorities. It was described
neuropsychiatric side-effects of AAS and their possible neurobiological correlates, with
particular emphasis on violent behaviour. Analytical methods and laboratories accredited by
the World Anti-Doping Agency can detect the misuse of all doping agents; although the
analysis of testosterone requires special techniques, and recently discovered interethnic
differences in testosterone excretion should be taken into account. The prevention of misuse
of doping agents should include random doping analyses, medical follow-ups, pedagogic
interventions, tougher legislation against possession of AAS, and longer disqualifications of
athletes who use AAS [08061].
Although androgen doping has been prohibited for over 3 decades with a ban enforced by
mass spectrometric-based urine testing for synthetic and exogenous natural androgens,
attempts continue to develop increasingly complex schemes to circumvent the ban. A
prominent recent approach has been the development of designer androgens. Such never-
marketed androgens evade detection because mass spectrometry relies on identifying
characteristic chemical signatures requiring prior knowledge of chemical structure. Although
once known, designer androgens are readily detected and added to the Prohibited List.
However, until their structures are elucidated, designer androgens can circumvent the ban on
androgen doping. To combat this, in vitro androgen bioassays offer powerful new possibilities
for the generic detection of unidentified bioactive androgens, regardless of their chemical
structure. Another approach to circumvent the ban on androgen doping has been the
development of indirect androgen doping, the use of exogenous drugs to produce a
sustained increase in endogenous testosterone production. Apart from estrogen blockers,
however, such neuroendocrine active drugs mostly provide only transient increases in blood
testosterone. Finally the ban on androgen doping must allow provision for rare athletes with
incidental, proven androgen deficiency who require testosterone replacement therapy. The
Therapeutic Use Exemption (TUE) mechanism makes provision for such necessary medical
treatment, subject to rigorous criteria for demonstrating a genuine ongoing need for
testosterone and monitoring of testosterone dosage. Effective deterrence of sports doping
requires novel, increasingly sophisticated detection options calibrated to defeat these
challenges, without which fairness in sport is tarnished and the social and health idealization
of sporting champions devalued [08062].
Anabolic-androgenic steroids (AASs) are synthetic derivatives of testosterone. The term

“androgenic” indicates masculinizing. Androgens are responsible for stimulating the growth of
the male reproductive tract and secondary sex characteristics. The term “anabolic” indicates
tissue building and is the component of a steroid which is responsible for thickening of the
vocal cords, enlargement of larynx, increasing libido, linear growth acceleration before
epiphyseal plate closure, increasing muscle bulk and strength through dose-dependent
hypertrophy, and decreasing body fat. The anabolic action is mediated by androgen
receptors on skeletal muscle. Testosterone increases muscle protein synthesis, thus
increasing the cross-sectional area of the fibers themselves, as well as increasing the
myonuclear number [07007].

814
kg bodyweight that may be attributed to an increase of the lean body mass, have been
with the body and low self-esteem may lead to the so-called 'reverse anorexia syndrome' that
predisposes to the start of AAS use. Many other adverse effects have been associated with
AAS misuse, including disturbance of endocrine and immune function, alterations of
[04049].
Anabolic-androgenic steroids (AAS) are synthetic derivatives of testosterone. According to

surveys and media reports, the legal and illegal use of these drugs is gaining popularity.
Testosterone restores sex drive and boosts muscle mass, making it central to 2 of society's
rising preoccupations: perfecting the male body and sustaining the male libido. The anabolic
effects of AAS have been questioned for decades, but recent scientific investigation of
supraphysiologic doses supports the efficacy of these regimens. Testosterone has potent
anabolic effects on the musculoskeletal system, including an increase in lean body mass, a
dose-related hypertrophy of muscle fibers, and an increase in muscle strength. For athletes
requiring speed and strength and men desiring a cosmetic muscle makeover, illegal steroids
are a powerful lure, despite the risk of subjective side effects. Recent clinical studies have
815
discovered novel therapeutic uses for physiologic doses of AAS, without any significant
adverse effects in the short term. In the wake of important scientific advances during the past
decade, the positive and negative effects of AAS warrant reevaluation. Guidelines for the
clinical evaluation of AAS users will be presented for sports medicine practitioners [04018].

kg bodyweight,that may be attributed to an increase of the lean body mass, have been
with the body and low self-esteem may lead to the so-called “reverse anorexia syndrome”
that predisposes to the start of AAS use. Many other adverse effects have been associated
with AAS misuse, including disturbance of endocrine and immune function, alterations of
[04050].
816
One article reviews the recent literature on the use of anabolic-androgenic steroids (AAS) for
performance enhancement. Recent studies utilizing supraphysiologic doses of testosterone
have demonstrated increases in strength and improvements in body composition, despite
earlier assertions by the medical community that steroids were ineffective as ergogenic aids.
Although data that support the theory of conversion of prohormones, such as androstenediol,
to testosterone in the body is available, support for testosterone precursors alone as
ergogenic aids is lacking. Drug testing laboratories are utilizing new techniques that analyze
carbon-13 levels of urinary steroids to detect exogenously administered steroids as well as
the use of urine-manipulating agents. Investigations that seek to refute athletes' various
claims for positive drug tests are ongoing. The recent discovery, characterization, and
development of a urine test for tetra-hydro-gestrinone, a designer steroid, has brought the
issue of performance enhancement once again into the public spotlight. Increasing attention
is also being paid to the long-term effects of AAS abuse, as more authors characterize the
changes to hematologic, hepatic, lipid, and hormone profiles as a result of years of steroid
use. Although the understanding of AAS and testosterone precursors as performance-
enhancing drugs continues to advance, there are likely to be more revelations as scientific
investigations continue [04051].
Anabolic-androgenic steroids (AAS) are synthetic derivatives of testosterone. According to

surveys and media reports, the legal and illegal use of these drugs is gaining popularity.
Testosterone restores sex drive and boosts muscle mass, making it central to 2 of society's
rising preoccupations: perfecting the male body and sustaining the male libido. The anabolic
effects of AAS have been questioned for decades, but recent scientific investigation of
supraphysiologic doses supports the efficacy of these regimens. Testosterone has potent
anabolic effects on the musculoskeletal system, including an increase in lean body mass, a
dose-related hypertrophy of muscle fibers, and an increase in muscle strength. For athletes
requiring speed and strength and men desiring a cosmetic muscle makeover, illegal steroids
are a powerful lure, despite the risk of subjective side effects. Recent clinical studies have
discovered novel therapeutic uses for physiologic doses of AAS, without any significant
adverse effects in the short term. In the wake of important scientific advances during the past
decade, the positive and negative effects of AAS warrant reevaluation. Guidelines for the
clinical evaluation of AAS users will be presented for sports medicine practitioners [04052].
Androgens are mainly prescribed to treat several diseases caused by testosterone

deficiency. However, athletes try to promote muscle growth by manipulating testosterone
levels or assuming androgen anabolic steroids (AAS). These substances were originally
synthesized to obtain anabolic effects greater than testosterone. Although AAS are rarely
prescribed compared to testosterone, their off-label utilization is very wide. Furthermore,
combinations of different steroids and doses generally higher than those used in therapy are
common. Symptoms of the chronic use of supra-therapeutic doses of AAS include anxiety,
depression, aggression, paranoia, distractibility, confusion, amnesia. Interestingly, some
studies have shown that AAS elicited electroencephalographic changes similar to those
observed with amphetamine abuse. The frequency of side effects is higher among AAS
abusers, with psychiatric complications such as labile mood, lack of impulse control and high
violence. On the other hand, AAS addiction studies are complex because data collection is
very difficult due to the subjects' reticence and can be biased by many variables, including
physical exercise, that alter the reward system. Moreover, it has been reported that AAS may
imbalance neurotransmitter systems involved in the reward process, leading to increased
sensitivity toward opioid narcotics and central stimulants [150004].
Androgen precursors are either inactive or weak androgens that the body converts into
potent androgens. These include naturally occurring precursors to testosterone, such as 4-
817
androstenediol, 5-androstenediol, 4-androstenedione, and dehydroepiandrosterone, as well
as precursors to synthetic AAS including 4-norandrostenedione, 4-norandrostenediol, and 5-
norandrostenediol, which the body converts to nandrolone. Other synthetic AAS, such as 17-
desmethylstanozolol, methylclostebol, and methyltrienolone have been recently introduced
into the market as dietary supplements. These “designed” steroids have not undergone
toxicological or safety testing in humans or animals. Thus, they potentially represent an even
more serious health risk than the more traditionally used AAS [150004].
Anabolic androgenic steroids (AASs) detected most often in international doping control tests
[150001]:
Practical explanations (definitions) of anabolic steroids
AAS are synthetic derivatives of the male hormone testosterone. In humans, testosterone is
produced in the Leydig cells in the testes. For many years it had been well known that
castration resulted in the loss of certain secondary male sex characteristics; therefore, in the
early 1900s several attempts were made to obtain a substance with the same potential as
testosterone. At the end of the 1920s an active extract became available and the production
of synthetic androgens was possible in 1935. Although the separation of androgenic and
anabolic properties was pursued, complete separation was not successful; however, there
are now products available with more androgenic and substances with more anabolic
properties. The androgenic actions primarily include the development of the male
characteristics, that is, increased strength, voice deepening and the typical male hair growth.
The anabolic action affects protein metabolism by stimulation of protein synthesis and
inhibition of protein breakdown. Neither oral nor parenteral administration of exogenous
testosterone exerts significant effects in the human body because they are rapidly
metabolised. To circumvent this problem, several chemical modifications of testosterone
have been developed. The variety of these modifications has led to substances with different
action modes. Three major modifications can be distinguished that have therapeutic
potential. First, alkylation at the 17-alpha-position with methyl or ethyl group. Alkylation was
important to create orally active compounds since this implies slower degradation of the drug
by the liver. Secondly, through esterification of testosterone and nortestosterone at the 17-
beta-position it was possible to administer these substances parenterally and the duration of
effectiveness could be prolonged. Agents soluble in oily vehicles used for injections may be
present in the body for several months. Finally, alterations of the ring structure of
818
testosterone were applied for both oral and parenteral agents and increased the activity of
these substances. Currently, the therapeutic use of AAS is limited and may vary between
steroids. The most important indications are endocrine dysfunction of the testes and of the
hypothalamus-pituitary-gonadal axis (i.e. male hypogonadism and growth retardation)
[04002].
Research activity
The consumption of anabolic steroids (AS) has been growing continuously in recent years. It
has gone beyond the sports world; AS are now widely used as drugs of abuse in connection
with bodybuilding. This study sets out to assess the state of scientific research in the area.
Bibliometrics were employed to evaluate the literature retrieved from the principal relevant
bibliographic databases: MEDLINE, SportDiscus, the Science Citation Index Expanded and
the Social Sciences Citation Index. The core journals were identified along with the leading
authors and research groups and their institutional affiliations. Techniques based on social
network analysis were applied in order to build up a concept map of research. 1325
documents were retrieved. They were produced by 3131 different researchers giving a
Collaboration Index of 3.32. The institutions with the most productive authors were Ball State
University (Muncie, IN, USA), the Ecole Nationale Vétérinaire de Nantes (ENVN), the Institut
Municipal dInvestigació Mèdica (IMIM) (Barcelona, Spain), the Institute of Biochemistry of the
German Sport University Cologne (DSHS), Iowa State University, Maastricht University and
the University of Iowa. It was concluded that there has been an upward trend in the number
of research projects. The sources used complemented one another, as 78 percent of the
documents retrieved were unique to one source. The productivity ranking was headed by
sports medicine journals, followed by journals of chemistry, physiology, endocrinology and
substance abuse. Besides sporting activities, the most important research clusters were
those connected with bodybuilding and with youth groups [07057].
Limitations of research on the effects of AAS in athletes
Scientific research on the effects of AAS in athletes started in the 1960s. Since then, a
number of studies have been published. The first studies focused on athletic performance,
with special attention to strength and endurance capacity alterations. Later, in part along with
the dramatic increase of AAS misuse by athletes for aesthetic purposes, the impact of AAS
on body composition became of interest. Concurrent with the increased misuse of AAS for
non-medical reasons, more attention was paid to the adverse effects. This led to an
overwhelming number of publications with large methodological and qualitative differences.
As a result, different conclusions and interpretations were drawn. Therefore, several
methodological considerations need to be addressed. Although a lot of AAS studies have
been published, only a few meet current scientific quality standards of randomised, double-
blind placebo-controlled study design. Furthermore, the design needs to follow AAS abuse
practices of real life to obtain insight into what really happens in sport. In investigating the
effects of AAS, the combination of both demands conflicts, especially because of ethical
considerations. It is not acceptable to expose healthy humans to potentially hazardous drugs
in supratherapeutic dosages for the single purpose of improving sports performance.
Therefore, different study designs have been used, with each possessing some benefits and
disadvantages [04002].
A number of cross-sectional studies have misinterpreted the observed differences between

AAS users and controls by attributing them to a causal relationship. Cross-sectional studies
are not designed to study causal relationships; at most, they are to observe an association.
819
For example, several cross-sectional studies concluded that differences of heart morphology
between AAS users and controls were attributed to AAS use. Although scientifically
preferable, randomised, placebo-controlled studies have several disadvantages for
investigating AAS effects in athletes. Because of ethical considerations, only relatively low
doses for a limited time period can be studied. However, such studies do not reflect sports
practices and may therefore provide only a glimpse of actual effects. Prospective,
observational studies can overcome several limitations of interventional studies, but have the
disadvantage of selection bias and are less controllable. To date, no studies investigating the
long-term effects of AAS are available. Only a few observational reports describing
alterations of body composition and health status in a single subject have been published.
Therefore, the long-term effects of AAS abuse are as yet unknown. Case reports are
commonly used to describe the association of abuse of AAS with the most unexpected,
severe and dramatic disease conditions. Such reports must be interpreted with caution. They
are characterised by describing a possible relationship between AAS administration and the
disease condition and, since evidence is lacking, may exaggerate the problem. Inclusion of
athletes of only one sport discipline may improve validity of the observations since athletes
from different sports have set different objectives to use AAS. For example, in a number of
studies “strength athletes” were the subjects of the investigation. “Strength athletes” is a term
comprising athletes from several disciplines with different goals. A powerlifter’s objective is to
lift weights, whereas bodybuilders are focused to enlarge muscle mass and dimensions. In
wrestlers, absolute power and muscle mass are not important at all. Studying a varsity group
of so-called “strength athletes” may influence study results of body composition, muscle
mass and strength. Another problem researchers are confronted with is the regimens used
by athletes, especially those involved in strength sports. Athletes use so-called stacking
regimens, characterised by the administration of several AAS simultaneously in huge
amounts in weekly changing dosages. Such regimens are applied based on the beliefs and
insights of the athlete, although no rationale for such a regimen is available yet. Another
problem is that since the substances are obtained from the black market quality is not
guaranteed. This may also induce the ongoing increase of doses administered since many
vials and tablets obtained on the black market only contain a fraction of the declared dosage.
Because of ethical considerations, many studies chose to study volunteers who had decided
to self-administer AAS and subjects who were not willing to take AAS at all. The inclusion of
participants without the possibility of randomisation may influence the study outcome as a
result of selection bias. To reduce this problem, strict inclusion and exclusion criteria have to
be set, especially with respect to training and training history, as well to health status and risk
factors of diseases. Furthermore, since most drugs are obtained from the black market,
quality is not guaranteed and polydrug use excludes the possibility of attributing the observed
effects to a single drug. Many laboratory studies have the disadvantage of not mimicking
actual AAS abuse habits among athletes, especially since the dosages self-administered
increased dramatically in the last few decades. For example, most of the early, well designed
studies assessed one drug in a therapeutic dose, while athletes are now used to self-
administering polydrug regimens in dosages that may be 5-20 times higher than in most
studies [04002].
Common dosage of anabolic steroids in dopers
The larger observational studies of AAS users indicate that drug regimens follow a typical
pattern. Combinations of different oral and injectable AASs are “stacked” to create a mega-
dose regimen that is self-administered during drug “cycles” lasting 4 to 12 weeks. In a survey
of 100 male AAS users, the drug dosages ranged from 250 mg to 3200 mg per week of
testosterone or its equivalent. Fifty percent of the AAS users in this sample reported using a
weekly dose of at least 500 mg. To achieve these supraphysiologic doses, 88 percent of
AAS users in this sample combined 2 or more different types of AAS – a process known as
820
stacking. Some bodybuilders who chose to be precise with their dosages reported calculating
their dosages using the following formula: 1 mg of steroid per kilogram of body weight per
day. In another field study of 88 AAS users, 28 percent reported using at least 1000 mg of
testosterone or its equivalent per week. In most surveys, the duration of steroid
administration or steroid cycle lasts between 4 and 12 weeks. The time interval between
steroid cycles is more variable. Regular users allow a 4- to 6-week drug holiday to “clear the
system,” whereas less frequent users may remain drug free for months. In 1 survey,
approximately half of the sample reported that their total annual AAS use was less than 6
months, whereas the other half used AAS for more than 6 months each year. Three of the
100 AAS users surveyed admitted to continuous steroid use for 52 weeks of the year. Two
recent surveys indicate that the majority (76-96 %) of AAS users self-administer injectable
(intramuscular) formulations of AAS. Drug use by AAS users is not confined to anabolic
steroids. Up to 90 percent of AAS users have a palate for polypharmacy, taking a mix of
muscle-shaping drugs, in addition to stacking different brands of steroids. These “steroid-
accessory” drugs are used for a variety of reasons and can be grouped according to their
desired effect. Some of these accessory drugs are potentially more dangerous than AAS; the
unsupervised use of insulin, diuretics, and thyroxine can precipitate a number of medical
emergencies [04018].
A variety of substances
Androgen precursors are either inactive or weak androgens that the body converts into
potent androgens. These include naturally occurring precursors to testosterone such as 4-
androstenediol, 5-androstenediol, 4-androstenedione, and dehydroepiandrosterone, as well
as precursors to synthetic AAS, including 4-norandrostenedione, 4-norandrostenediol, and 5-
norandrostenediol, which the body converts to nandrolone. The widespread, unregulated
sale of dietary supplements on the Internet has greatly increased the number of anabolic
steroids available. Of even greater concern is the introduction of synthetic anabolic steroids
such as 17-desmethylstanozolol, methylclostebol, and methyltrienolone into the market as
dietary supplements. Apartial list of steroids contained in dietary supplements can be found
at www.supplement411.org. The Steroid Control Act of 2004 banned most these substances.
However, there are now repeatedly presented novel synthetic designer androgens, such as
tetrahydrogestrinone and madol. Because these designer steroids have not undergone
toxicological or safety testing in humans or animals, they potentially pose an even more
serious health risk than the more traditionally used AAS, which have received some level of
animal or human testing [14017].
An androgen is a sex hormone that promotes the development and maintenance of the male
sex characteristics; testosterone is the principal secreted androgen in men. Androgens have
both androgenic (masculinizing) effects (development of male secondary sex characteristics,
including hair growth) and anabolic effects (increase in skeletal muscle mass and strength).
For decades, pharmaceutical companies have attempted to develop androgens that have
preferential anabolic activity and reduced or no androgenic activity; these compounds have
been referred to as anabolic steroids. While some steroidal compounds available to date are
preferentially anabolic, most generally have both androgenic and anabolic effects. Therefore,
for the sake of uniformity and accuracy, it was used the term androgenic-anabolic steroids to
describe these compounds that are structurally related to testosterone, bind to androgen
receptor, and exert masculinizing as well as anabolic effects to varying degrees. The
literature uses a number of terms (anabolic steroids, androgenic steroids, and androgens) to
describe these androgen derivatives. Testosterone remains popular, both among elite
athletes and nonathlete weightlifters, because of its low price, relatively ready access, and
821
the challenges in distinguishing exogenous from endogenous sources of testosterone.
Numerous AAS have been synthesized by structural modifications of testosterone molecule.
These structural modifications may alter the relative anabolic or androgenic activity, the
binding affinity for the androgen receptor, coactivator recruitment, metabolic clearance,
susceptibility to presystemic metabolism, and aromatization [14017].
Differences between polydrug regimens and single drug administration
The objective of this investigation was to study the effects of androgenic-anabolic steroid
(AAS) misuse on deltoid muscle fiber characteristics in experienced, male strength-trained
athletes. In a double-blind study, 15 volunteers were administered nandrolone decanoate
(ND) for 8 weeks (200 mg/week, intramuscularly). In an additional study, 12 subjects self-
administered various AASs at supratherapeutic dosages (AAS group), while 7 non-users
served as controls. In all subjects, a percutaneous needle biopsy sample of the deltoid
muscle was obtained at baseline and after 8 weeks. Muscle sections were pre-incubated at
pH 4.4, stained with adenosine triphosphatase and analyzed morphometrically. In each
biopsy sample, at least 150 fibers were classified for "gray level" and "lesser fiber diameter"
to determine the mean fiber size, the sizes of type I and type II fibers, and the fiber type
distribution. ND administration did not seem to affect any of those parameters. In the AAS
group, mean muscle fiber size (+ 12.6 %), and the size of type I (+ 10.8 %) and type II (+
14.6 %) muscle fibers increased. The fiber type distribution remained unaltered. We
conclude that polydrug regimens of AAS misuse at supratherapeutic dosages increased the
size of deltoid muscle fibers (especially type II fibers) in experienced strength-trained
athletes, while ND at a therapeutic intramuscular dose of 200 mg did not exert any effect
[02030].
Clinical use
The purpose of one study was to review the preclinical and clinical literature relevant to the
efficacy and safety of anabolic androgen steroid therapy for palliative treatment of severe
weight loss associated with chronic diseases. Data sources were published literature
identified from the Medline database from January 1966 to December 2000, bibliographic
references, and textbooks. Reports from preclinical and clinical trials were selected. Study
designs and results were extracted from trial reports. Statistical evaluation or meta-analysis
of combined results was not attempted. Androgenic anabolic steroids (AAS) are widely
prescribed for the treatment of male hypogonadism; however, they may play a significant role
in the treatment of other conditions as well, such as cachexia associated with human
immunodeficiency virus, cancer, burns, renal and hepatic failure, and anemia associated with
leukemia or kidney failure. A review of the anabolic effects of androgens and their efficacy in
the treatment of these conditions is provided. In addition, the numerous and sometimes
serious side effects that have been known to occur with androgen use are reviewed.
Although the threat of various side effects is present, AAS therapy appears to have a
favorable anabolic effect on patients with chronic diseases and muscle catabolism. We
recommend that AAS can be used for the treatment of patients with acquired
immunodeficiency syndrome wasting and in severely catabolic patients with severe burns.
Preliminary data in renal failure-associated wasting are also positive. Advantages and
disadvantages should be weighed carefully when comparing AAS therapy to other weight-
gaining measures. Although a conservative approach to the use of AAS in patients with
chronic diseases is still recommended, the utility of AAS therapy in the attenuation of severe
weight loss associated with disease states such as cancer, postoperative recovery, and
822
wasting due to pulmonary and hepatic disease should be more thoroughly investigated
[01030].
Anabolic-androgenic steroids were developed to treat hypogonadism, a condition in which

the testes do not produce sufficient testosterone for normal growth, development, and sexual
functioning. Clinical studies suggest that the use of AASs (e.g.nandrolone) or testosterone
improves lean bodymass in patients with mild to moderate cachexia secondary to chronic
illness (e.g. human immune deficiency virus, HIV). The use of testosterone and AAS has not
been effective for the treatment of the catabolic states associated with severe burns or
muscle- wasting diseases. Historical uses of AASs include anemia, hereditary angioedema,
metastatic breast cancer, protein deficiencies following trauma or severe infections, and
certain psychiatric disorders (involutional psychoses and depression) [13002].
Androgens are used clinically to treat a range of different human disorders. Among these are
several catabolic conditions such as obstructive pulmonary disease, severe burn injuries,
and also HIV-related muscle wasting. It can also be used to treat a number of conditions
resulting from deficiencies in androgen production, such as constitutional growth retardation
and hypogonadism. Androgen therapy can be administered orally, by intramuscular injection,
and as gels and creams. Synthetic alkyl esters of androgens have been used therapeutically
for decades due to their high potency and prolonged action. The realization that androgen
administration can augment muscle hypertrophy has led to the abuse of androgens to
increase muscle size, strength and sport performance [13084].
The anabolic properties of AASs have proven beneficial for some therapeutic applications.
They have been used in clinical practice since the 1940s for the treatment of trauma, burns,
extensive surgery, radiation therapy, and chronic debilitating illnesses. Before the advent of
bone marrow transplantation and synthetic erythropoietin, AASs were used often in the
treatment of various types of anemias. AASs have shown promise in treating short stature,
as in Turner's syndrome, or constitutional growth and puberty delay. 1985-2006, the clinical
use of AASs has increased 400 percent, mostly due to the management of AIDS-associated
wasting syndrome. AASs may enhance the effects of the increased caloric intake and
exercise regimen. A pilot study in malnourished HIV-infected children as young as 4 years
old showed that oxandrolone treatment was well-tolerated and improved nutritional status.
After 3 months of treatment, the study subjects experienced an accelerated rate of weight
gain, increased body mass index, increased muscle mass, and decreased fat stores as
compared with pretreatment values. The results were supported further by the improved
serum albumin levels noted during the course of treatment. Future studies using a larger
study population and longer- or higher-dose AAS administration would strengthen the current
data. In patients with severe burns, AASs may play an important role in reversing the
catabolic state. A small prospective randomized study of patients who had burns showed that
those receiving oxandrolone in addition to a high-protein diet experienced a significantly
greater increase in weight and physical therapy index than did patients who were treated with
diet alone. AAS therapy seems to be promising in the treatment of malnutrition and muscle
wasting seen in patients who have end-stage renal disease. In addition to the increase in
lean body mass, these patients also benefit from a stimulated erythropoiesis resulting from
the administration of AASs [07008].
Critical illness precipitates a marked catabolic response, with protein wasting and loss of lean
body mass. Prolongation of this response leads to impaired immunity, poor wound healing,
loss of intestinal barrier function and muscle weakness, thereby increasing morbidity and
perhaps mortality. Conventional nutritional support only partially ameliorates this process.
Disappointingly, specific anabolic and anticatabolic strategies have so far met with only
limited success, although recent findings, in particular studies demonstrating the potential
823
value of aggressive insulin therapy and the administration of growth hormone secretagogues,
have been encouraging [02028].
From a clinical standpoint, AAS are commonly prescribed to treat several disorders, such as
the androgen deficiency syndromes, hereditary angioedema, hematological disorders,
catabolic conditions, such as some types of cancer-related cachexia, metabolic dysfunctions
induced by severe burn, inflammatory pulmonary diseases, radiation therapy, and AIDS-
associated malnutrition. Less common medical uses of AAS deal with heart and renal failure.
Contrasting data exists in the literature regarding the use of AAS in the treatment of
androgen deficiency in aging males, infertility, sexual dysfunctions or impotence, as well as
post-menopausal syndrome in women. Thus, while a review points toward therapeutic effects
on libido and menopause-induced sarcopenia, another consider their therapeutic application
in these pathological conditions as “misuse of androgens”. Hence, according to the state of
the art presented there is no indication for androgen therapy in male infertility because of its
suppressing effect on spermatogenesis. Importantly, there is no evidence in available
literature that AAS abuse or dependence might develop from the legitimate medical use of
AAS [150004].
One article addressed the use of anabolic androgenic steroids (AAS), synthetic drugs whose
abuse has been characterized as a public health problem, operated in the opposition
between "medical" and "non-medical" uses. A qualitative approach was used to analyze the
text in 76 biomedical articles published from 2002 to 2012. The discourse shows a persistent
ban on non-medically regulated use of AAS by young people, while the limits on clinically
qualified use appear to expand among older people, even given the contradictions straining
the argument on the prevention of health risks. Moralizing biopolitical stances appear, based
on gender distinctions or under the aegis of criminalizing drug use [150141].
Hereditary angiooedema
Androgen derivatives are regarded as standard in the long-term prophylaxis of swelling

attacks in patients with hereditary angioedema (HAE). Because of their relatively slow onset
of action, they are not suitable for acute therapy. Long-term prophylaxis with androgen
derivatives must be regarded critically, especially on account of their androgenic and
anabolic effects, some of which are severe. The risk of adverse events increases with the
daily dose and the duration of treatment. Thus, treatment always calls for close monitoring of
patients with regard to potential adverse events. In addition, androgens are subject to
numerous contraindications and they show interactions with a large number of other drugs.
Off-label use, doping issues, clarification of reimbursement and the need to import the
androgen derivatives, which are no longer marketed in Germany, result in additional effort for
the treating physician in terms of logistics and time involved. In symptomatic treatment of
acute attacks the intravenous substitution of C1-INH and – since 2008 – subcutaneous
administration of icatibant are available. The two substances are well tolerated and their
effect occurs rapidly and, when the diagnosis has been confirmed, reliably. In the light of
these two treatment options for controlling acute attacks, prophylactic treatment of HAE
patients with androgen derivatives such as danazol should be reassessed. Patients might
benefit from a dose reduction or the withdrawal of androgen prophylaxis and attacks can be
controlled with demand-oriented acute treatment using C1-INH or icatibant [11064].
To provide an objective basis for evaluating the risk-benefit ratio of long-term androgen use
in patients with hereditary angioedema (HAE) PubMed was searched with no time limitations
using the keywords hereditary angioedema or angio-oedema combined with danazol,
stanozolol, and androgen. Qualifying articles were English-language reports of androgen use
824
in patients with HAE, with relevant safety and/or efficacy information. Reports were
categorized according to level of evidence (LOE). The search process identified 153
citations, 63 of which contained relevant information; 2 additional publications were identified
while other citations were being reviewed. Fifteen LOE 2 studies and multiple LOE 4 reports
provided efficacy data, confirming a high level of prophylactic efficacy for androgen therapy
in HAE, with occasional reports of poor prophylactic response. Common adverse events
include weight gain, menstrual irregularities, virilization, headaches, myalgias or cramps,
mood changes, and elevations in creatine phosphokinase level, liver function test results,
and serum lipid level. The risk of adverse events is often correlated with dose and/or
treatment duration. Rare cases of hepatic adenomas and hepatocellular carcinoma
associated with long-term androgen use often had no preceding changes in liver function test
results. It was concluded that androgen therapy may be effective for most patients with HAE;
however, potential risks and adverse effects must be carefully considered and discussed with
patients when considering options for long-term HAE prophylaxis [150142].
Cystic fibrosis
The use of non-prescribed anabolic agents amongst non-athletes is increasing with young,
adult males with cystic fibrosis (CF) in the highest risk demographic. There is evidence that
anabolic agents increase weight and muscle mass in adults with a variety of catabolic
conditions but there is no evidence for their use in hormone sufficient adults with CF. It was
reported a case of anabolic agent use in a male adult with CF and review the clinical features
of anabolic agent use with a focus on adults with CF [150143].
Reduced risk of atrophy after tendon rupture
Chronic rotator cuff tendon tearing is associated with irreversible atrophy, fatty infiltration,
and interstitial fibrosis of the corresponding muscle. Anabolic steroids can prevent
musculotendinous degeneration during retraction and/or can reverse these changes after
operative repair of the retracted musculotendinous unit in sheep. The infraspinatus tendon
was released in 18 alpine sheep. All sheep underwent repair of the retracted
musculotendinous unit after 16 weeks and were sacrificed after 22 weeks; 6 sheep served as
controls, 6 sheep were treated with weekly intramuscular injection of 150 mg of nandrolone
decanoate after infraspinatus (ISP) repair (group N6W), and 6 sheep were treated with 150
mg of nandrolone decanoate immediately after tendon release (group N22W). Muscle biopsy
specimens were taken before tendon release and after 16 and 22 weeks. Muscle volume and
fatty infiltration (on MRI), myotendinous retraction, and muscle density (on computed
tomography) were measured immediately after ISP release, after 6 weeks, and before ISP
repair and sacrifice. Muscle volume on MRI decreased to a mean (±SD) of 80 ± 8 percent of
the original volume after 6 weeks, remained stable at 78 ± 11 percent after 16 weeks, and
decreased further to 69 ± 9 percent after 22 weeks in the control group. These findings were
no different from those in group N22W (72 % ± 9 % at 6 weeks, 73 % ± 6 % at 16 weeks,
and 67 % ± 5 % at 22 weeks). Conversely, the N6W group did not show a decrease in ISP
volume after repair; this finding differed significantly from the response in the control and
N22W groups. Fatty infiltration (on MRI) continuously increased in the control group (12 % ±
4 % at tendon release, 17 % ± 4 % after 6 weeks, 50 % ± 9 % after 16 weeks, and 60 % ± 8
% after 22 weeks) and the N6W group. However, application of anabolic steroids at the time
of tendon release (N22W group) significantly reduced fatty infiltration after 16 (16 % ± 5 %)
and 22 weeks (22 % ± 7 %). It was concluded that in a sheep model of rotator cuff tendon
tear, further muscle atrophy can be prevented with the application of anabolic steroids
starting immediately after tendon repair. In addition, fatty muscle infiltration can largely be
prevented if the steroids are applied immediately after tendon release. The study findings
825
may lead to the development of treatment strategies to prevent or reduce muscle
degeneration caused by rotator cuff tendon tearing [150144].
Effect of AAS after repair of tendons
Chronic rotator cuff tendon tearing is associated with irreversible atrophy, fatty infiltration,
and interstitial fibrosis of the corresponding muscle. It was hypothesed that anabolic steroids
can prevent musculotendinous degeneration during retraction and/or can reverse these
changes after operative repair of the retracted musculotendinous unit in sheep. The
infraspinatus tendon was released in 18 alpine sheep. All sheep underwent repair of the
retracted musculotendinous unit after 16 weeks and were sacrificed after 22 weeks; 6 sheep
served as controls, 6 sheep were treated with weekly intramuscular injection of 150 mg of
nandrolone decanoate after infraspinatus (ISP) repair (group N6W), and 6 sheep were
treated with 150 mg of nandrolone decanoate immediately after tendon release (group
N22W). Muscle biopsy specimens were taken before tendon release and after 16 and 22
weeks. Muscle volume and fatty infiltration (on MRI), myotendinous retraction, and muscle
density (on computed tomography) were measured immediately after ISP release, after 6
weeks, and before ISP repair and sacrifice. Muscle volume on MRI decreased to a mean
(±SD) of 80 ± 8 percent of the original volume after 6 weeks, remained stable at 78 ± 11
percent after 16 weeks, and decreased further to 69 ± 9 percent after 22 weeks in the control
group. These findings were no different from those in group N22W (72 % ± 9 % at 6 weeks,
73 % ± 6 % at 16 weeks, and 67 % ± 5 % at 22 weeks). Conversely, the N6W group did not
show a decrease in ISP volume after repair; this finding differed significantly from the
response in the control and N22W groups. Fatty infiltration (on MRI) continuously increased
in the control group (12 % ± 4 % at tendon release, 17 % ± 4 % after 6 weeks, 50 % ± 9 %
after 16 weeks, and 60 % ± 8 % after 22 weeks) and the N6W group. However, application of
anabolic steroids at the time of tendon release (N22W group) significantly reduced fatty
infiltration after 16 (16 % ± 5 %) and 22 weeks (22 % ± 7 %). It was concluded thatn in a
sheep model of rotator cuff tendon tear, further muscle atrophy can be prevented with the
application of anabolic steroids starting immediately after tendon repair. In addition, fatty
muscle infiltration can largely be prevented if the steroids are applied immediately after
tendon release [150145].
Andropause and somatopause
Aging is accompanied by gradual but progressive reductions in the secretion of testosterone

and growth hormone in men, and by alterations in body composition and functional capacity
that, to some degree, undo the effects of puberty. Preventing or reversing these changes
with the use of trophic factors, including androgens, growth hormone, and growth hormone
secretagogues, is an appealing prospect, but documenting the effectiveness of these
interventions and their benefits and risks has proven to be a difficult undertaking that is far
from complete. Small-scale clinical studies have shown that it is practicable to boost growth
hormone and IGF-1 levels for periods of up to 12 months, and testosterone for up to 36
months, to reverse at least some age-related changes in body composition. Information
regarding the effects of these interventions on strength, exercise capacity, and the ability to
perform activities of daily living is still sparse, and additional reports from recently completed
or currently ongoing clinical trials will not provide sufficient data to make firm conclusions.
From the limited information currently available, androgen supplementation may be of benefit
in some men aged more than 65 years, particularly in men with low serum testosterone
levels (< 2 ng/mL). In this group, supplemental androgen therapy would be expected to
increase lean body mass, bone mass, and possibly strength. In older men with testosterone
levels between 2 and 3.5 ng/mL, some benefit might result from androgen supplementation,
826
but it is not yet clear whether the benefits outweigh the risks. For men in this category, one
might consider a 6- to 12-month trial of therapy after a full discussion and explicit consent,
followed by a reassessment of the value of ongoing treatment. The even more limited data
on growth hormone or growth hormone secretagogue interventions in aging do not support
their general clinical use in healthy older men. Growth hormone is much more expensive
than testosterone and is not covered by insurance for off-label uses. Patients who
persistently seek a trial of therapy should be encouraged to enroll in a study if one is locally
available. All of the growth hormone studies reported to date have focused, generally for
reasons of safety, on healthy and robust groups of older subjects, men in whom the need for
intervention is least compelling and in whom the functional effects of treatment may be the
most difficult to observe. Phase II studies of intermediate size and duration examining prefrail
groups of elderly who are at greater risk for functional loss and who stand to benefit the most
from either preventive or restorative interventions are underway but are limited to the
intermediate outcomes of body composition, strength, and function. Trials designed to
assess clinically relevant final outcomes, such as falls, fractures, and institutionalization, are
of necessity large-scale, long-term, and expensive. Support for larger phase III studies of
growth hormone is unlikely to be forthcoming until the phase II studies are completed and
show further promise. A multicenter clinical trial of testosterone is currently being planned
under the joint sponsorship of the National Institute on Aging, the Veterans Health
Administration, and industry, aimed at assessing the effects of testosterone on the risk for
falls and fractures. The results of this trial and other large clinical trials should help to better
define the balance of benefits and risks of trophic factor intervention in normal older men
[01033].
Rehabilitation of hip fractures
Hip fracture occurs predominantly in older people, many of whom are frail and
undernourished. After hip fracture surgery and rehabilitation, most patients experience a
decline in mobility and function. Anabolic steroids, the synthetic derivatives of the male
hormone testosterone, have been used in combination with exercise to improve muscle
mass and strength in athletes. They may have similar effects in older people who are
recovering from hip fracture. To examine the effects (primarily in terms of functional outcome
and adverse events) of anabolic steroids after surgical treatment of hip fracture in older
people it was searched the Cochrane Bone, Joint and Muscle Trauma Group Specialised
Register (10 September 2013), the Cochrane Central Register of Controlled Trials
(CENTRAL) (The Cochrane Library, 2013 Issue 8), MEDLINE (1946 to August Week 4
2013), EMBASE (1974 to 2013 Week 36), trial registers, conference proceedings, and
reference lists of relevant articles. The search was run in September 2013. Two review
authors independently selected trials (based on predefined inclusion criteria), extracted data
and assessed each study's risk of bias. A third review author moderated disagreements.
Only very limited pooling of data was possible. The primary outcomes were function (for
example, independence in mobility and activities of daily living) and adverse events,
including mortality. It was screened 1290 records and found only three trials involving 154
female participants, all of whom were aged above 65 years and had had hip fracture surgery.
All studies had methodological shortcomings that placed them at high or unclear risk of bias.
Because of this high risk of bias, imprecise results and likelihood of publication bias, we
judged the quality of the evidence for all primary outcomes to be very low.These trials tested
two comparisons. One trial had three groups and contributed data to both comparisons.
None of the trials reported on patient acceptability of the intervention.Two very different trials
compared anabolic steroid versus control (no anabolic steroid or placebo). One trial
compared anabolic steroid injections (given weekly until discharge from hospital or four
weeks, whichever came first) versus placebo injections in 29 "frail elderly females". This
827
found very low quality evidence of little difference between the two groups in the numbers
discharged to a higher level of care or dead (one person in the control group died) (8/15
versus 10/14; risk ratio (RR) 0.75, 95 % confidence interval 0.42 to 1.33), time to
independent mobilisation or individual adverse events. The second trial compared anabolic
steroid injections (every three weeks for six months) and daily protein supplementation
versus daily protein supplementation alone in 40 "lean elderly women" who were followed up
for one year after surgery. This trial provided very low quality evidence that anabolic steroid
may result in less dependency, assessed in terms of being either dependent in at least two
functions or dead (one person in the control group died) at six and 12 months, but the result
was also compatible with no difference or an increase in dependency. The trial found no
evidence of between-group differences in individual adverse events.Two trials compared
anabolic steroids combined with another nutritional intervention (“steroid plus”) versus
control (no “steroid plus”). One trial compared anabolic steroid injections every three weeks
for 12 months in combination with daily supplement of vitamin D and calcium versus calcium
only in 63 women who were living independently at home. The other trial compared anabolic
steroid injections every three weeks for six months and daily protein supplementation versus
control in 40 "lean elderly women". Both trials found some evidence of better function in the
steroid plus group. One trial reported greater independence, higher Harris hip scores and
gait speeds in the steroid plus group at 12 months. The second trial found fewer participants
in the anabolic steroid group were either dependent in at least two functions, including
bathing, or dead at six and 12 months. Pooled mortality data (2/51 versus 3/51) from the two
trials showed no evidence of a difference between the two groups at one year. Similarly,
there was no evidence of between-group differences in individual adverse events. Three
participants in the steroid group of one trial reported side effects of hoarseness and
increased facial hair. The other trial reported better quality of life in the steroid plus group. It
was concluded that the available evidence is insufficient to draw conclusions on the effects,
primarily in terms of functional outcome and adverse events, of anabolic steroids, either
separately or in combination with nutritional supplements, after surgical treatment of hip
fracture in older people. Given that the available data points to the potential for more
promising outcomes with a combined anabolic steroid and nutritional supplement
intervention, it was suggested that future research should focus on evaluating this
combination [14746].
Effect of androgen deprivation
Indices of body composition and muscular strength were compared between men with
prostate cancer (PCa) treated with androgen deprivation therapy (ADT) and asymptomatic
matched men. Nine subjects aged 63-83 years with PCa who received ADT (PCa+ADT;
duration 6-180 months) and 11 asymptomatic aged-matched eugonadal men (HM) aged 59-
80 years were assessed for prostate-specific antigen (PSA) and total testosterone (TT). Total
body non-osseous lean mass (TBLM) and right thigh non-osseous fat-free mass (RTLM)
were assessed using dual-energy X-ray absorptiometry. Peak torque of the right knee
extensors at 0° s-1 and 60° s-1, maximal handgrip strength of the dominant hand (MHS) and
whole-body strength (WBS) were assessed. ISO and CON per unit mass of RTLM and MHS
and WBS per unit mass of TBLM were calculated. Age, height, mass, body mass index and
prostate-specific antigen were comparable between groups, while TT was lower in
PCa+ADT. RTLM was similar between groups. Absolute peak torque of the right knee
extensors were lower for PCa+ADT as were extensor effects per unit of RTLM. Absolute
MHS, WBS and MHS per unit of TBLM and WBS per unit of TBLM were lower for PCa+ADT.
Men with PCa who receive ADT experience significant losses in whole-body muscular
strength compared with asymptomatic age-matched men, which may impair functional
828
capacity. These losses in muscular strength appear to involve neuromuscular mechanisms
that are yet to be identified [13124].
Abuse
One of the earliest accounts of androgen abuse dates back to the 1950s where Soviet
weightlifters were allegedly taking testosterone. While the abuse of androgens is more
commonly associated with the weightlifting industry, their use is widespread amongst many
sports, both at the elite and amateur levels. Alarmingly, androgen use is not limited to adults,
with reports that school-aged children use androgens. For bodybuilding and enhanced
athletic performance enhancement, it is common that very high doses of androgens are
consumed. Moreover, it is common practice to employ “stacking” regimes where a number of
different androgens and/or metabolism inhibitors are simultaneously consumed. This can
lead to serious clinical consequences such as abnormal liver function, gynocomastia, severe
psychological or psychiatric disorders, increased risk of cardiovascular disease; and in
females, menstrual disorders and virilization. Many of the androgens that are used are 17-
methylated compounds and are associated with high-liver toxicity [13084].
Methods of abuse
The pattern of AAS abuse among athletes is quite variable, and the dosing intervals are not
usually regular. These patterns include stacking, tapering, plateauing, cycling, and
pyramiding. Many AAS abusers are poly-drug users including the abuse of traditional
recreational drugs and misuse of prescription drugs. Athletes typically administer AAS
intramuscularly with or without oral preparations in cycles lasting 6-12 weeks with periods of
abstinence between these cycles as a mean store duce adverse effects. The abuse of
transdermal patches, sublingual tablets, dermatologic gels, and nasal sprays is rare. In an
attempt to maximize anabolic gains and minimize side effects, some AAS users start with low
doses at the beginning of a cycle and then steadily increase the dose until a gradual
‘‘tapering’’ phase at the end of a cycle (i.e. ‘‘pyramid’’ regimen). Frequently, these athletes
use more than 1 steroid simultaneously (i.e. ‘‘stacking’’) or use several AASs in overlapping
patterns to avoid the development of tolerance (i.e. ‘‘plateauing’’). In an internet study of 207
weight lifters and bodybuilders using AASs, the steroid regimens included a mean of 3
agents with cycles ranging from 5 to 10 weeks and AAS doses 5-29 times above physiologic
replacement doses. Often these athletes ingest other drugs (i.e. ‘‘array’’) as a mean store
duce adverse effects and enhance the effect of AASs. These additional medications include
human chorionic gonadotrophin, anti-acne drugs, oral hypoglycemic agents, analgesics,
ketoconazole shampoo, stimulant aminoacids, erythropoietin, aminoglutethimide, diuretics,
and estrogen antagonists. The abuse of AASs continues, in part, because of the
effectiveness of these regimens with training programs and the perceived difficulty detecting
AAS use, particularly in weight lifters. Commonly abused steroid supplements (i.e.
precursors of testosterone and related hormones) include androstenedione and DHEA. The
latter compound is an endogenous hormone secreted b ytheadrenal cortexin response to
adrenocorticotropin (ACTH) [13003].
Dietary supplements and ergogenic agents, including anabolic steroids, are common
components of present-day bodybuilder and weightlifter training regimens. Prior reports of
anabolic steroid use suggest polypharmacy and high doses of injectable agents. To provide
an updated description of anabolic steroid regimens employed by weightlifters and
bodybuilders and to determine the extent to which anabolic steroid-associated behaviors are
consistent with substance dependence a web-based survey was done. Links to the Web-
829
based survey instrument were established from leading bodybuilding and fitness web pages.
The questionnaire included demographic information, anabolic drug use history, adverse
effects, information sources, and steroid use behavior consistent with criteria for a substance
dependence disorder. A total of 207 subjects provided a detailed anabolic steroid drug
history. Steroid regimens included a mean of 3.1 agents, involved cycles ranging from 5 to
10 weeks, and often included doses 5 to 29 times greater than physiologic replacement
doses. Behavior consistent with a substance dependence disorder was endorsed by 33
percent of respondents. It was concluded that these findings suggest that anabolic steroid
use among weightlifters and bodybuilders continues, generally involving multiple steroids and
additional dietary supplementary agents. The adverse effects, polypharmacy, large dosages,
and risk of substance abuse are all major health care concerns that require further study.
The survey findings provide sports medicine practitioners a reasonable estimate of the
expected drug history among bodybuilders and weightlifters for the use of performance-
enhancing agents [05032].
Abuse dosage
Athletes usually consume supraphysiologic doses of AAS. Steroids are generally taken in 4-
to 12-week cycles. Athletes often ‘‘stack’’ multiple steroids simultaneously and ‘‘pyramid’’ the
dosing schedule, beginning with low dosage and increasing amounts during the middle and
end of a cycle. Between dosing cycles, it is typical for users to have a period of abstinence,
known as a ‘‘drug holiday,’’ which usually occurs during the competition phases. This method
provides 50 to 100 times the physiologically needed dose of steroids, resulting in levels that
are far higher than physiological levels [12119].
Anabolic steroids may be taken orally or injected intramuscularly and are grouped into three
main classes. Testosterone esters, such as testosterone propionate, are injected compounds
and constitute class I. Class II agents include the nortestosterone derivatives (eg, nandrolone
decanoate and nandrolone phenpropionate). Class I and II AASs exert effects at androgen
receptors as well as at estrogen receptors by way of aromatization to estradiol. The third
class of AASs are those alkylated at C-17 and are the orally administered compounds
oxymetholone, methandrostenolone, and stanozolol. Alkylation of these compounds involves
the addition of a methyl or ethyl group to the carbon at position 17 of the steroid backbone.
The alkylation slows the hepatic metabolism of these agents. A typical pattern of use
consists of a combination of injectable and oral steroids taken during 6- to 12-week cycles.
Injectable forms tend to be favored by users because they are less hepatotoxic than the oral
forms. Because oral preparations are cleared from the system more quickly, they are the
preferred form of steroids when drug testing is anticipated. The simultaneous use of multiple
steroids is referred to as “stacking.” A pattern of increasing a dose through a cycle is called
“pyramiding.” Pyramiding can lead to doses 10 to 40 times greater than the dose
recommended for medical indications. By stacking and pyramiding doses, the user hopes to
maximize steroid receptor binding, thereby reducing toxic side effects. These patterns have
remained popular, despite the lack of scientific evidence of a benefit. Some users take other
drugs concurrently in an effort to minimize side effects. These “accessory” medications
include clomiphene and human chorionic gonadotropin and are administered to reverse the
endogenous testosterone production. Additionally, tamoxifen and antiaromatase drugs can
prevent or decrease gynecomastia by limiting estrogenic effects and the metabolism of
excess testosterone derivatives to estradiol. It is not uncommon for users to take other legal
performance-enhancing substances and dietary supplements, such as creatine, glutamine,
and protein, while using AASs [07008].
It is worthy to note that the classification of anabolic steroids (AS) covers a number of
structural variants. Classically, AS are classified as water-soluble orally active forms (17-
830
alpha-alkylated) and lipid-soluble parenteral forms (17-beta-esterified). In addition, they are
often also classified as either testosterone-based, dihydrotestosterone-based (DHT) or 19-
nortestosterone-based (Nandrolone) all of which have differing properties and expected side
effects. The situation is further complicated by belief among users, often stemming from
anecdotal advice, that some AS are better for predominantly “bulking” (e.g. Deca-Durabolin)
while others are better suited to losing body fat or “cutting” (e.g. Winstrol). Users will often
use these different forms of AS in varying quantities. The use of AS is also characterised by
periods of use followed by periods of abstinence, or “cycles”. This helps to maximise the
effects of the drugs while also limiting the negative consequences and allowing the body to
normalise following an “on” cycle. Furthermore, users will often supplement their cycles with
additional pharmaceutical agents both when bulking (e.g. insulin, human growth hormone)
and when losing body fat (clenbuterol, cytomel, 2,4, dinitrophenol). Finally, there are a
surprising number of drugs used to attempt to limit side effects of AS use or normalise the
hypothalamo-pituitary-gonadal (HPG) axis following an AS cycle. These include estrogen
receptor antagonists (tamoxifen), selective estrogen receptor inhibitors (clomifene),
aromatase inhibitors (arimidex), 5-alpha reductase inhibitors (finesteride) and HPG axis
stimulators such as HCG [12114].
There are no specific guidelines on the appropriate dosing of AAS to mimic or supplement,
normal physiologic hormone levels. It has been conducted only a few randomized studies to
look at the effects of supraphysiologic doses of AAS. In one trial, patients received weekly
intramuscular doses of 600 mg of testosterone enanthate or placebo for 10 weeks. However,
the doses utilized were still substantially lower than those commonly utilized by athletes, who
additionally tend to stack multiple AAS preparations in an attempt to increase the desired
effects while decreasing potential adverse effects. Therefore, case reports and retrospective
studies have been the principal contributors in identifying common, as well as life-
threatening, complications, which may potentially arise from AAS abuse [150011].
Consumption of high doses of AAS typically consists in 6-12 week cycles, followed by a 6-12
week period of wash-out. These patterns of AAS use may easily precipitate in periods of
continuous consumption without any AAS-free intervals due to the fact that abusers try to
assure their muscle gains while avoiding withdrawal symptoms [150004].
Combination with other drugs

Several other drugs are frequently associated with the use of supra-pharmacological doses
of AAS by abusers that are designed to increase their effects, diminish side effects or avoid
detection by urine testing. The abuse of other illicit drugs, such as amphetamines and
opioids, has also been shown to be strengthened by AAS use. Moreover, such abuse might
reinforce the occurrence of adverse substance interactions. In particular, in the case of AAS
and amphetamine association, the overdose potential appears to be increased, due to
cardiotoxicity. The contemporary consumption of AAS and bromocriptine, used to rapidly
reduce body fat and total weight, has been described as the cause of a syndrome
characterized by syncopal episodes and atrial fibrillation [150004].
Dose effects
The dose of AAS used by athletes depends on individual needs and the athletic
requirements of the particular sport. Endurance athletes use AAS doses near or slightly
below physiologic replacement concentrations (i.e. about 7 mg testosterone daily) as a
means to block catabolism, while sprinters typically use 1.5- 2 times replacement
concentrations. Traditional strength athletes use much higher doses (i.e.10-100 times
replacement concentrations) to ‘‘bulk up.’’ Generally, the dose of AASs is lower in women
831
than in men. Data from human test participants indicate that AAS produce a dose-dependent
and gender- dependent increase in lean body mass and strength, but the changes are highly
variable and relatively small without an accompanying conditioning and strength program.
The administration of AAS to men participating in weight training consistently produces
increased strength when compared with controls (i.e. weight training alone). The
endogenous testosterone production during male adolescenc eproduces a sex-differential in
lean body mass similar to the increment in lean body mass caused by the administration of
exogenous AAS to adults. However, different androgen-dependent processes have different
testosterone-dose-response relationships. In a study o f61 eugonadal men receiving a long-
acting gonadotropin-releasing hormone agonist to suppress endogenous testosterone
secretion, changes in leg press strength, leg power, thigh and quadriceps muscle volumes,
hemoglobin, and insulin-like growth factor 1 (somatomedin C) positively correlated to
testosterone concentrations. Changes in fat mass and plasma high-densitylipoprotein (HDL)
cholesterol were negatively correlated to the testosterone dose. Although adverse effects
following AAS administration are usually dose related, thereare few data on th elong-term
physiologic effects of chronic AAS use, particularly in women. In addition to dose and
duration of use, long-term toxicity depends on the age of initiation, gender, steroid structure,
and concurrent illicit use of other drugs. The daily production of testosterone in healthy men
is about 4-10 mg compared with about 1 mg in healthy women. Psychotic symptoms
associated with AAS abuse typically occur in individuals using 41 g testosterone weekly, but
the development to psychologic changes is highly variable. In a randomized, placebo-
controlled, crossover trial of 56 healthy men agged 20-50 years, the administration of
testosterone cypionate for 6 weeks in doses increasing to 600 mg/week caused little
psychologic change in most participants (i.e. 84 %). The regimen produced mild hypomania
in 12 percent and marked hypomania in 4 percent of the men [13003].
Different actions due to ways of administration
The route of administration has been found to have an effect on the detection of anabolic
steroids since quantification of testosterone in urine samples is reliable only when the drug
has been administered intramuscularly. Oral administration, by contrast, is more problematic
since it results in rapid pharmacokinetics, this requiring urine samples to be collected as
soon as possible following administration to enable reliable quantification. AAS bind with
variably affinity in the cytoplasm to the androgen receptor (AR), a member of the steroid
hormone receptor family, where they exert potent anabolic and endocrine activities. The AR’s
binding to the androgen response element triggers its potential to act as a transcriptional
modifier of various genes. The enzyme 5-alpha-reductase seems to possess an essential
role by converting AAS into dihydrotestosterone (androstanolone), which acts in the cell
nucleus of target organs, while the enzyme aromatase converts AAS into female sex
hormones (estradiol and estrone). By displacing cortisol from its receptors, they antagonize
the catabolic effects of glucocorticoids. AAS increase strength (by about 5-20 %) and body
weight (by about 2-5 kg) due to an increase of the lean body mass without reduction of fat
mass, although no effects have been observed on endurance performance [12011].
Different anabolic androgenic steroids with different specific actions
Stanozolol is an anabolic steroid compound particulary favoured among athletes and body
builders since it boosts strength without weight gain, while it is not converted to estradiol.
Metribolone (methyltrienolone) is a potent anabolic steroid, a non-aromatizable androgen,
the 17-methylated derivative of trenbolone, which is characterized by high potential for
hepatotoxicity [12011].
832
Recognizing steroid abuse
Early recognition and intervention may prevent adverse and potentially irreversible
consequences. New-onset acne on the back and chest, temporal hair loss, and alopecia are
common signs. Subtle personality or mood changes are sometimes the only manifestation
[07031]:
Cardiac disease in absence of risk factors

Thrombotic events in absence of risk factors
Alopecia
Male pattern baldness in women
Needle marks on buttocks and thighs
New-onset acne affecting the chest and back
Glucose intolerance
Lipid abnormalities
Abnormal liver function tests
Hepatic masses
Jaundice
Deep abscesses in the thighs or buttocks
Human immunodeficiency virus infection or hepatitis
Rapid and pronounced muscle hypertrophy
Tendon injury
Strokes in absence of risk factors
Unexplained syncope
Irritability, hostility
Mood changes (mania or depression)
Personality changes
Psychosis
Breast atrophy in women
Clitoromegaly
Gynecomastia in men
Testicular volume decrease
Virilization in women with voice changes
Training euphoria due to anabolic steroids
Anabolic-androgenic steroids (AASs) are abused primarily in the context of intense exercise
and for the purposes of increasing muscle mass as opposed to drug-induced euphoria. AASs
also modulate the HPA axis and may increase the reinforcing value of exercise through
changes to stress hormone and endorphin release. To test this hypothesis, 26 adult males
drawn from a larger study on AAS use completed a progressive ratio task designed to
examine the reinforcing value of exercise relative to financial reinforcer. Sixteen experienced
and current users (8 on-cycle, 8 off-cycle) and 10 controls matched on quantity×frequency of
exercise, age, and education abstained from exercise for 24h prior to testing and provided
24-h cortisol, plasma cortisol, ACTH, beta-endorphin samples, and measures of mood,
compulsive exercise, and body image. Between group differences indicated that on-cycle
AAS users had the highest beta-endorphin levels, lowest cortisol levels, higher ACTH levels
than controls. Conversely, off-cycle AAS users had the highest cortisol and ACTH levels, but
the lowest beta-endorphin levels. Exercise value was positively correlated with beta-
endorphin and symptoms of AAS dependence. It was concluded that the HPA response to
AASs may explain why AASs are reinforcing in humans and exercise may play a key role in
the development of AAS dependence [14006].
833
Possible use in children and adolescents
The dietary supplements androstenedione, dehydroepiandrosterone, and androstenediol are

precursors in the endogenous production of testosterone. The efficacy and safety of these
prohormones are not well established but are promoted to have the same androgenic effects
on building muscle mass and strength as anabolic-androgenic steroids. Studies have
demonstrated repeatedly that acute and long-term administration of these oral testosterone
precursors does not effectively increase serum testosterone levels and fails to produce any
significant changes in lean body mass, muscle strength, or performance improvement
compared with placebo. Testosterone precursors are banned by most major sports
organizations [07083].
Anabolic steroids, derivatives of testosterone used to enhance anabolic, muscle-building

properties, have important medical uses. For patients with severe osteoporosis, AIDS
wasting syndrome, and occasionally other chronic medical disease states including
hypogonadism, anabolic steroids are important components of treatment. More commonly,
however, these compounds are used by athletes looking to gain the “extra edge” in
competition. Use of steroids has been well described in the medical literature, starting in the
1960s and continuing until the present day. The sanguine approach from the medical
community to anabolic steroids in the 1960s and 1970s has been replaced by a slowly
developing appreciation for the significant medical problems associated with use, including
impotence, cardiovascular disease, mood instability, and hepatic cancer. In children and
teens, anabolic steroid use has been well documented, with various studies showing a
national user rate between 3 and 9 percent in high school students. The major reason for the
failure to convince young athletes to abstain from anabolic steroid use is that these
substances work; they make athletes stronger and faster. This advantage, however, comes
at a very dangerous price. More needs to be done to actively discourage steroid use in
pediatric and adolescent athletes. From a medical perspective, the complete ramifications of
anabolic steroid use in a developing body are not known. Aside from the adult problems
associated with usage, including a significantly increased risk of cardiovascular disease, the
issue of mood instability in teens, whose minds are already in a precarious state of flux,
seems more acute. The upcoming American Academy of Pediatrics policy statement on
performance-enhancing drugs will help, as will the Anabolic Steroid Control Act, which was
signed in October 2004 and bans the sale of over-the-counter presteroid supplements.
These steps will aid the concerted efforts of those who work with young athletes, but the true
momentum needs to come from the grass-roots level, team by team and community by
community. This type of effort can include medical professionals creating educational
seminars for parents and coaches on the issue of performance-enhancing drugs [05035].
In response to the controversy over the use of anabolic steroids, the American Academy of
Pediatrics has condemned their use for bodybuilding or performance enhancement in
adolescents. However, abuse of anabolic steroids, such as androstenedione and
dehydroepiandrosterone, to increase muscle mass is a serious problem not only among
professional athletes but also among bodybuilders and teenagers. Among high school
students, 3 to 12 percent of males and 1 to 2 percent of females admit to anabolic steroid
use at some time. Long-term effects and fatalities due to anabolic steroid abuse have been
reported, including liver tumours, myocardial infarction, stroke and severe arteriosclerosis. A
significant black market has been established in the gym culture, but studies of anabolic
steroids bought on the black market have shown that 35 percent do not contain the expected
ingredients [05008].
834
Pubertal androgen therapy in boys
Anabolic-androgenic steroids (AAS) are necessary for normal male sexual differentiation and
development and pubertal development. Androgen therapy is appropriate for boys with
delayed development (constitutional delay of growth and puberty, CDGP) as well as those
with primary or secondary hypogonadism. The principal goal is to restore the serum
testosterone (T) level to the normal range at each stage of adolescent development and then
to the normal adult range if the hypogonadism is permanent. In addition the levels of
dihydrotestosterone and estradiol should also be within the normal range. One should be
able to do that with a wide variety of androgen preparations-injectable, implantable, and
cutaneous patches or gels. However, during the transition from prepubertal to adult it is
difficult to reliably deliver the relatively small doses of T necessary for adolescent
development using any of the cutaneous preparations. Androgen therapy should permit
normal linear growth (including the adolescent growth spurt), adolescent sexual
development, and the attainment of normal body composition including lean body mass,
bone and the appropriate regional distribution of body fat as well as the psychological
development appropriate for the stage of adolescent development [05036].
Puberty is associated with an increasing production of androgenic steroids. Adrenal

androgen formation, termed adrenarche, may precede gonadal testosterone synthesis. Both
adrenal and gonadal androgens exert their biological effects via the androgen receptor, a
nuclear transcription factor modulating a specific transcription regulation of largely unknown
genes. During puberty, virilizing actions such as genital enlargement and sexual hair growth
can be distinguished from anabolic action such as the gain in muscle strength and general
changes in body composition. Furthermore, androgens play a major role in the initiation and
maintenance of spermatogenesis. Thus, different androgenic steroids play an important role
in the process of puberty. The control of their biosynthesis, their possible differential action
on the molecular level, as well as the different target organs in males and females are
discussed [02027].
Clinical effects
Children seem to be the most susceptible to the adverse effects of AAS use. Children and
adolescents experience accelerated maturation associated with changes in physique and
earlier development of secondary sexual characteristics. An additional concern with
adolescents is premature closure of growth plates in long bones, leading to a decrease in
final height; this likely is due to aromatization to estrogens. Precocious puberty in boys and
contrasexual precocity in girls also can occur. With adolescents, some of the effects may
become irreversible with chronic use, particularly the virilizing effects in young women
[07058].
One article was part of a Special Issue "Puberty and Adolescence". Puberty is a critical
period for brain maturation that is highly dependent on gonadal sex hormones. Modifications
in the gonadal steroid environment, via the use of anabolic androgenic steroids (AAS), have
been shown to affect brain development and behavior. Studies in both humans and animal
models indicate that AAS exposure during adolescence alters normal brain remodeling,
including structural changes and neurotransmitter function. The most commonly reported
behavioral effect is an increase in aggression. Evidence has been presented to identify
factors that influence the effect of AAS on the expression of aggression. The chemical
composition of the AAS plays a major role in determining whether aggression is displayed,
with testosterone being the most effective. The hormonal context, the environmental context,
835
physical provocation and the perceived threat during the social encounter have all been
found to influence the expression of aggression and sexual behavior. All of these factors
point toward an altered behavioral state that includes an increased readiness to respond to a
social encounter with heightened vigilance and enhanced motivation. This AAS-induced state
may be defined as emboldenment. The evidence suggests that the use of AAS during this
critical period of development may increase the risk for maladaptive behaviors along with
neurological disorders [13123].
It was presented a case of a 16-year-old male who presented reporting a 6-month history of
lowered mood, fatigue, anhedonia, disturbed sleep and heightened anxiety. On further
questioning he reported restricted eating and weightlifting for at least 1 h on a daily basis.
Investigations revealed findings compatible with secondary hypogonadism. The potential
causes of secondary hypogonadism including structural lesions, muscle dysmorphia and use
of illicit anabolic steroids were discussed [14055].
Aggresiveness
In a double-blind, placebo-controlled, crossover trial, it was given injections of testosterone in
increasing doses to 35 boys and oral doses of conjugated estrogens to 14 girls with delayed
puberty. Both treatment groups had more physical aggressive behavior and aggressive
impulses than those receiving placebo. In another study it was compared plasma levels of
hormones in 15 boys with conduct disorders and in 25 normal controls. The boys with
conduct disorders had significantly higher levels of dehydroepiandrosterone sulfate,
marginally significantly higher levels of androstenedione, and no differences in testosterone
levels [07031].
Epiphyseal plate closure in adolescents

Premature closure of the epiphyseal plates during chronic AAS use can stunt bone growth
[13003].
Prevalence of adolescent anabolic-androgenic steroid use
The first reported adolescent use of AASs was in 1959 by a high school football player.
Estimates of high school steroid usage in 2007 ranged from 4 to 11 percent in boys and up to
3.3 percent in girls. A landmark study of prevalence involved a nationwide survey of more
than 3000 boys. They found that 6.6 percent of male high school seniors had tried steroids,
with 67 percent initiating use by 16 years of age and 40 percent using multiple cycles. These
results have been confirmed in later studies and a 2003 Centers for Disease Control and
Prevention report finding of a 6.4 percent use of steroids by 12th-grade boys. The largest
nationwide cohort of nearly 50,000 students is being examined in the Monitoring the Future
study. As of 2004, results of this ongoing study indicated a 1.3, 2.3, and 3.3 percent annual
prevalence of male AAS users in the eighth, 10th, and 12th grades, respectively. Girls in the
12th grade had a 1.7 percent use rate in this study, whereas the Centers for Disease Control
and Prevention reported a 3.3 percent lifetime prevalence in 12th-grade girls. AAS use by
adolescents is not limited to the United States. Three Canadian studies, two Swedish
surveys, two South African investigations, one British study, and one Australian investigation
reported an overall prevalence range between 1 and 3 percent. Although slightly lower, these
rates approximate those reported in the United States, demonstrating that the impact of
AASs on athletic performance and physical appearance reaches across cultures A
considerable percentage of adolescents turn to AAS use to help them achieve an attractive
physique. This is the second most popular reason for using AASs. One study of bodybuilders
suggests that the drive for a muscular physique sometimes reaches an unhealthy extreme
and likens the use of AAS to the “unhealthy extremes” that are characteristic of anorexic and
bulimic individuals. Just as eating disordered women see their bodies as larger than they
836
actually are, some men perceive themselves as smaller than they actually are. This
phenomenon has been referred to as “bigamerexia” and suggests that this misperception
may be a contributory factor in AAS use. This misperception is likely evident in many ninth-
grade boys, who – in the early stages of puberty – are impatient with their muscular
development. Perceiving themselves smaller than their peers, these boys may engage in
AAS use as a shortcut to increasing muscle strength and size. Exposure to the media may
intensify this body dysmorphia. Adolescent AAS use has been associated with the use of
other harmful drugs, including cigarettes, smokeless tobacco, marijuana, alcohol, cocaine,
and injected drugs. Thus, AAS use would be considered a part of this cluster rather than an
isolated behavior [07008].
It was examined the prevalence, persistence, secular and longitudinal trends, and predictors
of steroid use in a diverse sample of adolescents. Data are from Project EAT-II (Eating
Among Teens), a 5-year longitudinal study of eating, activity, weight, and related variables in
2516 middle and high school students. Data were collected in 1999 (time 1) and 2004 (time
2). Approximately 1.5 percent of adolescents reported steroid use at time 2. Use differed by
ethnicity but not socioeconomic status. Steroid use was not stable across time, although the
risk of use at time 2 was higher for girls and (marginally) for boys who used steroids at time
1. No secular trends were noted in middle adolescents' steroid use between 1999 and 2004.
Developmentally, steroid use decreased as adolescents grew older. Predictors of use for
male adolescents included wanting to weigh more and reporting higher use of healthy
weight-control behaviors. Female time 2 steroid users had higher BMIs and were less
satisfied with their weight, had poorer nutrition knowledge and concern for health, and were
marginally more likely to have participated in weight-related sports at time 1. It was
concluded that the prevalence of steroid use in adolescents was low but of concern.
Although use was not persistent over 5 years, time 1 use was a risk factor for time 2 use in
female adolescents. There was no change in the prevalence of steroid use by middle
adolescents between 1999 and 2004 despite a great deal of public interest in steroids during
this time period. Steroid use decreased as adolescents grew older. Weight-related variables
predicted adolescents' steroid use 5 years later, and health and nutrition knowledge and
concern and (marginally) participation in weight-related sports further predicted use in female
adolescents. These findings suggest that early preventive efforts may be most useful
[07084].
Girls
Recent media reports have portrayed an alarming increase in apparent anabolic-androgenic
steroid (AAS) use among American teenage girls; Congress even held hearings on the
subject in June 2005. It was questioned whether AAS use among teenage girls was as
widespread as claimed. It was reviewed four large national surveys and many smaller
surveys examining the prevalence of AAS use among teenage girls. Virtually all of these
surveys used anonymous questionnaires. It was asked particularly whether the language of
survey questions might generate false-positive responses among girls who misinterpreted
the term "steroid." It was also reviewed data from other countries, together with results from
the only recent study in which investigators personally interviewed female AAS users. The
surveys produced remarkably disparate findings, with the lifetime prevalence of AAS use
estimated as high as 7.3 percent among ninth-grade girls in one study, but only 0.1 percent
among teenage girls in several others. Upon examining the surveys reporting an elevated
prevalence, it appeared that most used questions that failed to distinguish between anabolic
steroids, corticosteroids, and over-the-counter supplements that respondents might confuse
with "steroids." Other features in the phrasing of certain questions also seemed likely to
further bias results in favor of false-positive responses. It was concluded that many
anonymous surveys, using imprecise questions, appear to have greatly overestimated the
lifetime prevalence of AAS use among teenage girls; the true lifetime prevalence may well be
837
as low as 0.1 percent. Future studies can test this impression by using a carefully phrased
question regarding AAS use [07085].
Use of testosterone precursors by adolescents
The extent of the use of testosterone precursors, such as androstenedione and DHEA, in the
pediatric and adolescent population is unknown. The initial over-the-counter dietary
supplement status and availability more than likely led to a large increase in the number of
adolescents using testosterone precursors. A 2002 survey of 475 high school students by
revealed that 4 percent of athletes and nonathletes admitted to using steroid precursors in
the past year. Surveys by the National Collegiate Athletic Association (NCAA) revealed that 5
percent of athletes admitted to using DHEA or androstenedione, 33 percent admitted to
using nutritional supplements, and 1 percent admitted to using anabolic steroids. With the
passage of the Anabolic Steroid Control Act of 2004, androstenedione became illegal to
purchase; the only available source is through the black market or acquaintances. One would
surmise that the use of testosterone precursors among adolescents would be higher than the
use of AASs [07085].
Use in women
Prevalence
Anabolic-androgenic steroids (AAS) are synthetic derivatives of testosterone that maximize

anabolic and minimize androgenic effects. Exogenous testosterone is rapidly metabolized
and has no significant effect on performance. Men have traditionally used AAS for muscle
gain. Women have also used AAS for increased strength and lean muscle mass. AAS use by
women was first sensationalized when East German athletes routinely received steroids from
team doctors. Surveys analyzing AAS prevalence in adolescent girls have found a range
from 0.1 to 4.8 percent. The Centers for Disease Control and Prevention (CDC) reported an
overall prevalence of lifetime steroid use in females, grades 9 to 12, of 3.2 percent. In
another study surveying boys and girls between 9 and 13 years old, found that 2.8 percent of
girls and 2.6 percent of boys reported AAS use. Steroid use by girls was found across
various sports, with the highest prevalence in weight training. Steroid users were more likely
to think that steroids improve athletic performance or enhance appearance Steroid use
prevalence has also been studied in women collegiate athletes. A National Collegiate Athletic
Association (NCAA) survey of more than 19,000 collegiate men and women found that
between 2001 and 2005 AAS use increased in women's ice hockey, gymnastics, and
volleyball]. Between 2001 and 2005, AAS use increased in women's ice hockey from 0.8 to
2.4 percent, in women's gymnastics from 0 to 1 percent, and in volleyball from 0.1 to 0.6
percent [07086].
Effects
Studying AAS effects is difficult due to an incomplete understanding of how athletes use
steroids. Athletes often combine AAS with other supplements, or use doses several times
higher than recommended. Data on the performance-enhancing effect of AAS on women is
limited. However, most studies in men found an increase in muscle mass and body weight by
an average of 2 to 5 kg. Steroids have also been shown to increase strength, with
improvements ranging from 5 to 20 percent. Clinical trials have not demonstrated improved
endurance performance. A double-blind, placebo-controlled trial gave AAS to men 12 times
over 1 month, showing no improvement in treadmill testing [07086].
838
Side effects
Women taking AAS are at risk for androgenic side effects, including deepened voice, acne,
male pattern baldness, clitoromegaly, menstrual irregularities, and increased facial hair. A
survey of steroid-using women bodybuilders found that 64 percent reported an adverse
psychological effect, including labile mood, irritability, or aggressive behavior most frequently.
More serious side effects include liver damage. Transient increases in hepatic enzymes that
return to normal after steroids are discontinued have occurred. Other significant adverse
effects include cholestasis, hepatic peliosis, and hepatocellular hyperplasia that may lead to
hepatic carcinoma [07086].
In one study it was evaluated 75 female bodybuilders and weightlifters and found that 33
percent reported current or past anabolic-androgenic steroid use. Among steroid users, 56
percent reported hypomanic symptoms during use, and 40 percent reported depression
when the steroids were discontinued. Some users developed a body image distortion similar
to “reverse anorexia,” in which they felt they were too small. Ten of the 75 weightlifters had
been raped as teenagers or adults, and most started or increased their weightlifting activities
as a defense strategy. Seven of the 10 rape victims used anabolic steroids [07031].
Steroid precursores (androstenedione and dehydroepiandrosterone)
Steroid precursors include androstenedione and dehydroepiandrosterone (DHEA). These

substances are closely related to testosterone. Due to poor surveillance, the prevalence of
steroid precursor use by girls and women is difficult to determine. The NCAA's study of
substance use habits of college student-athletes found that 4.7 percent of men and women
athletes surveyed had tried DHEA or androstenedione products at least once in the past
year. The survey data, however, did not organize the percentages in terms of men and
women. The CDC's survey on youth risk behavior does not ask specifically about steroid
precursors. One study of high school students found that androstenedione was the second
most popular supplement, with 4 percent of students using it at least once in the past year.
Again, the authors did not isolate a number for girls using androstenedione. It is also
believed that these substances may have an androgenizing effect in female athletes.
Androstenedione, produced in the adrenal glands and gonads, is more potent than DHEA,
which is naturally produced in the adrenal cortex. The actual physiologic effects and effects
on athletic performance have not been consistent. Several studies in women have shown
increased serum androstenedione and testosterone after androstenedione supplementation.
No trials have been published examining steroid precursors' performance-enhancing effects
in women. Further, studies in men have not consistently found an improvement in athletic
performance with androstenedione or DHE. Steroid precursor use carries the same risks as
testosterone use. Common adverse effects in women include virilization. There is also a risk
of testing positive for testosterone or androstenedione [07086].
Anabolic steroids in young females
Women who take AAS can experience androgenic effects including changes in libido, male-
pattern baldness, deepening of the voice, acne vulgaris, and other masculinizing effects in
early use. Longer-term use causes clitoromegaly, changes in pubic hair growth, menstrual
irregularities, and even breast reduction [06031].
Media reports have portrayed an alarming increase in apparent anabolic-androgenic steroid

(AAS) use among American teenage girls; Congress even held hearings on the subject in
June 2005. It was questioned whether AAS use among teenage girls was as widespread as
839
claimed. It was reviewed four large national surveys and many smaller surveys examining
the prevalence of AAS use among teenage girls. Virtually all of these surveys used
anonymous questionnaires. It was asked particularly whether the language of survey
questions might generate false-positive responses among girls who misinterpreted the term
"steroid." It was also reviewed data from other countries, together with results from the only
recent study (to our knowledge) in which investigators personally interviewed female AAS
users. The surveys produced remarkably disparate findings, with the lifetime prevalence of
AAS use estimated as high as 7.3 percent among ninth-grade girls in one study, but only 0.1
percent among teenage girls in several others. Upon examining the surveys reporting an
elevated prevalence, it appeared that most used questions that failed to distinguish between
anabolic steroids, corticosteroids, and over-the-counter supplements that respondents might
confuse with "steroids." Other features in the phrasing of certain questions also seemed
likely to further bias results in favor of false-positive responses. It was concluded that many
anonymous surveys, using imprecise questions, appear to have greatly overestimated the
lifetime prevalence of AAS use among teenage girls; the true lifetime prevalence may well be
as low as 0.1 percent. Future studies can test this impression by using a carefully phrased
question regarding AAS use [06097].
Pregnancy
During pregnancy, however, female athletes encounter much more dramatic changes.
Controlled longitudinal studies of steroid profile during pregnancy are scarce, but according
to available data, significant alterations occur not only in the production of progesterone and
oestriol, but also in androgen concentrations. For the status of the steroid profile and its
interpretation, the most significant factors are pregnandiol (PD) and T itself. The PD
concentration may increase up to 10–100 fold (to 10 000 ng/mL) from the baseline levels
during the early pregnancy, and despite being quite theoretical in performance-sport context,
the levels of 20 000 ng/mL concentration can be reached just before delivery. In a recent
work it was conducted a longitudinal study in three pregnant women, and focused on
cysteine-conjugated androgens and glucuronide-conjugated androgens and oestrogens
during different trimesters of pregnancy. From a steroid profile perspective, there was a
significant increase in urinary oestrogen levels and moderate decrease in urinary androgen
concentration, and thus alteration in general profiles due to pregnancy. Interesting results
were obtained for E glucuronide concentrations, which were elevated during the first
trimester, and thus a feature to take into account in interpreting of T/E in steroid profiles of
female athletes. The results of this study were also well in accordance with the earlier ones
describing the formation of norandrosterone, a nandrolone metabolite, during pregnancy
[14450].
Estrogen antagonists
Estrogen antagonists, such as tamoxifen, clomiphene, and anastrozole, have been used to
block estrogen and increase testosterone effects. In the 1970s, the first reports of men using
tamoxifen along with AAS occurred. Bodybuilders claimed that it decreased fat over muscle
and produced a leaner appearance. An Internet-based survey of 500 bodybuilders found that
53 percent of AAS users were also using tamoxifen. This study included 494 men and 6
women. One case report and one survey have captured tamoxifen use for performance
enhancement in women. All published reports have been in bodybuilders. In 2002, it was
published a case report detailing a female competitive bodybuilder using tamoxifen based on
a recommendation from another bodybuilder. She claimed it was easier to lose fat but noted
side effects such as night sweats, hot flashes, and breast-mass loss. A recent survey of
health club members to determine the prevalence of nontherapeutic medication use found
840
that 22 percent had used tamoxifen. Seven percent of respondents to the survey were
women. Tamoxifen use had increased from its previous levels. Tamoxifen's effect on athletic
performance is unknown. Anecdotal reports of fat loss and leaner physique exist, but no
studies have evaluated their validity. This medication involves multiple adverse effects for
women, including endometrial cancer, thromboembolic events, teratogenic effects, ovarian
cysts, bone-density loss, cataracts, hot flashes, and vaginal discharge [07086].
Genetic influence
In 1982, the International Olympic Committee accepted a T/E ratio greater than six as proof
of testosterone doping, based on the log-normal distribution of the ratio established from the
first population studies. A testosterone over epitestosterone (T/E) ratio exceeding 4.0 has
later been considered as suspicious of testosterone administration, irrespectively of
individual heterogeneous factors such as the athlete’s ethnicity. In antidoping laboratories,
the urinary steroid profile usually encompasses the concentration levels of testosterone (T);
its inactive epimer, epitestosterone (E); four testosterone metabolites, androsterone (A),
etiocholanolone (Etio), 5α-androstane-3α,17β-diol (α-diol) and 5β-androstane-3α,17β-diol (β-
diol); and a testosterone precursor, dehydroepiandrosterone (DHEA). The following cut-off
concentration levels of endogenous steroids equivalent to the glucuronide: T>200 ng/ml,
E>200 ng/ml, A>10 000 ng/ml, Etio>10 000 ng/ml and DHEA>100 ng/ml are considered as
putative markers of androgen administration. In contrast to absolute steroid concentrations,
ratios such as T/E, A/Etio, A/T, α-diol/Ε and α-diol/β-diol are robust to circadian rhythm or
changes in physiological conditions such as exercise workload for athletes. On the other
hand, these parameters may be significantly altered according to the administered steroid
and its application mode. However, a T/E higher than 4.0 no longer constitutes proof of
testosterone misuse, but requires a subsequent confirmation analysis by gas
chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). GC/C/IRMS
allows measurement of slight differences in 13C/12C ratio of testosterone metabolites. A
discrimination is feasible since exogenous testosterone or its precursors contain less 13C
than their endogenous homologues. A deletion polymorphism in the UGT2B17 gene was
demonstrated to account for a significant part of the interindividual variability in the T/E
between Caucasians and Asians. The variability of urinary steroid profiles was examined in a
widely heterogeneous cohort of professional soccer players. The steroid profile of 57
Africans, 32 Asians, 50 Caucasians and 32 Hispanics was determined by gas
chromatography–mass spectrometry. Significant differences were observed between all
ethnic groups. After estimation of the prevalence of the UGT2B17 deletion/deletion genotype
(African: 22 %; Asian: 81 %; Caucasian: 10 %; Hispanic: 7 %), ethnic-specific thresholds
were developed for a specificity of 99 percent for the T/E (African: 5.6; Asian: 3.8;
Caucasian: 5.7; Hispanic: 5.8). Finally, another polymorphism could be hypothesised in
Asians based on specific concentration ratio of 5α-/5β-androstane-3α,17β-diol in urine.
These results demonstrate that a unique and non-specific threshold to evidence testosterone
misuse is not fit for purpose. An athlete’s endocrinological passport consisting of a
longitudinal follow-up together with the ethnicity and/or the genotype would strongly enhance
the detection of testosterone abuse. Finally, additional genotyping studies should be
undertaken to determine whether the remaining unexplained disparities have an
environmental or a genetic origin [09049].
Androgens are an essential part of endocrinological homeostasis in human body and their
dual effects are associated mainly to masculinisation (androgenic effects) and protein
synthesis (anabolic effects). There are several mechanisms and functions which mediate the
androgen action, control the transport and binding of T and other androgens or activate the
expression of androgen-responsive genes. In human genome, two or more variants can be
841
encountered for a particular DNA sequence. In its simplest form, this natural variation,
polymorphism, involves not only a single nucleotide (SNP), but also longer DNA stretches
can be involved. The outcome of the complex network of these bioprocesses and
interindividual as well as interethnic variations within them leads to a steroid profile with an
individual baseline of endogenous steroids. Massive amounts of research results are
available on the clinical and pathological relevance of androgens and the factors contributing
to the phenotype of an individual. For example, low serum T concentration is associated to
several pathological conditions, for example, cardiovascular morbidity, type 2 diabetes and
increased risk of mortality. The studies indicate strong heritability of serum T levels and
clinical studies have focused on T as a biomarker of male health status and on the effects of
genetic variants on serum T concentrations. Although sports and doping control involve only
minor fraction of population, the atypical patterns, anomalies and pathological conditions are
factors to keep in mind when evaluating individual athlete profiles [14450].
Phase II metabolism, usually known as conjugation reactions, generate metabolites that are,
in most cases, biologically inactive and subsequently excreted in bile or urine. Androgenic
and estrogenic sex steroids are mainly inactivated by sulfation or glucuronidation by the
enzyme families’ sulfotransferases (SULTs) or uridine diphospho glucuronosyltransferases
(UGTs). Genetic variations in UGTs and SULTs have been implicated to play a role in
hormone-dependent diseases and in the outcome for doping test results. Moreover, the use
of drugs may interact with UGTs and SULTs and hence affect the phase II metabolism. The
aim of a research topic forum was to highlight the progress made in this field via review
papers and original articles as well as to promote future research with the aim to further
understand the consequences of inter-individual difference in phase II metabolism and
regulation of sex steroids. Thus, the UGT2B17 deletion polymorphism impact on
testosterone replacement therapy was studied. There was no association with UGT2B17
genotype and testosterone serum peak levels, whereas the increase in testosterone from
week 8 to 18 differed, subjects homozygous for deletion (del/del) had a smaller increase as
compared to UGT2B17 carriers. Moreover, an association between LH levels and UGT2B17
genotype was found; del/del subjects exhibit lower levels of LH during treatment period.
Testosterone is not only being therapeutically used but also a common drug together with
synthetic androgens, i.e, nandrolone to abuse (doping). It was also shown that a well
characterized single nucleotide polymorphism in UGT2B15 is associated with the
glucurunidation activity of 19-noradrosterone, the nandrolone metabolite analyzed at the
WADA accredited doping labs. Moreover, it was confirmed that UGT2B17 deletion
polymorphism is an important determinant of T/E (a biomarker for testosterone doping) in
women. Moreover, the use of hormonal contraceptives was shown to affect the T/E ratio,
which will be important to consider in doping test programs. The fact that drug use can affect
the phase II metabolism and consequently doping test results were also investigated and
found that the use of NSAIDs (diclofenac and ibuprofen) did not interact with the urinary
excretion of testosterone- and epitestosterone-glucuronides, and hence have no impact on
T/E ratio. It was also noted that dietary compounds such as white and green tea inhibit
testosterone UGT2B17-derived glucuronidation activity in vitro. Hence, it is possible that in
addition to drug use, the diet may influence the UGTs and alter the risk of hormone-related
diseases and impacting doping test results; however, this needs to be verified in vivo. Even
though most androgens are excreted as glucuronides, some are also subject to sulfate
conjugations. It has been identified a new copy number variation polymorphism in the
SULT2A1 gene. This CNV alters the capacity to excrete testosterone and some of its
metabolites in the urine. Insertions are associated with higher excretion rate, both at basal
level and after the administration of 500 mg testosterone enanthate. It is possible that this
polymorphism may alter the risk of hormone-related diseases [150146].
Covariation with BMI

842
A number of candidate gene and genome-wide association studies have identified significant
associations between single nucleotide polymorphisms, particularly in FTO and MC4R, and
body weight. However, the association between copy number variation and body weight is
less understood. Anabolic androgenic steroids, such as testosterone, can regulate body
weight. In humans, UDP-glucuronosyltransferase 2B17 (UGT2B17) metabolizes testosterone
to a metabolite, which is readily excreted in urine. It was investigate the association between
genetic and phenotypic variation in UGT2B17 and body weight. UGT2B17 deletion was
genotyped and in-vivo UGT2B17 enzymatic activity (as measured by the 3-hydroxycotinine
glucuronide to free 3-hydroxycotinine ratio) was measured in 400 Alaska Native individuals
and 540 African Americans. In Alaska Native people, UGT2B17 deletion was strongly
associated with lower BMI in male individuals, but not in female individuals, consistent with
testosterone being a male dominant steroid. The sex-specific association between UGT2B17
deletion and lower BMI was also observed in African Americans. In both populations,
UGT2B17 deletion was significantly associated with lower measured in-vivo UGT2B17
activity. In male individuals, lower in-vivo UGT2B17 activity was associated with lower BMI,
as observed in the sex-specific genotypic association. These data suggest that UGT2B17
deletion leads to reduced UGT2B17 activity, and lower BMI in male individuals. This is
consistent with the hypothesis that reduced UGT2B17-mediated testosterone excretion
results in higher testosterone levels [150147].
UGT2B17 gene deletion
Testosterone is mainly excreted as a glucuronide conjugate after its metabolism by uridine

diphosphate (UDP) and glucuronosyl transferase (UGT). UGT2B7, UGT2B15, and UGT2B17
are known to be the main glucuronidation catalysts of androgens and their metabolites in
humans. Testosterone is mainly conjugated by UGT2B17 and to a lesser extent by
UGT2B15. The main androgen substrate of UGT2B15 is androstane‐3alpha 17beta‐diol. The
actions of UGT2B17 are 96 percent in common with those of UGT2B15. The enzyme
UGT2B7 also has the capacity to conjugate epitestosterone, while testosterone is a poor
substrate for this enzyme. It has been established that a deletion polymorphism in the gene
that codes for UGT2B17 correlates highly with testosterone levels in urine. Thus, subjects
lacking this gene have been found to show a T/E ratio lower than 0.4. This polymorphism is
much more common in Asian subjects than Caucasian subjects and its prevalence has been
estimated at 66.7 percent in Asians versus 9.3 percent in Caucasians. The measurement of
the testosterone to epitestosterone ratio (T/E ratio) in urine is often used as a marker for
testosterone administration in the doping control field. One study examines the frequencies
of the different expression forms of the UGT2B17 gene, and assesses their effects on this
marker in volunteer subjects. The sample for this descriptive study was composed of male
and female athletes aged between 16 and 55 years old who practiced different sports
disciplines. All participants underwent a sports-medical physical examination, and
subsequently provided 10 urine samples consecutively over a period of 48 h. The dependent
variable examined was T/E and the main independent variable was the UGT2B17 gene
polymorphism. During 1 year, 1410 urine samples were obtained from 141 athletes. The
frequencies of the three genotypes were as follows: wt homozygotes (ins/ins) 48 percent
(n=68), mutant homozygotes (del/del) 12 percent (n=17), and heterozygotes (ins/del) 0
percent (n=56). Genotype distributions varied significantly according to ethnicity, 80 percent
of Asian subjects being homozygous for the gene deletion (del/del) compared to 6.9 percent
of Caucasian subjects. A multivariate analysis adjusted for genotype, age, sex, and sports
discipline revealed that athletes with the del/del polymorphism showed a significantly lower
mean T/E than heterozygotes (ins/del). In contrast, homozygous athletes for the gene
insertion (ins/ins) showed higher mean T/E ratios than heterozygotes (ins/del). UGT2B17
843
gene deletion has a strong influence on the T/E ratio in urine, which is the most efficient
indicator of testosterone prohormone misuse. Others factors studied seem not to have such
an impact. The genotyping of UGT2B17 is an important source of information for
understanding steroid profiling in the doping control field; therefore it is suggested that it be
included in the Athletes Biological Passport [150148].
Doping tests and genetic confounders
So far it is known of only genetic variations in three enzyme (UGT2B17, CYP17 and PDE7B)
genes with a substantial influence on the T/E ratio. The major part of testosterone is excreted
as the glucuronide but it was found a 100- or more -fold variation in excretion rate, both in a
Caucasian and an Asian study group. A majority (75 %) of Asian subjects falls within the “low
excretion” group compared with 9 percentr among the Caucasians, and the widely different
distribution into the two modes indicated a monogenic background of the excretion pattern.
The conspicuous intra- and interethnic differences in testosterone excretion gave cause for
concern about how to interpret the classical urinary T/E screening test as an indicator of
exogenous administration of the hormone. The future test programme for testosterone ought
to adopt a Bayesian inference technique for analysis of consecutive T/E samples in the
individual, as has been applied in the detection of blood doping with the “blood pass”
[12192].
In summary, large genetic variation in the disposition and effects of anabolic androgenic
steroids is a reality that must be considered in the doping control strategy in sports as well as
in society. Polymorphisms in three crucial enzyme genes have hitherto been shown to have
an impact on the disposition of testosterone and the T/E ratio. Further genetic confounders in
other genes cannot be excluded at present time. False negative and false positive results
with the current principles for interpretation of the T/E screening test as described will lead to
unnecessary and expensive follow-up analyses of many samples [12192].
UDP-glucuronosyltransferase and UGT2B17 genotype
Synthetic anabolic androgenic steroids are a group of testosterone derivatives that are widely
misused in sport to improve the physical performance of skeletal muscle and to balance the
catabolic condition after stress. These compounds are extensively modified in the body by
phase I and phase II metabolic reactions before excretion in the urine, and their phase I
metabolism has been described previously. Glucuronidation, a typical reaction of phase II
metabolism, is probably the main pathway of conjugation with anabolic androgenic steroid
metabolites, but detailed information about the factors that determine and affect this
biotransformation is still very limited. More is known, however, on glucuronidation of clinically
important endogenous steroids, which have been taken as an estimate for the total
androgenic pool in men, or for the production of dihydrotestosterone in extrahepatic tissues.
UDP-glucuronosyltransferases (UGTs; EC 2.4.1.17) are a family of membrane-bound
enzymes of the endoplasmic reticulum. They catalyze the glucuronidation of various
endogenous and exogenous compounds, including steroids, thereby converting the substrate
molecules (the aglycones) into a less toxic and more polar beta-D-glucuronides. The human
genome encodes at least 16 different UGTs, and they are divided into families (1 and 2) and
subfamilies (2A and 2B) according to the degree of sequence identities and genomic
organization. Most of the UGTs are expressed in the liver, the organ that is considered to be
the major site of glucuronidation. However, some UGTs are extrahepatic enzymes, and
many of the liver UGTs are also found in other tissues. The involvement of UGTs of the 2B
subfamily in steroid glucuronidation is well documented, and evidence of regio- and
stereoselective conjugation of endogenous androgens and pregnanes for these enzymes
844
has been presented. The activity of UGT1A isoforms toward steroids was also studied,
particularly for aglycones having a C18 structure. In addition, conjugation capabilities toward
C19 steroids were described for UGT1A3, UGT1A4, and UGT1A10. Nevertheless, a
systematic approach that will help to underline the structure-function relationships in this
activity, both with respect to the steroid and the individual human UGT, is still missing. In one
study it was examined the activity of recombinant human UGTs in glucuronidation of a set of
11 exogenous anabolic steroids and their phase I metabolites to gain insight into the
structural factors that affect the enzyme-aglycone interactions. The analyses were performed
using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with electrospray
ionization, which allowed direct determination of steroid glucuronides. The results reveal
interesting differences in substrate specificity among the human UGTs, particularly those of
the 1A family. Thus, a multidimensional study on the glucuronidation of anabolic androgenic
steroids and their phase I metabolites by 11 recombinant human UDP-glucuronosyl-
transferases (UGTs) was carried out using liquid chromatographic-tandem mass
spectrometric analyses. Large differences between the enzymes with respect to the
conjugation profiles of the 11 tested aglycones were detected. Two UGTs, 1A6 and 1A7, did
not exhibit measurable activity toward any of the aglycones that were examined in this study.
Regioselectivity was demonstrated by UGTs 1A8, 1A9, and 2B15 that preferentially
catalyzed hydroxyl glucuronidation at the 17beta-position. Most of the other enzymes
glucuronidated hydroxyl groups at both the 3alpha- and the 17beta-positions. Clear
stereoselectivity was observed in glucuronidation of diastereomeric nandrolone metabolites
(5alpha-estran-3alpha-ol-17-one and 5beta-estran-3alpha-ol-17-one), whereas such
specificity was not seen when analogous methyltestosterone metabolites were assayed.
UGTs 1A1, 1A3, 1A4, 1A8, 1A9, 1A10, 2B4, 2B7, and 2B15 readily glucuronidated 5alpha-
androstane-3alpha,17beta-diol, but none of them exhibited methyltestosterone
glucuronidation activity. In agreement with the latter observations, we found that the
methyltestosterone glucuronidation activity of human liver microsomes is extremely low,
whereas in induced rat liver microsomes it was significantly higher. The homology among
UGTs 1A7 to 1A10 at the level of amino acid sequence is very high, and it was thus
surprising to find large differences in their activity toward this set of aglycones. Furthermore,
the high activity of UGT1A8 and 1A10 toward some of the substrates indicates that
extrahepatic enzymes might play a role in the metabolism of anabolic androgenic steroids
[03043].
It has thus been demonstrated that genotype-based cutoff values may improve the sensitivity
and specificity of the test, this demonstrating that genetic variation in androgen disposition is
of importance in those instances when androgen urinary excretion profile should be tested. A
deletion polymorphism in the UGT2B17 gene can lead to misinterpretation of T/E ratio, this
accounting for a significant part of ethnic interindividual variability. UGT2B17 genotype
information is therefore crucial to the decision as to which initial cutoff ratio is to be employed
for an individual as well as for enhancement of the sensitivity of the Bayesian analysis (a
method of interpretation of probabilities). On the basis of these data, the proposition has
been made that a Bayesian interpretation of consecutive tests in the same individual should
be adopted to replace the epitestosterone ratio [12011].
The anabolic steroid testosterone can be used by athletes to enhance athletic performance
and muscle growth. UDP-glucuronosyltransferase (UGT2B17) is the key enzyme involved in
the glucuronidation of testosterone to testosterone glucuronide, which also serves as a
marker for the testosterone/epitestosterone (T/E) ratio used to detect testosterone abuse in
sport. Inhibitors of testosterone glucuronidation could have an impact on circulating
testosterone levels, thus aiding performance, as well as potentially affecting the urinary T/E
ratio and therefore masking testosterone abuse. Previous reports have revealed that non-
steroidal, anti-inflammatory drugs, diclofenac and ibuprofen, inhibit the UGT2B17 enzyme.
845
The aim of one study was to analyse dietary tea samples for inhibition of testosterone
glucuronidation and, where inhibition is present, to identify the active compounds. Analysis of
testosterone glucuronidation was conducted by performing UGT2B17 assays with detection
of un-glucuronidated testosterone using high performance liquid chromatography. The
results from this study showed that testosterone glucuronidation was inhibited by the green
and white tea extracts, along with specific catechin compounds, notably: epicatechin,
epigallocatechin gallate (EGCG) and catechin gallate. The IC50 inhibition value for EGCG
was determined, using a Dixon plot, to be 64 μM, equalling the most active NSAID inhibitor
diclofenac. Thus, common foodstuffs and their constituents, for the first time, have been
identified as inhibitors of a key enzyme involved in testosterone glucuronidation. Whilst these
common compounds are not substrates of the UGT2B17 enzyme, we showed that they
inhibit testosterone glucuronidation which may have implications on current doping control in
sport [12193].
The large variation in disposition known for most drugs is also true for anabolic androgenic
steroids. Genetic factors are probably the single most important cause of this variation.
Further, there are reasons to believe that there is a corresponding variation in efficacy of
doping agents. Doped individuals employ a large variety of doping strategies in respect of
choice of substance, dose, dose interval, duration of treatment and use of other drugs for
enforcement of effects or correction of side effects. Metabolic steps up-stream and down-
stream of testosterone are genetically variable and contribute substantially to the variation in
disposition of testosterone, the most common doping agent in sports and in society. Large
inter- and intra-ethnic variation in testosterone glucuronidation and excretion is described as
well as the pit-falls in evaluation of testosterone doping test results. The hydrolysis and
bioactivation of testosterone enanthate is also genetically variable yielding a 2-3 fold
variation in excretion rate and serum concentration, thereby implicating a substantial
variation in ”efficacy” of testosterone. Given this situation it is logical to adopt the new
findings in the doping control programme. The population based cut-off level for the
testosterone:epitestosterone ratio should be replaced by a Bayesian interpretation of
consecutive tests in the same individual. When combined with the above genetic information
the sensitivity of the test is considerably improved. The combination of the three approaches
should reduce the rate of falsely negative or positive results and the number of expensive
follow-up tests, stipulated by the World Anti-Doping Agency [12192].
In the light of the continuously growing knowledge on the relevance of steroid conjugation
(i.e. phase-II metabolism reactions), various new studies were initiated and reported
concerning steroid glucuronides and sulfates, factors arguably confining their urinary
concentrations, and the accurate and sensitive determination of these steroid conjugates for
potential evaluation as a complement to the current steroid profiling approaches. It was
investigated the utility of combining analytical results of 7 steroid glucuronides and 5 steroid
sulfates for the detection of transdermal and oral administrations of testosterone and
testosterone undecanoate, respectively. A total of 19 volunteers were subjected to
genotyping concerning the insertion/deletion of the UGT2B17 gene yielding 7 ins/ins, 7
ins/del, and 5 del/del genotypes. All participants underwent a transdermal testosterone
application (via patches providing 2.4 mg of testosterone/24 h) and, after washout, oral
testosterone undecanoate administration (2 x 40 mg), and urine samples were collected over
a period of 96 h. By means of LC-MS/MS, relevant steroid conjugates were quantified,
corroborating the issue of common GC-MS-based steroid profile approaches that population-
based reference ranges barely allow the identification of topical and oral testosterone
administration. Employing an intra-individual profiling strategy, the administration of
testosterone via patches was identified, particularly by means of the ratios of testosterone
glucuronide (TG)/epitestosterone glucuronide (epiTG) as well as androsterone glucuronide
(AG)/etiocholanolone glucuronide (EG). The ingestion of testosterone undecanoate was
846
detectable predominantly by means of etiocholanolone sulfate (ES), especially in UGT2B17
del/del genotypes [13009].
UGT2B17 deletion genotypes are especially prevalent among Asian athletes, and the
traceability of intramuscularly administered testosterone enanthate to female Japanese
volunteers was therefore investigated with a cohort consisting of six del/del, three ins/del,
and one ins/ins genotype. As expected, the T/epiT ratio of the del/del group did not exceed
the limit of 4 at any time of the 16 days of the study period, thus no follow-up analyses by
IRMS would have been triggered. Consequently, the authors suggested employing subject-
based reference ranges and/or genotype-specific thresholds for steroid profile parameters
such as the T/epiT ratio [13009].
Emphasis was put for instance on UDP-glucuronosyltransferase UGT2B17, a key enzyme in

testosterone glucuronidation. In an in vitro experiment it was shown that UGT2B17 was be
negatively influenced by catechins (epicatechin, epigallocatechin gallate, and catechin
gallate) commonly found in dietary green and white teas. Since tea consumption can lead to
pharmacologically relevant concentrations of these catechins, it is conceivable that steroid
profiles can vary due to such licit dietary products; however, in vivo data remain to support
this assumption and to assess the relevance for sports drug testing. Concerning the same
key enzyme UGT2B17, the role of androgen sulfation was studied in volunteers with two,
one, or no allele of the respective gene, who received a single oral dose of testosterone
enanthate. While sulfates of urinary steroids were found to be inadequate for monitoring
purposes in this scenario, the increased excretion of androsterone (A) glucuronide was
considered helpful (especially when evaluated in relation to epitestosterone (EpiT)
glucuronide), which is in agreement with earlier studies outlining the relevance of the A/EpiT
ratio in steroid profiling. Deletion polymorphism concerning UGT2B17 is of great importance
when interpreting steroid profile data; hence, the availability of a test assay for its
determination from doping control urine sample was desirable and established in 2011. A
total of 674 urine samples was phenotyped, corresponding T/EpiT ratios were determined
and significant correlations between homozygote gene-deletion and low T/EpiT ratios
confirmed [13012].
It was investigated the androgen receptor (AR) bioluminescense response in serum and
urine before and after testosterone challenge in different genotypes of the UGT2B17
enzyme, which catalyses testosterone glucuronidation. The androgen receptor activity was
determined using a yeast-based bioluminescence assay. The androgens were analysed
using LC-MS/MS, and the individuals were genotyped for UGT2B17 deletion polymorphism
using real-time polymerase chain reaction. The serum concentrations of testosterone and
dihydrotestosterone (DHT) were markedly elevated on days 2 and 4 and were still above
baseline on day 15 after a dose of 500 mg testosterone enanthate. The androgenic activity in
serum increased in parallel and correlated with the hormone concentrations and remained
above baseline on day 15. The urinary androgenic activity increased 4-5-fold and was closely
related to the unconjugated testosterone and independent of the UGT2B17 genotype. The
AR assay may serve as a complement to the urinary testosterone/epitestosterone (T/E)
doping test, because this is profoundly influenced by the UGT2B17 deletion polymorphism. It
may also be useful for detection of other illicit androgens in sports, or in the society, or for
monitoring and diagnostics of androgen-related disorders [13217].
Genetic differences in testosterone metabolism can alter T/E ratio and result in a false
negative test. Studies have linked deletion polymorphisms of uridine diphospho-glucurosyl
transferase 2B17 (UGT2B17) (the major enzyme for testosterone glucuronidation) with
significantly lower T/E ratios. Because of the high frequency of this polymorphism among
East Asian populations, the likelihood of a false negative test is higher in these populations
847
than in Caucasian populations. Additionally, studies have shown variations in UGT2B17 copy
number, which may affect T/E ratios among populations from Africa, Europe, and East Asia
[14017].
Glucuronidation of androgens by UDP-glucuronosyltransferases (UGTs), i.e. phase II

metabolism, is the major route for androgen inactivation and excretion. UGT2B7 has been
identified as the enzyme responsible for epitestosterone conjugation whereas testosterone is
a poor substrate for this enzyme. Testosterone is mainly conjugated by UGT2B17 and to a
minor extent by UGT2B15. It has been shown that testosterone glucuronidation activity in the
liver is significantly higher in men homozygous for the insertion (ins/ins) of UGT2B17 than in
women with the same genotype [14007].
Detection of doping with endogenous steroids, such as testosterone, has been and continues
to be a challenge. To overcome the problem of separating testosterone doping from
endogenous testosterone the ratio between the glucuronides of testosterone and
epitestosterone (T/E) is used. This T/E ratio was introduced in doping tests with an
authorized upper limit of 4. Interestingly, the mean T/E ratio in Caucasian men is
approximately 1, whereas in Asians, the mean ratio is considerably lower. It has also been
shown that the ethnic disparity in the T/E ratio is strongly associated with a deletion
polymorphism in the UGT2B17 gene. Individuals homozygous for this deletion (del/del) may
not reach a T/E ratio of 4 when doped with testosterone. The deletion polymorphism is much
more common in East Asian populations as compared to Caucasians and Africans. There
are also individuals that have naturally high T/E ratios due to decreased excretion of
epitestosterone. In males, part of this low epitestosterone excretion can be explained by a
promoter polymorphism in the CYP17 gene, resulting in 64 percent higher T/E ratios in men
homozygous for the T allele. However, this polymorphism in relation to epitestosterone
excretion has not been studied in women. Women show a greater individual variation in T/E
ratio than men due to concentrations near the detection limit for the method of analysis as
well as variations during the menstrual cycle. Furthermore, the use of hormonal
contraceptives (HC) has been suggested to suppress the production of epitestosterone and
thus lead to an increase in the T/E ratio. In Sweden, a study has shown that almost half of
female elite athletes are using HC, a frequency comparable to the general population of the
same age group. HC consist of a progesterone derivative (progestogens) or a combination of
a progestogen and synthetic or natural estrogen. Their main mechanism of action is inhibition
of ovulation. Both progesterone and estrogen are negative regulators of the hypothalamic–
pituitary–gonadal (HPG) axis, meaning that an increase in these sex hormones results in a
decrease in gonadotropin releasing hormone (GnRH), luteinizing hormone (LH), and follicle
stimulating hormone (FSH) [14007].
UGT2B17 deletion genotypes are especially prevalent among Asian athletes, and the
traceability of intramuscularly administered testosterone enanthate to female Japanese
volunteers was therefore investigated with a cohort consisting of six del/del, three ins/del,
and one ins/ins genotype. As expected, the T/epiT ratio of the del/del group did not exceed
the limit of 4 at any time of the 16 days of the study period, thus no follow-up analyses by
IRMS would have been triggered. Consequently, the authors suggested employing subject-
based reference ranges and/or genotype-specific thresholds for steroid profile parameters
such as the T/epiT ratio [14009].
Japan
Ethnicity has been found to influence urinary testosterone glucuronide to epitestosterone
glucuronide (T/E) ratios among athletes. Uridine diphospho-glucuronosyltransferase 2B17
(UGT2B17) is the most active enzyme in testosterone glucuronidation. UGT2B17
polymorphism analysis is rarely performed in Japanese athletes, and the influence of
848
testosterone administration on steroid profiles and carbon isotope ratios, according to gene
polymorphisms, in Asians remains unknown. The prevalence of UGT2B17 genotypes and
urinary androgenic steroid profiles, classified according to UGT2B17 genotypes, was
investigated in Japanese athletes (255 male and 256 female). Testosterone enanthate (100
mg) was administered intramuscularly to Japanese female volunteers (del/del: n=6, del/ins:
n=3, ins/ins: n=1). The distribution rates of the UGT2B17 del/del genotype in Japanese male
and female athletes were 75 percent and 60 percent, respectively. The ins/ins genotype was
detected in only three male (1.2 %) and seven female (2.7 %) athletes. The prevalence of the
UGT2B17 deletion genotype was extremely high in Japanese athletes. The T/E ratio in the
del/del group was significantly lower than that in the other groups. After testosterone was
administered to female volunteers, the T/E ratios for the del/del individuals failed to reach the
positivity criterion of 4. By contrast, in all of the del/del subjects, the gas chromatography/
combustion/isotope ratio mass spectrometry (GC-C-IRMS) analysis successfully fulfilled the
positivity criterion. The overall result has demonstrated the limited effectiveness of
population-based T/E ratios in screening tests for testosterone use. Subject-based steroid
profiling with UGT2B17 genotyping will be an effective strategy for detecting testosterone
misuse [12194, 13218].
Genetic polymorphism
Emphasis has been put on UDP-glucuronosyltransferase UGT2B17, a key enzyme in

testosterone glucuronidation. In an in vitro experiment it was shown that UGT2B17 was be
negatively influenced by catechins (epicatechin, epigallocatechin gallate, and catechin
gallate) commonly found in dietary green and white teas. Since tea consumption can lead to
pharmacologically relevant concentrations of these catechins, it is conceivable that steroid
profiles can vary due to such licit dietary products; however, in vivo data remain to support
this assumption and to assess the relevance for sports drug testing. Concerning the same
key enzyme UGT2B17, the role of androgen sulfation was studied in volunteers with two,
one, or no allele of the respective gene, who received a single oral dose of testosterone
enanthate. While sulfates of urinary steroids were found to be inadequate for monitoring
purposes in this scenario, the increased excretion of androsterone (A) glucuronide was
considered helpful (especially when evaluated in relation to epitestosterone (EpiT)
glucuronide), which is in agreement with earlier studies outlining the relevance of the A/EpiT
ratio in steroid profiling. Deletion polymorphism concerning UGT2B17 is of great importance
when interpreting steroid profile data [12017].
Aiming at the identification of new, complementary biomarkers for endogenous steroid

abuse, the utility of a steroidomic approach using UHPLC-HRMS was assessed. In a
controlled elimination study with orally administered testosterone undecanoate (80 mg), urine
samples were subjected to a holistic steroid analysis followed by chemometric/statistical data
evaluation. Here, numerous glucuronidated or sulfated steroids, the deconjugated analogs,
of which mostly constitute the established steroid profile, were found to support the
discrimination of the groups having received either placebo or testosterone undecanoate.
The study demonstrated the principle of modern analytical approaches commonly referred to
as “-omics” strategies and its potential application to issues of doping controls; in order to
consider the whole (holistic) picture of such approaches, complementary analyses (e.g. by
means of GC-HRMS) might be required to strengthen the outcome and value [12017].
Urinary steroid profiling is used in doping controls to detect testosterone abuse. A

testosterone over epitestosterone (T/E) ratio exceeding 4.0 is considered as suspicious of
testosterone administration, irrespectively of individual heterogeneous factors such as the
athlete's ethnicity. A deletion polymorphism in the UGT2B17 gene was demonstrated to
account for a significant part of the inter-individual variability in the T/E between Caucasians
849
and Asians. Now it was examined the variability of urinary steroid profiles in a widely
heterogeneous cohort of professional soccer players. The steroid profile of 57 Africans, 32
Asians, 50 Caucasians and 32 Hispanics was determined by gas chromatography-mass
spectrometry. Significant differences have been observed between all ethnic groups. After
estimation of the prevalence of the UGT2B17 deletion/deletion genotype (African 22 %;
Asian 81%; Caucasian 10 %; Hispanic 7 %), ethnic-specific thresholds were developed for a
specificity of 99 percent for the T/E (African 5.6; Asian 3.8; Caucasian 5.7; Hispanic 5.8).
Finally, another polymorphism could be hypothesized in Asians based on specific
concentrations ratio of 5a-/5b-androstane-3a,17b-diol in urine. These results demonstrate
that a unique and nonspecific threshold to evidence testosterone misuse is not fit for
purpose. An athlete's endocrinological passport consisting of a longitudinal follow-up together
with the ethnicity and/or the genotype would strongly enhance the detection of testosterone
abuse. Finally, additional genotyping studies should be undertaken to determine if the
remaining unexplained disparities have an environmental or a genetic origon [09050].
Genetic variation has a large impact on androgen disposition. This variation is of the utmost
importance for the interpretation of doping test results and may modulate the effects of
testosterone replacement therapy and testosterone doping [09051].
Association with renal disease and gene polymorphism
With prolonged use of anabolic androgenic steroids (AAS), occasional incidents of renal
disorders have been observed. Independently, it has also been established that there are
considerable inter-individual and inter-ethnic differences, in particular with reference to the
uridine diphosphate-glucuronosyltransferase 2B17 (UGT2B17) gene, in metabolising these
compounds. One report postulated the association of deletion polymorphism in the UGT2B17
gene with the occurrence of renal disorders on chronic exposure to AAS. The major
deactivation and elimination pathway of AASs is through glucuronide conjugation, chiefly
catalyzed by the UGT2B17 enzyme, followed by excretion in urine. Excretion of steroids is
affected in individuals with a deletion mutation in the UGT2B17 gene. It was hypothesize that
UGT2B17 deficient individuals are more vulnerable to developing renal disorders with
prolonged use of AAS owing to increases in body mass index and possible direct toxic
effects of steroids on the kidneys. Elevated serum levels of biologically active steroids due to
inadequate elimination can lead to prolonged muscle build up. An increase in body mass
index may cause renal injuries due to sustained elevated glomerular pressure and flow rate.
In the absence of controlled clinical trials in humans, observational studies can be carried
out. Real time PCR with allelic discrimination should be employed to examine the prevalence
of different UGT2B17 genotypes in patients with impaired renal function and AAS abuse. In
individuals with the UGT2B17 deletion polymorphism, blood tests, biofluid analyses,
urinalysis, and hair analyses following the administration of an anabolic steroid can be used
to determine the fate of the substance once in the body. If the hypothesis is upheld, anabolic
steroid users with a deletion mutation in the UGT2B17 gene may be exposed to an increased
risk of developing renal disorders. In the current detecting – sanctioning anti-doping system,
athletes motivated by the potential to evade detection owing to their unique genetic make-up
could subject themselves to a serious health consequence. More research on AAS
metabolism in the presence of UGT2B17 gene deletion is required. Benefit or harm
evaluations in therapeutic use of anabolic steroids should also consider this potential link
between UGT2B17 gene deletion polymorphism and renal disorders [10057].
Chemistry
850
Androgenic and anabolic steroids (AASs) are a class of chemical substances closely related
to testosterone in molecular structure. They can be abused to enhance performances in
human and equine athletes, and are banned by the sports authorities. To assist with method
development for doping analyses of AASs, investigations were conducted to correlate their
product ion profiles with the molecular structures. Although very similar in chemical structure,
AASs generated noticeably different product ion profiles from collision-induced dissociation
(CID). On the basis of both outlines of the product ion profiles and molecular structures,
AASs studied were classified into six subclasses. In each subclass, the product ion profiles
were identical or similar. However, the product ion profiles in one subclass were remarkably
different from those in another. The classification reveals that the position and number of
double bond(s) in conjugation with the 3-carbonyl group in the molecular structure of an AAS
have significant effects on product ion profile. The presence or absence of the 19-methyl
group in an AAS also has a remarkable influence on its product ion profile. A substitution in
the A-, B- or D-ring of an AAS may cause a shift in mass value of the product ions. The
correlation of product ion profiles with molecular structures of AASs has the implication that
each AAS can be characterized by a combination of its [M + H]+ ion and product ion profile
and as a result be identified with specificity. The classified product ion pattern may be useful
in the identification of unknown AASs [10323].
An androgen is a sex hormone that promotes the development and maintenance of the male
sex characteristics; testosterone is the principal secreted androgen in men. Androgens have
both androgenic (masculinizing) effects (development of male secondary sex characteristics,
including hair growth) and anabolic effects (increase in skeletal muscle mass and strength).
For decades, pharmaceutical companies have attempted to develop androgens that have
preferential anabolic activity and reduced or no androgenic activity; these compounds have
been referred to as anabolic steroids. Although some steroidal compounds available to date
are preferentially anabolic, most generally have both androgenic and anabolic effects.
Therefore, for the sake of uniformity and accuracy, we have used the term AAS to describe
these compounds that are structurally related to testosterone, bind to androgen receptor, and
exert masculinizing as well as anabolic effects to varying degrees. The literature uses a
number of terms (anabolic steroids, androgenic steroids, and androgens) to describe these
androgen derivatives [14426].
Testosterone remains popular, both among elite athletes and nonathlete weightlifters,
because of its low price, relatively ready access, and the challenges in distinguishing
exogenous from endogenous sources of testosterone. Numerous AASs have been
synthesized by structural modifications of the testosterone molecule. These structural
modifications may alter the relative anabolic or androgenic activity, the binding affinity for the
androgen receptor, coactivator recruitment, metabolic clearance, susceptibility to
presystemic metabolism, and aromatization. Testosterone is metabolized rapidly in the body;
however, esterification of the 17beta-hydroxyl group renders the molecule more hydrophobic.
When these esters of testosterone (such as testosterone enanthate and cypionate) are
administered in an oily suspension, they are released very slowly into the aqueous plasma
because of their hydrophobicity. This extends their duration of action. These esters are
readily de-esterified to testosterone in the body. Investigations of the structure-activity
relationships have established that removal of the 19-methyl group increases the anabolic
activity; thus, 19-nortestosterone (nandrolone) is a potent AAS and a very popular training
drug that accounts for a large number of positive tests. 7alpha-Alkyl substitutions of the 19-
nortestosterone molecule may further increase the anabolic to androgenic activity. 17alpha-
Alkyl substitutions render the molecule resistant to degradation; thus, 17alpha-alkylated
androgens can be administered orally. Stanozolol is a 17alpha-alkylated androgen that can
be taken orally or by injection. Orally administered 17alpha-alkylated androgens are
hepatotoxic. Stanozolol is also nonaromatizable. Other substitutions in the steroid A ring may
851
alter the susceptibility of the steroid molecule to aromatization. A number of nonsteroidal
SARMs, which display tissue-specific activation of androgen signaling, are in development
[14426].
The mechanisms by which AASs improve athletic performance are not fully understood.
Testosterone administration increases skeletal muscle mass by inducing the hypertrophy of
both type 1 and 2 fibers (98); testosterone does not change the absolute number or the
relative proportion of type 1 and 2 fibers. Testosterone administration increases the number
of muscle progenitor cells (satellite cells), which contribute to muscle fiber hypertrophy.
Testosterone promotes myogenic differentiation of muscle progenitor cells. Upon binding to
its cognate androgen receptor, the liganded androgen receptor associates with beta-catenin
and other proteins, and the complex translocates into the nucleus where it binds transcription
factor-4 and activates a number of Wnt target genes, including follistatin. Follistatin blocks
the effects of a number of TGF-beta family members, including myostatin and activins, and
plays an essential role in mediating testosterone's effects on myogenic differentiation. Most
of the anabolic effects of testosterone appear to be mediated through androgen receptor
signaling. Testosterone stimulates circulating GH and IGF-1, although circulating GH is not
essential for mediating testosterone's effects on muscle mass. However, im IGF-1 receptor
signaling plays an important role in mediating the effects of testosterone on myogenesis. The
conversion of testosterone to dihydrotestosterone by steroid 5alpha-reductase is not
essential for mediating its effects on the muscle. Testosterone increases maximal voluntary
strength and leg power but does not increase specific force. Testosterone also promotes
mitochondrial biogenesis and quality control and increases net oxygen delivery to the tissue
by increasing red cell mass and tissue capillarity. Testosterone also increases the circulating
levels of 2,3-biphosphoglycerate, which shifts the oxygen:hemoglobin curve to the left,
thereby facilitating oxygen unloading from oxyhemoglobin (HbO2). The observations that
testosterone improves neuromuscular transmission and upregulates acetyl cholinesterase
expression in the frog hind limb model have led to speculation that testosterone may reduce
reaction time, which may contribute to improved performance in sprint events or in sports
requiring a high level of hand-eye coordination, such as baseball [14426].
Testosterone administration may also affect mood and motivation, which may indirectly affect
athletic performance [14426].
Steroid hormones
Steroid hormones include the sex hormones, glucocorticoids, and mineralocorticoids. Within
the family of sex hormones are the androgens, estrogens, and progestogens. All of the
steroid hormones bind to their own specific receptor, which may be cytosolic or nuclear, to
induce changes within a cell. All natural steroid hormones are synthesized from cholesterol in
the adrenal glands and/or gonads. Some steroid hormones are further metabolized in the
liver, peripheral and/or target tissues. As their precursor is cholesterol, they are hydrophobic
in nature which allows them to pass easily through cell membranes. Once synthesized, the
steroid hormones are carried in the blood stream bound to carrier proteins such as albumin,
steroid hormone-binding globulin (SHBG) or corticosteroid-binding globulin to target tissues.
The androgen produced in the highest concentration in the body is testosterone (T). This is a
19-C steroid that has androgenic and anabolic effects within the body. T is primarily
produced in the gonads but a small amount is produced in the adrenal cortex or from the
peripheral conversion of androstenedione. T production is much greater in males than in
females (5000-7000 microg/day vs 300 microg/day). In males, T is primarily produced by the
Leydig cells in the testes whereas in females, the primary production of T occurs in the
Theca cells of the ovaries. In both sexes, small amounts come from the adrenal cortex and
852
the peripheral conversion of androstenedione. T acts in the body by acting directly through
the AR or indirectly via metabolism to other sex steroids. T can be aromatized to estradiol
(E2) which activates ER-alpha and/or ER-beta. Alternatively, T can be irreversibly converted
to the more potent 5alpha-dihydrostestosterone (5alpha-DHT) by the enzyme 5alpha-
reductase. T has many physiological actions in the body. It acts on muscles to stimulate
growth and maintenance, it promotes bone development while inhibiting bone resorption, and
it increases red blood cell and hemoglobin levels, augments libido and erectile function,
enhances mood and cognition, and induces lipolysis. Low testosterone levels or deficit in
androgen action induces frailty, sarcopenia, poor muscle quality, muscle weakness,
hypertrophy of adipose tissue and impaired neurotransmission [13084].
Testosterone and its modifications
Testosterone is the most important androgen in the human body. The effects of androgens
are most evident during puberty, as they elicit dramatic physiological changes in the male
body, including the onset of secondary male characteristics, hair growth pattern, sebaceous
gland activity and maturation of sperm and libido. These are considered the virilizing or
“androgenic” effects. Daily testosterone synthesis ranges from 2.1 to 11.0 mg in individual
males, with normal plasmatic levels of 300-1000 ng/dL, which progressively decline with age.
Testosterone has several possible metabolic fates. First, it binds to the androgen receptor
(AR) in target tissues to exert its effects. Second, it is reduced to 5alpha-dihydrotestosterone
(5DHT), which also acts on the AR. Following a different path, testosterone may be
aromatized to estradiol to exert estrogenic effects, typically water retention, breast tissue
growth and an increase in body fat deposition. Along with the androgenic changes comes the
“anabolic” effect. Anabolism is defined as any state in which nitrogen is differentially retained
in lean body mass through the stimulation of protein synthesis and/or a reduction in protein
breakdown. It includes growth promotion, protein and collagen synthesis and an increase in
muscle size and bone metabolism. Characteristically, steroids that are more anabolic present
weaker AR bindings, and those that are more androgenic strongly bind the AR, exerting a
more potent effect. A “myotrophic-androgenic index”, based on the association between
anabolic and androgenic bioassays in rats has been previously described. Since
testosterone is the basic AAS, it has a 1:1 anabolic-androgenic ratio. Structural modifications
have been made to the testosterone molecule in an attempt to maximize the anabolic effects
and minimize the androgenic ones; however, all AASs are virilizing if administered for long
enough, at high enough dosages. AASs therefore include synthetic derivatives of
testosterone, and not only testosterone itself. The AAS structural base is the steran nucleus,
consisting of three condensed cyclohexan rings, in nonlinear junction, and a cyclopentane
ring. The anabolic effects are dose-dependent, and usually occur when supraphysiological
testosterone levels (>1000 ng/dL) are found, which generally requires weekly doses of 300
mg or more. Traditionally, AASs are classified according to the route of administration and
their carrier solvent and fall into two categories: oral and parenteral. Alkyl substitution
prevents deactivation of the steroid by hepatic first-pass metabolism (necessitating hepatic
monitoring), which promotes oral activity. They usually have short half-lives, making several
daily doses necessary to maintain appropriate blood levels. This class includes the very
common stanozolol and oxandrolone, as well as methyltestosterone and others. If the
17beta-hydroxyl group is esterified with an acid moiety it prevents rapid release from the oily
vehicle. Roughly, the longer the chain length of the acid moiety, the more slowly the
preparation is released into the blood stream. Once in the circulation, hydrolysis rapidly
occurs yielding the active compound. They usually have a longer half-life and a slower
absorption rate, bringing much less hepatic stress than the orally taken steroids. Pain at
injection sites is common, because of the oily base. There are several basic active
compounds: testosterone, bound to esters such as undecanoate, cypionate, propionate and
others; and 19-nortestosterone (or nandrolone), also bound to different esters. Nandrolone is
853
extremely popular, owing to its high anabolic to androgenic ratio. In contrast to testosterone,
nandrolone is converted to a less potent metabolite after 5alpha-reduction. This, in addition
to nandrolone’s lesser affinity to AR, explains the higher myotrophic to androgenic ratio.
Other group of compounds is headed by boldenone, bound to ester undecylenate; and
trenbolone, bound to ester acetate [11028].
Two modifications made to the testosterone molecule alter the androgenic/anabolic profile,
and thus the type of side effects seen from usage of the various AAS. Class A modifications
are formulated by esterification of the 17-alpha-hydroxyl group, which increases the lipophilic
properties, allowing a slow, delayed absorption as an injectable form. This is the most
common form of modification because it allows the injection to be administered as
infrequently as once every 2 to 6 weeks. This pharmacokinetic alteration also results in
increased and unwanted androgenic effects. Class B modifications result from alkylation of
the 17-alpha portion of the molecule, which decreases hepatic metabolism. This allows
increased oral absorption and slower hepatic degradation. Slower clearance from the liver
results in greater hepatic toxicity [06031].
General metabolism
The major elimination and deactivation pathway of AAS and their phase I metabolites is
through glucuronide conjugation (phase II metabolism), mainly catalysed by the enzyme
UGT2B17, followed by excretion in urine. However, inter-individual and inter-ethnic variations
in the prevalence of deletion polymorphism in the gene coding of the UGT2B17 enzyme have
been reported, which eventually influence the urinary excretion of AAS and potentially lead to
false-negative doping results. It has also been reported that the glucuronidation activity of
UGT2B17 and other UGTs towards AAS is inhibited by commonly used anti-inflammatory
drugs like diclofenac and ibuprofen, in vitro. Common dietary substances such as red wine,
white tea and green tea have also shown similar inhibitory effects in in vitro studies. Although
the inhibitory effect is yet to be examined and reported in vivo, these in vitro results indicate
that concomitant use of such over-the-counter medication or common dietary products with
AAS may lead to impaired urinary excretion of AAS and their metabolites [12116].
Considering that such genetic and metabolic variations may limit the efficacy of urinalysis in
testing doping, it can be suggested that urinalysis, if used as a stand-alone test, is
susceptible to confounding doping results. Owing to the growing number of doping cases
with AAS, there is an ever-increasing need to develop new methods to detect drug doping.
The current anti-doping regime can be reinforced by employing additional biological samples
like blood and hair analysed in tandem with urine. Since impaired glucuronidation leads to
reduction in the urinary excretion rate of AAS, it can be assumed that the levels of
unconjugated AAS and their phase I metabolites in the systemic circulation will be elevated
and thus higher levels of AAS and their phase I metabolites will be available to get
incorporated into hair and other body tissues. Hair analysis has been used in the past for
detecting drug use as it predominantly favours the direct detection of parent AAS and
determines a retrospective history of drug use. Thus, hair analysis and blood analysis can
provide complementary information to urinalysis to prevent false doping results [12116].
Effect of oxidizing adulterants on human urinary steroid profiles

Steroid profiling is the most versatile and informative technique adapted by doping control
laboratories for detection of steroid abuse. The absolute concentrations and ratios of
endogenous steroids including testosterone, epitestosterone, androsterone, etiocholanolone,
5alpha-androstane-3alpha,17beta-diol and 5beta-androstane-3alpha,17beta-diol constitute
the significant characteristics of a steroid profile. In one study we report the influence of
854
various oxidizing adulterants on the steroid profile of human urine. Gas chromatography-
mass spectrometry analysis was carried out to develop the steroid profile of human male and
female urine. Oxidants potassium nitrite, sodium hypochlorite, potassium permanganate,
cerium ammonium nitrate, sodium metaperiodate, pyridinium chlorochromate, potassium
dichromate and potassium perchlorate were reacted with urine at various concentrations and
conditions and the effect of these oxidants on the steroid profile were analyzed. Most of the
oxidizing chemicals led to significant changes in endogenous steroid profile parameters
which were considered stable under normal conditions. These oxidizing chemicals can cause
serious problems regarding the interpretation of steroid profiles and have the potential to act
as masking agents that can complicate or prevent the detection of the steroid abuse [12117].
Biodegradation
For the steroidal growth promoters trenbolone acetate (TBA) and melengestrol acetate
(MGA) neither the complete spectrum of biological activities nor the potential endocrine
disrupting activity of their excreted metabolites in the environment is fully understood. The
potency of these substances in [3H]dihydrotestosterone ([3H]-DHT) displacement from the
recombinant human androgen receptor (rhAR) and from human sex-hormone binding
globulin (hSHBG) was evaluated. In addition, the potency for [3H]-ORG2058 displacement
from the bovine uterine progestin receptor (bPR) was tested. For comparison, different
anabolics and synthetic hormones were also tested for their binding affinities. For 17beta-
trenbolone (17beta-TbOH), the active compound after TBA administration, an affinity the
rhAR similar to dihydrotestosterone (DHT) and a slightly higher affinity to the bPR than
progesterone were demonstrated. The affinity of the two major metabolites, 17alpha-
trenbolone and trendione, was reduced to less than 5 percent of the 17beta-TbOH-value.
The affinity of these three compounds and of MGA to the hSHBG was much lower compared
with DHT. MGA showed a 5.3-fold higher affinity than progesterone to the bPR but only a
weak affinity to the rhAR. The major MGA metabolites have an affinity to the bPR between
85 and 28 percent of the affinity of progesterone. In consequence, MGA and TBA
metabolites may be hormonally active substances, which will be present in edible tissues and
in manure. It was concluded that detailed investigations on biodegradation, distribution and
bio-efficacy of these substances are necessary [00036].
Steroids from musk deer
The administration of musk extract, that is, ingredients obtained by extraction of the liquid
secreted from the preputial gland or resulting grains of the male musk deer (e.g. Moschus
moschiferus), has been recommended in Traditional Chinese Medicine (TCM) applications
and was listed in the Japanese pharmacopoeia for various indications requiring
cardiovascular stimulation, anti-inflammatory medication or androgenic hormone therapy.
Numerous steroidal components including cholesterol, 5alpha-androstane-3,17-dione, 5beta-
androstane-3,17-dione, androsterone, etiocholanolone, epiandrosterone, 3beta-hydroxy-
androst-5-en-17-one, androst-4-ene-3,17-dione and the corresponding urea adduct 3alpha-
ureido-androst-4-en-17-one were characterised as natural ingredients of musk over several
decades, implicating an issue concerning doping controls if used for the treatment of elite
athletes. In the present study, the impact of musk extract administration on sports drug
testing results of five females competing in an international sporting event is reported. In the
course of routine doping controls, adverse analytical findings concerning the athletes' steroid
profile, corroborated by isotope-ratio mass spectrometry (IRMS) data, were obtained. The
athletes' medical advisors admitted the prescription of TCM-based musk pod preparations
and provided musk pod samples for comparison purposes to clarify the antidoping rule
violation. Steroid profiles, IRMS results, literature data and a musk sample obtained from a
living musk deer of a local zoo conclusively demonstrated the use of musk pod extracts in all
855
cases which, however, represented a doping offence as prohibited anabolic-androgenic
steroids were administered [12120].
Influence of pharmacological interventions on the serum and urine steroid profile
The influence of pharmacological interventions on the serum and urine steroid profile was
studied by investigating the effect of administrations of the superactive gonadotropin
analogue leuprolide to men. Serum T, dihydrotestosterone (DHT), and 5alpha-androstane-
3alkpha,17beta-diol (Adiol) were significantly increased along with urinary T, EpiT, and
androsterone (A) concentrations upon five days of leuprolide administration, resulting in
modest (if any) changes in T/EpiT ratios. Urinary steroid levels returned to baseline values at
day 10, and a detection strategy involving both the direct analysis of leuprolide and its main
metabolite as well as using the ratio of luteinizing hormone (LH)/T were suggested. Further
to the indirect stimulation of T secretion via releasing factors, the option of direct
enhancement of serum T concentrations by transdermal applications remains a challenging
doping control analytical task. By means of LC-MS/MS targeting 12 urinary steroid glucuro-
and sulfoconjugates, it was assessed the possibility to complement routine steroid profiling
protocols by measuring intact phase-II metabolites. Due to substantial inter-individual
variabilities, only intra-individual profiles demonstrated the capability of uncovering topical
(transdermal) T applications by targeting specifically the ratios of the glucuronides of T and
EpiT as well as A and etiocholanolone (E). For oral T undecanoate administrations, E sulfate
was found to be a promising marker to complement the currently employed steroid profile
[14715].
Physiology
The mechanism of action of AAS may differ between compounds because of variations in the
steroid molecules. These variations are responsible for differences in the specificity of
binding to receptor proteins or to interaction with various steroid-metabolising enzymes. With
respect to interactions with intracellular steroid receptor proteins, several pathways can be
distinguished. First, binding with high affinity to androgen receptors – these steroids are
therefore recognised as strong androgens (e.g. 19-nortestosterone, metenolone). Secondly,
several compounds are characterised by binding with low affinity to androgens and therefore
are weak androgenic substances (e.g. stanozolol, fluoxymesterone). Thirdly, some AAS (e.g.
oxymetholone) do not bind to the androgen receptor at all. These steroids are supposed to
act after biotransformation to more active compounds or via alternative mechanisms of
action. Furthermore, it has been established for AAS that other mechanisms may also be
involved [04002].
Testosterone supplementation acts via numerous mechanisms as a highly potent anabolic

agent to skeletal muscle. Although growth hormone (GH) strongly affects collagen synthesis
and lipolysis, as well as increasing lean body mass, it is not anabolic toward the contractile
(ie, myofibrillar) muscle tissue in healthy individuals. However, there is a persistent belief
(both in scientific literature and among recreational weightlifters) that exercise-induced
release of GH and testosterone underpins muscular hypertrophy with resistance training.
This is a premature assumption because although pharmacological GH supplementation can
increase muscle strength or size in individuals with clinical GH deficiency, there is no
evidence that transient exercise-induced changes in GH have the same effects in individuals
with normal GH levels. Exercise paradigms are designed based on the assumption (not
necessarily evidenced-based mechanisms) that GH and testosterone facilitate anabolic
processes that lead to skeletal muscle protein accretion and hypertrophy. Recent work
856
disputes this assumption. Instead, data indicate that exercise-induced hormonal elevations
do not enhance intracellular markers of anabolic signaling or the acute postexercise
elevation of myofibrillar protein synthesis. Furthermore, data from our training study
demonstrate that exercise-induced increases in GH and testosterone availability are not
necessary for and do not enhance strength and hypertrophy adaptations. Instead, our data
lead us to conclude that local mechanisms that are intrinsic to the skeletal muscle tissue
performing the resistive contractions (ie, weightlifting) are predominant in stimulating
anabolism. Clarifying both the role of hormones in regulating muscle mass as well as the
underlying basis for adaptation of skeletal muscle to resistance exercise will hopefully
enhance and support the prescription of resistance exercise as an integral component of a
healthy lifestyle [10444].
Dimethandrolone (DMA: 7α,11beta-dimethyl-19-nortestosterone) and 11beta-methyl-19-

nortestosterone (MNT) are potent androgens in development for hormonal therapy in men.
As 5alpha-reduced androgens, such as 5alpha-dihydrotestosterone (DHT), may raise the risk
of benign prostate hyperplasia, accelerate the development of prostate carcinoma, and
increase male pattern baldness and acne, we investigated the role of 5alpha-reduction in the
androgenic activity of DMA and MNT. The authentic 5alpha-reduced metabolites, 5alpha-
dihydroDMA (5alpha-DHDMA) and 5lpha-dihydroMNT (5alpha-DHMNT), were prepared by
chemical synthesis and compared in vitro and in vivo to the parent compounds. Both 5alpha-
reduced androgens bound with high affinity to the rat androgen receptor (AR) and were
potent inducers of transactivation of 3XHRE-LUC in CV-1 cells cotransfected with a human
AR expression plasmid. To examine in vivo androgenic (stimulation of ventral prostate and
seminal vesicle weights) and anabolic (stimulation of levator ani muscle weights) activity, 22-
day-old castrate male rats were treated sc for 7 days with various doses of DMA, 5α-
DHDMA, or testosterone (T) or MNT, 5α-DHMNT, or T and necropsied on day 8. 5α-DHDMA
was at least threefold more potent than T in stimulating growth of the ventral prostate but
only 30-40 percent as potent as DMA. 5α-DHMNT was four- to eightfold more potent than T,
whereas MNT was approximately equipotent to T. To assess the possible role of 5α-
reduction in ventral prostate and seminal vesicle growth, castrate immature rats were treated
with maximally effective doses of T, DHT, DMA, MNT, or the related 19-norandrogen, 7α-
methyl-19-nortestosterone (MENT), or vehicle, with or without dutasteride (DUT), an inhibitor
of 5α-reductases types 1 and 2. In rats treated with T+DUT, serum T was significantly higher
than in rats treated with T alone, and serum DHT was decreased to levels observed in
castrate vehicle-treated rats. DUT significantly reduced both ventral prostate and seminal
vesicle weights in T-treated rats, whereas there was no significant effect of DUT on weights
of these accessory sex glands in rats treated with DMA, MNT, DHT, or MENT. These results
indicate that inhibition of 5α-reductase activity in vivo does not affect the androgenic potency
of DMA, MNT, or MENT [10445].
Over a period of 10 to 20 weeks, a supraphysiologic dose of testosterone administered to

healthy young men can increase lean body mass, as well as muscle size and strength with or
without exercise. These significant increases are dose dependent and only occur with doses
of 300 mg per week and higher. The most profound effects are noted when supraphysiologic
doses accompany a training program and are used in conjunction with a diet adequate in
protein and calories. Testosterone-induced muscle hypertrophy and increases in muscle
strength are the result of increases in the cross-sectional area of muscle fibers and
myonuclear number. Research suggests that these anabolic effects are mediated by
testosterone-influenced increases in muscle protein synthesis, creating a positive nitrogen
balance. Androgen receptors in skeletal muscle regulate the transcription of the target genes
that control the accumulation of DNA needed for muscle growth. Complementary effects
include glucocorticoid antagonism, which minimizes the catabolic actions of corticosteroids
released during the stress of athletic activity. Similarly, stimulation of the growth hormone
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insulin-like growth factor-1 axis and enhanced collagen synthesis and bone mineral density
are additional anabolic effects [07008].
Testosterone is produced in several areas within the human body. In men, most of it is
synthesized in the Leydig cells of testes and in the adrenal glands; in women, testosterone is
produced in the ovaries and adrenal glands, with a smaller fraction produced in other
peripheral sites. Its synthesis involves a cholesterol precursor and a series of enzymatic
reactions. Secretion is determined by a negative feedback mechanism that involves the
anterior pituitary gland. It is here that luteinizing hormone (LH) and follicle-stimulating
hormone (FSH) are stored. Elevated levels of testosterone affect the hypothalamus and
pituitary. At high levels of testosterone, LH tends to be reduced (affecting sperm and
endogenous testosterone production). Studies by the World Health Organization examined
the use of anabolic steroids as a form of male birth control, but the results were not
promising [07007].
The AAS are a family of lipophilic hormones derived from cholesterol that includes the
natural male hormone, testosterone, together with numerous synthetic testosterone
derivatives [150140].
Generally, supra-pharmacological doses of AAS act either by a direct mechanism, promoting

an increase in mass, force, speed of muscular contraction, and recovery after intense
physical exercise or by an indirect pathway through erythropoietic stimulation, leading to
increased synthesis of 2,3-diphospholglycerate and tissutal oxygen transfer facilitation
[150003].
During puberty, the increase in testosterone levels contributes to linear growth augmentation,
as well as muscle mass accumulation by inducing hypertrophy without changes in the
absolute number of both Type 1 and 2 muscle fibers. Testosterone also acts by increasing
the number of muscle progenitor cells and promoting their myogenic differentiation.
Testosterone promotes mitochondrial biogenesis, improves net oxygen delivery to the tissue
by increasing red cell mass and tissue capillarity, and facilitates oxygen unloading from
oxyhemoglobin [150004].
Complexities of androgen action
Androgens mediate a wide range of processes during embryogenesis and in the adult. In
mammals, the principal androgens are testosterone and its 5alpha-reduced metabolite,
5alpha-dihydrotestosterone (DHT). Although these androgenic hormones are diverse in
character, it is believed that their effects are mediated via the protein products of a single
androgen receptor gene encoded on the X-chromosome. A great deal of information has now
accumulated pertaining to the mechanisms by which nuclear receptors, such as the
androgen receptor, modulate the activity of responsive genes. The studies have
demonstrated the participation of a number of ancillary proteins in modulating activation or
repression by nuclear receptors. In addition to studies focused on the mechanisms of nuclear
receptor function, additional work has illuminated the mechanism by which androgens are
metabolized in selected tissues. This information provides a perspective on the number of
levels of complexity by which differential gene regulation by androgens may occur in different
tissues and in different cell types [01027].
Effect on glucocorticoid receptors
Androgens are hormones that are based on the structure of testosterone and act on various
858
tissues via androgen receptors to produce anabolic effects (i.e. increasing protein synthesis).
In addition, they displace glucocorticoids from their respective receptors, exerting an anti-
catabolic effect, which also promotes protein synthesis. Androgen receptors are found in
bone, adipose tissue, skeletal muscle, brain, prostate, liver, kidney, and reproductive tissues,
which accounts for both their desired and adverse effects [150011].
Mechanism of action
Testosterone is both an active hormone and a prohormone for the formation of the more
active androgen, 5alpha-dihydrotestosterone (DHT) via 5alpha-reductase...The production of
testosterone occurs predominantly (i.e. 95 %) in the Leydig cells of the testes and, to a
lesser, extent in the adrenal glands. These creations of androgens from the adrenal cortex
are insufficient to maintain male sexuality. Women also secrete small amounts of
testosterone from the ovaries and adrenal glands including DHEA and androstenedione.
Testosterone acts as an androgen either directly by binding to the androgen receptor or
indirectly by conversion to DHT. This latter compound binds more avidly to the androgen
receptor than testosterone. Dihydrotestosterone amplifies the action of androgen and
conveys specific function to the androgen–androgen receptor complex. The conversion of
testosterone to DHT is especially important for the appearance of virilization in female AAS
users because high levels of 5alpha-reductase activity in the male accessory sex glands
limits the effects of DHT. Activation of the intracellular, ligand-dependent androgen receptor
complex by testosterone and AAS results in the production RNA, DNA, and the subsequent
enhancement of protein synthesis, including increased amounts of actin and myosin in
skeletal muscles. The androgen receptor belongs to the nuclear receptor family that contains
a DNA-binding domain, a ligand-binding domain, and at least 2 transcriptional activation
domains. The enzyme, aromatase controls theandrogen-estrogen ratio by catalyzing the
conversion of testosterone to estradiol in other tissues (e.g. adipose tissue and brain).
Anabolic-androgenic steroids (e.g. methandienone, nandrolone, stanozolol) with the most
potent anabolic effects also possess the relatively greatest androgenic effects. The
interactions of AAS with the androgen receptors in various tissues vary between compounds
in this group, and these variations account for differences in the anabolic andandrogenic
effects of these compounds. The use of AAS increases skeletal lmuscle mass and strength
when used in combination with intensive strength training and high-protein, high-caloriediets.
Endogenous testosterone is responsible for sexual maturation at all stages of development
throughout the life of males. Increased AAS plasma concentrations suppress gonadotropin-
releasing hormone, endogenous testosterone secretion, luteinizing hormone, and follicle-
stimulating hormone by a negative-feedback testosterone causes a down regulation of
luteinizing hormone and is duction in the endogenous formation of steroids via a negative
feedback mechanism. Endogenous androgens stimulate RNA-polymerase, resulting in an
increased production of proteins. These proteins are responsible for normal male sexual
development, including the growth and maturation of the prostate, seminal vesicle, penis,
and scrotum. During puberty, androgens cause a sudden increase in growt hand
development of muscle along with redistribution of body fat and deepening of the voice.
Continued endogenous testosterone secretion produces increased hair (beard, and body
hair), fusion of the epiphyses, termination of growth, and the maintenance of
spermatogenesis. Testosterone also affects the formation of erythropoietin, thebalance of
calcium, and blood glucose concentrations [13002].
The anabolic effects of androgens are mediated via the androgen receptor (AR), a 110 kDa
protein with three functional domains: the transactivation, DNA binding, and ligand binding
(LBD) domains. Most of the unliganded AR is localized in the cytoplasmic compartment of
target cells, in which it is sequestered as a multiprotein complex with heat-shock proteins and
859
immunophilins. During ligand binding, the receptor dissociates from the multiprotein complex,
recruits coactivators, and translocates to the nucleus. Subsequently, it modulates the
expression of androgen-responsive genes that regulate cell fate determination. It has been
shown that androgens promote myogenic commitment and differentiation of mesenchymal
multipotent cells. The promyogenic effects of androgens in mesenchymal multipotent cells
are associated with up-regulation of myogenic determination transcription factor (MyoD),
myogenin, and myosin heavy chain II (MHC-II) [05030].
Affinity to androgen receptors

Anabolic steroids are synthetic derivatives of testosterone and are characterized by their
ability to cause nitrogen retention and positive protein metabolism, thereby leading to
increased protein synthesis and muscle mass. There are disagreements in the literature in
regards to the interaction of anabolic steroids with the androgen receptor (AR) as revealed
by competitive ligand binding assays in vitro using cytosolic preparations from prostate and
skeletal muscle. By use of tissue extracts, it has been shown that some anabolic steroids
have binding affinities for the AR that are higher than that of the natural androgen
testosterone, while others such as stanozolol and methanedienone have significantly lower
affinities as compared with testosterone. In the study it was shown that stanozolol and
methanedienone are low affinity ligands of the rat recombinant AR as revealed by a ligand
binding assay in vitro, however, based on a cell-based AR-dependent transactivation assay,
they are potent activators of the AR. It was also shown that a single injection of stanozolol
and methanedienone causes a rapid cytosolic depletion of AR in rat skeletal muscle. Based
on these results, it was conclude that anabolic steroids with low affinity to AR in vitro, can in
fact in vivo act on the AR to cause biological responses [05031].
Anabolic effects
Anabolism is defined as any state in which nitrogen is differentially retained in lean body
mass through the stimulation of protein synthesis and/or a reduction in protein breakdown.
There is a growing body of evidence that AASs have positive anabolic actions on the
musculoskeletal system, influencing lean body mass, muscle size, strength, protein
metabolism, bone metabolism, and collagen synthesis. Skeletal muscle is a primary target
tissue for the anabolic effects of AAS. Supraphysiologic doses of testosterone enanthate
administered to healthy young men over periods lasting 10 to 20 weeks increase lean body
mass, muscle size, and strength, with or without exercise. The anabolic effect of testosterone
is dose dependent, and significant increases in muscle size and strength occur only with
doses of 300 mg per week and higher. Such supraphysiologic doses elevate mean serum
testosterone concentrations above normal values to more than 1000 ng/dL. The observed
effects of testosterone and strength training are additive. The testosterone-induced increase
in muscle size is due to a dose-dependent hypertrophy that results from an increase in cross-
sectional areas of both type I and type II muscle fibers and an increase in myonuclear
number. Evidence suggests that these morphometric effects are the result of a testosterone-
induced increase in muscle protein synthesis. The testosterone-induced increase in strength
may be the result of muscle fiber hypertrophy. However, strength increases may also reflect
changes in muscle architecture because testosterone-treated muscles exhibit an increase in
muscle pennation – a finding typically associated with high-force, low-velocity contractions.
AASs have also been shown to improve exercise tolerance and the adaptability of muscle to
overload by protecting against muscle fiber damage and increasing the rate of protein
synthesis during recovery. Collagen and bone are also target tissues for the anabolic actions
of AAS. In soft connective tissues, AASs enhance collagen synthesis in a dose-dependent
manner. In bone, testosterone supplementation increases bone mineral density via a direct
suppressive effect on osteoclasts [04018].
860
Wasting disease states can be characterized either by decreased or accelerated protein
turnover. Slow protein turnover conditions include some chronic disease states characterized
by low protein-energy intake, immobilization, tissue hypoxia, or moderate liver or kidney
failure. Rapid protein turnover conditions include the acute hypercatabolic states and some
chronic disease characterized by systemic inflammation. Anabolic hormones, such as growth
hormone and androgenic steroids, act by stimulating protein turnover especially in skeletal
muscle. These therapies appear to be safe and efficacious in chronic diseases states where
they tend to increase and normalize a rate of protein turnover which is already depressed. In
critically ill patients with a preexisting condition of accelerated protein turnover, it might
therefore not be appropriate to further accelerate the rate of protein turnover by using these
anabolic agents. Chronically uremic patients often exhibit a low protein turnover that may
increase progressively with the decline of renal function. Thus, anabolic agents can
normalize protein metabolism in stable patients without complications, but they should be
used carefully in advanced renal failure especially during intercurrent infections [01028].
The anabolic effect of AAS is mediated primarily by ARs in skeletal muscle. The AR
regulates the transcription of target genes that may control the accumulation of DNA required
for muscle growth. It was previously thought that ARs are saturated at physiologic levels of
testosterone and that providing supplemental exogenous testosterone offered no additional
benefit. However, recent studies demonstrate that ARs can be up-regulated by exposure to
AAS and that AR number is increased by strength training. This suggests a possible
mechanism by which supraphysiologic doses of AAS combined with exercise might
complement each other [04018].
An anticatabolic mechanism has also been proposed for the anabolic effects of AAS, but
because testosterone can increase net protein synthesis without slowing protein degradation,
the specific contribution of glucocorticoid antagonism has not been demonstrated
equivocally. Testosterone may also influence anabolism via a direct induction of GH and
IGF-1 [04018].
Previous studies have shown that strength exercise improves memory and increases
expression of a myriad of proteins involved on neuronal survival and synaptic plasticity in the
hippocampus. Conversely, chronic exposure to supraphysiological levels of anabolic
androgenic steroids (AAS) can induce psychiatric abnormalities, cognitive deficits, impair
neurotransmission, alter the levels of neurotrophic factors, decrease cell proliferation and
neurogenesis, and enhance neuronal cell death. In the present study, we investigated the
effects of the AAS nandrolone decanoate (ND) administration during a strength exercise
program on cell proliferation, apoptotic status and brain-derived neurotrophic factor (BDNF)
expression in the rat hippocampus. Adult male Wistar rats were subjected to 4 weeks of
progressive strength exercise in a vertical ladder apparatus with or without daily doses (5.0
mg/kg, SC) of ND. Immunohistochemistry analysis revealed that strength exercise increased
significantly the number of Ki-67-positive cells (a cell proliferation marker) in dentate gyrus
(DG) of hippocampus. However, this effect was abrogated when strength exercise was
combined with ND. Although western blot analysis of whole hippocampus showed no
significant differences in Bax and Bcl-2 protein expression among groups, the
immunoreactivity of the pro-apoptotic protein Bax was significantly increased in DG, CA1 and
CA3 hippocampal subfields of sedentary rats treated with ND. Moreover, the increase in the
immunoreactivity of anti-apoptotic protein Bcl-2 (DG and CA3) induced by strength exercise
was diminished by ND. There were no significant differences in BDNF expression among
experimental groups. Therefore, the present findings suggest that the beneficial effects of
861
strength exercise on hippocampal cell proliferation and apoptotic signaling are impaired by
ND [14636].
Chemical structure versus function
Steroids are organic molecules with a tetracyclic ring system; all steroids with the exception
of retinoic acid are derived from cholesterol. There are 4 major classes of natural steroid
hormones (androgens, corticoids, estrogens, and progestogens) with testosterone being the
principal male androgenic steroid. AASs are synthetic compounds similar in chemical
structure to testosterone (molecular weight 288 g/mol). There are 3 major classes of AASs
(oral, injectableoil-based, and injectablewater-based) andat least 30 anabolic-androgenic
steroid compounds. Abuse of other forms of AASs includes the use of buccal (Striant),
sublingual (tetrahydrogestrinone), and transdermal (testosterone cream) preparations; these
short-acting preparations are typically testosterone-based. The advantage of the buccal,
sublingual, and transdermal preparations is the rapid clearance within 1 week after even
large doses compared with 2-14 days for oral preparations and 4 weeks for water-soluble
parenteral preparations. Non-steroidal selective androgen receptor modulators (SARMs) are
experimental substances (e.g. bicyclic hydantoin, analogs of arylpropionamide, quinoline,
tetrahydroquinoline) that may offer betterd issociation of thebiologic and anabolic effects of
steroids; although these substances are not routinely available, the World Anti-Doping
Agency (WADA) added these substances to the prohibited list in 2008. Up-to-date
information on the list of banned substances is available on the WADA website
(http://www.wada-ama.org/en/). Testosterone is the prototype for all AASs. Modification of
testosterone during the synthesis of AASs involves the following three methods: alkylationof
the 17beta-hydroxyl group, esterification at the 17alpha-position, or modification of the
steroid nucleus to enhance anabolic properties. Although all currently available anabolic
steroids have androgenic properties, the anabolic properties are greater for synthetic AASs
than for testosterone. Some structura lmodifications improve bioavailability and prolong the
duration of action, whereas other modifications reduce anabolic effects while enhancing
androgenic effects. Esterification at the 17beta-hydroxy position increases lipophilic and
androgenic properties while improving intramuscular bioavailability. In general, alkylation of
the 17alpha-hydroxy position retards hepatic degradation and improves oral bioavailability,
but these compounds typically are less potent than 17beta-esters. Weaker formulations of
AASs are often marketed as prohormones in dietary supplements, particularly
dehydroepiandrosterone (DHEA), 19-norandrostenediol, androstenedione, 19-norandro-
stenedione, 1-testosterone, and prostanozol. The lack of the 17alpha-alkyl moiety results in
extensive first-pass metabolism, and the anabolic effect of DHEA and androstenedione are
limited by their weakbinding to the androgen receptor despite some conversion to
testosterone. The effect of these AASs is substantially greater on women than men because
of therelatively larger increase in testosterone in the former. Tetrahydrogestrinone (THG;13-
ethyl-17hydroxy-18,19-dinor-17alphapregn-4,9,11-trien-3-one), norbolethone, and madol
(17alpha-methyl-5alpha-androst-2-en-17beta-ol) are designer anabolic steroids, which were
synthesized to avoid detection during use. However, THG is a nonspecific androgen agonist
that binds many steroid hormone receptors, particularly the glucocorticoid receptors [13002].
Effect on human skeletal muscle
The myotrophic effects of androgens on muscle strength and mass are the main reason for
their popularity among androgen users. Androgens also increase lean body mass, decrease
fat mass, enhance performance, sustain intensive training periods, and can improve
appearance. The effects on lean body mass were shown by treating young men with a
gonadotropin-releasing hormone (GnRH) analogue that suppressed endogenous T
862
production. These men showed decreased rates of whole body protein synthesis, muscle
strength and fat oxidation, together with an increased fat mass. When T was replaced
through supplementation, there was a restoration of muscle size and strength with a
concomitant reduction in fat mass. Androgens including T increase muscle fiber hypertrophy
in human skeletal muscle by enhancing protein synthesis. This occurs via the activation of
satellite cells and the promotion of myonuclear accretion when existing myonuclei become
unable to sustain further enhancement of protein synthesis. The use of androgen therapy
during aging is primarily to promote muscle strength by improving or maintaining muscle
mass [13084].
The effects of long-term (over several years) anabolic androgen steroids (AAS)
administration on human skeletal muscle are still unclear. In this study, seventeen strength
training athletes were recruited and individually interviewed regarding self-administration of
banned substances. Ten subjects admitted having taken AAS or AAS derivatives for the past
5 to 15 years (Doped) and the dosage and type of banned substances were recorded. The
remaining seven subjects testified to having never used any banned substances (Clean). For
all subjects, maximal muscle strength and body composition were tested, and biopsies from
the vastus lateralis muscle were obtained. Using histochemistry and immunohistochemistry
(IHC), muscle biopsies were evaluated for morphology including fiber type composition, fiber
size, capillary variables and myonuclei. Compared with the Clean athletes, the Doped
athletes had significantly higher lean leg mass, capillary per fibre and myonuclei per fiber. In
contrast, the Doped athletes had significantly lower absolute value in maximal squat force
and relative values in maximal squat force (relative to lean body mass, to lean leg mass and
to muscle fiber area). Using multivariate statistics, an orthogonal projection of latent structure
discriminant analysis (OPLS-DA) model was established, in which the maximal squat force
relative to muscle mass and the maximal squat force relative to fiber area, together with
capillary density and nuclei density were the most important variables for separating Doped
from the Clean athletes (regression = 0.93 and prediction = 0.92). In Doped athletes, AAS
dose-dependent increases were observed in lean body mass, muscle fiber area, capillary
density and myonuclei density. In conclusion, long term AAS supplementation led to
increases in lean leg mass, muscle fiber size and a parallel improvement in muscle strength,
and all were dose-dependent. Administration of AAS may induce sustained morphological
changes in human skeletal muscle, leading to physical performance enhancement [14604].
Transcriptional regulation of myotrophic actions by testosterone

Androgens regulate body composition and skeletal muscle mass in males, but the molecular
mechanisms are not fully understood. Recently, we demonstrated that trenbolone (a potent
synthetic testosterone analogue that is not a substrate for 5-alpha reductase or for
aromatase) induces myotrophic effects in skeletal muscle without causing prostate
enlargement, which is in contrast to the known prostate enlarging effects of testosterone.
These previous results suggest that the 5alpha-reduction of testosterone is not required for
myotrophic action. It was now reported differential gene expression in response to
testosterone versus trenbolone in the highly androgen-sensitive levator ani/bulbocavernosus
(LABC) muscle complex of the adult rat after 6 weeks of orchiectomy (ORX), using real time
PCR. The ORX-induced expression of atrogenes (Muscle RING-finger protein-1 [MuRF1]
and atrogin-1) was suppressed by both androgens, with trenbolone producing a greater
suppression of atrogin-1 mRNA compared to testosterone. Both androgens elevated
expression of anabolic genes (insulin-like growth factor-1 and mechano-growth factor) after
ORX. ORX-induced increases in expression of glucocorticoid receptor (GR) mRNA were
suppressed by trenbolone treatment, but not testosterone. In ORX animals, testosterone
promoted WNT1-inducible-signaling pathway protein 2 (WISP-2) gene expression while
trenbolone did not. Testosterone and trenbolone equally enhanced muscle regeneration as
shown by increases in LABC mass and in protein expression of embryonic myosin by
863
western blotting. In addition, testosterone increased WISP-2 protein levels. Together, these
findings identify specific mechanisms by which testosterone and trenbolone may regulate
skeletal muscle maintenance and growth [14260].
To highlight recent data demonstrating direct anabolic effects of androgens on the

mammalian skeletal muscle and review the mechanisms by which testosterone regulates
body composition. Testosterone increases lean body mass and decreases fat mass in young
men; the magnitude of the changes induced by testosterone in lean and fat mass are
correlated with testosterone dose and the prevalent testosterone concentrations. Older men
are as responsive to the anabolic effects of testosterone on the muscle as young men, but
have increased frequency of adverse events with higher testosterone doses. This reciprocal
change in lean and fat mass induced by androgens is best explained by the hypothesis that
androgens promote the commitment of mesenchymal pluripotent cells into myogenic lineage
and inhibit adipogenesis through an androgen receptor mediated pathway. Resident muscle
satellite cells increase in number with testosterone administration forming myoblasts leading
to greater numbers of myonuclei in larger myofibers. Testosterone administration is
associated with increased size of motor neurons. The roles of 5-alpha reduction and
aromatization of testosterone into dihydrotestosterone and estradiol, respectively, in
mediating testosterone effects on body composition are poorly understood. Thus,
testosterone induces skeletal muscle hypertrophy by multiple mechanisms, including its
effects in modulating the commitment of pluripotent mesenchymal cells. These changes in
skeletal muscle lead to improved muscle strength and leg power; however, further studies
are needed to determine the effects of testosterone on physical function and health-related
outcomes in sarcopenia associated with aging and chronic illness [04069].
Short term AAS administration

Even though athletes using AAS claim significant gain in performance, a large number of
academic studies investigating the performance-enhancing effects of AAS have described
discordant and often contradictory outcomes. Some studies revealed significant gains in
strength and muscle mass/girth whereas others reported no effects of AAS on muscle
mass/girth and/or muscle strength. Such conflicting results have been attributed to poor
study design including non-blinded condition, no placebo control, small sample size and AAS
dose variation. Above all, in most studies, out of ethic consideration, AAS administration was
usually no longer than 6 months. Such a short period of AAS administration obviously could
not reflect the reality of AAS abuse in athletes and sport enthusiasts. In reality, AAS usage
was estimated to sustain for several years or the whole competition period in athletes. Thus,
the difference in AAS administration period between AAS abusers and subjects in most
academic studies might be one of the major reasons leading to the different conclusions.
Short term AAS administration has been shown to induce muscle strength enhancement. It
has been proposed that the effects of AAS on muscle are dose-dependent. Twenty weeks of
testosterone administration increases skeletal muscle mass, leg strength and power in a
dose-dependent fashion, but did not improve muscle fatigability or physical function. The
increased muscle strength has been attributed to increased muscle mass which was
associated with muscle fiber hypertrophy of both type I and type II fibers [14604].
Long term AAS administration

Effects of long term AAS administration on muscle morphology in relation with muscle
strength as well as with body composition are, however, still unclear. In an early studies on
strength training subjects with long period AAS self-administration (9 ± 3 years), analysis of
muscle biopsies revealed significant increases in mean fiber area for both type I and type II
fibers, number of myonuclei and proportion of central nuclei in the steroid users compared to
the non-steroid users. In addition, in the steroid users, significant increase in frequency of
fibers expressing developmental myosin heavy chain (MyHC) isoforms was also observed
864
compared to the non-steroid users. On basis of the results, we concluded that intake of
anabolic steroids in combination with strength training induced both fiber hypertrophy and
fiber hyperplasia (formation of new muscle fibres), in which the activation of satellite cells is a
key process. However, the studies did not reveal whether the changes in muscle morphology
were accompanied by improvement in muscle strength as well as body composition [14604].
The main findings of one study of long term AAS administration were that the doped athletes
had higher lean mass, capillary density and myonuclei density, but lower maximal squat
force relative to muscle mass and to fiber area, compared to the clean athletes. The Doped
group also had a tendency towards larger fibers, although not significant, most likely due to
large variations in fibre area. Low levels of LH and FSH, and high levels of prolactin in some
individuals indicate a disturbed pituitary gland function with possible negative effects on
reproductive function. High levels of ALAT, ASAT and CK in some individuals suggest that
long term use of AAS could damage both liver and muscle tissue. However, no correlations
between AAS intake and hormone levels was observed. Thus, testosterone levels at time of
sampling cannot explain alternations in these variables, rather concentrations outside clinical
limits must stem from long-term supplementation of AAS. Multivariate statistics showed that
a combination of eight morphological parameters could clearly separate the doped from the
clean athletes. Correlation analysis revealed significant positive correlations between AAS
dosage and relative muscle force. The results support previous findings that AAS
administration could induce enhancement in both muscle mass and muscle strength, and
that the improvements are AAS dose-dependent [14604].
Despite abundant studies on the effects of AAS on skeletal muscle, many results are
contradictory. Some studies have shown gains in body weight, girth, fat-free mass or lean
body mass, but not in muscle strength, whereas others have shown gains in both muscle
mass/girth and muscle strength, or in neither muscle mass/girth nor muscle strength after
short term (from 17 days to 16 weeks) or long term (2 years) AAS administration. Increased
muscle mass in subjects using AAS has been proposed to result from muscle hypertrophy
alone or from both muscle hypertrophy and hyperplasia. Muscle hypertrophy is often evident
by increased muscle fiber size and increased number of myonuclei. The latter is associated
with satellite cell activation and myoblast infusion with the existing muscle fibers, leading to
greater numbers of myonuclei in larger myofibers. In previous studies on subjects with long
term AAS supplementation (9 ± 3 years), it was observed significant higher frequency of
newly formed myofibers in AAS users than in the non-AAS users, indicating that steroid can
induce both muscle hypertrophy and hyperplasia. In the next study, long term AAS
supplementation was only associated with higher lean leg mass, but not with larger fiber size,
indicating that muscle fiber hyperplasia may play a role in the muscle mass enhancement.
Coincidently, the number of myonuclei in type I fibers in the doped athletes was significantly
higher than in the clean athletes, which may indicate satellite cell activation for muscle fiber
hyperplasia [14604].
Not many studies have examined the effects of AAS on muscle capillaries. In a previous
study of 20 weeks of graded testosterone enanthate injection (25, 50, 125, 300, or 600 mg),
it was not observed significant difference in capillary density among the five treatment
groups. The authors suggested that it is not unlikely that a significant increase in capillaries
takes longer than 20 weeks. In the present study, we observed more capillaries around both
type I and type IIa fibers in the Doped athletes compared to the Clean group. Importantly,
when the parameter of capillaries per fiber (CAF) was calculated by fiber area (CAFA), the
significant difference in CAF between the two groups disappeared, indicating proportional
and simultaneous increases in number of capillary around each fiber and in muscle fiber size
in the Doped group. These are the first results demonstrating an association between long
term AAS supplementation and muscle capillarization. Consequently, AAS will enhance not
865
only muscle strength, but also muscle endurance [14604].
It has been shown that combined administration of androgens and resistance training is
associated with greater gains in lean body mass, muscle size, and maximal voluntary
strength than either intervention alone. The greater increase in maximal voluntary strength is
often attributed to greater increase in lean body mass and/or muscle size. However, some
studies using lower AAS doses and shorter supplementation times have shown no gains in
muscle strength, regardless if lean body mass and muscle size were increased or not. Thus,
for long-term AAS abusers, increase in muscle mass/lean body mass may be not directly
associated with muscle strength improvement. It is worth to notice that compared to a not
doped group, the doped group presented larger variations in many of the measurements like
leg lean mass (Doped, 24.6-32.6 kg vs Clean, 22.8-26.9 kg), leg maximal strength (Doped,
1823-3242 N vs Clean, 2799-3570 N) and muscle fibre size (Doped, 6055-16330 microm2 vs
Clean, 5668-8567 microm2). It has been reported that much of the gains in body weight and
maximum bench press obtained during, and immediately after, 12 weeks of steroid
administration and resistance training was lost during a subsequent 12 week period when
androgens were not administered. However, not all doped athletes are in the same “AAS
cycle”, indicating that during a study, some of the doped subjects are taking AAS whereas
the others were in a “clean” period, i.e. AAS effects on muscles were stacking in some
subjects but diminishing in the others. This may explain, among other factors, the large
variations in some measurements [14604].
Previous studies have shown that testosterone administration was associated with a dose-
dependent increase in skeletal muscle mass, leg strength and power. However, similar
correlation between AAS dosage and leg lean mass (or fat free body mass) was not
observed in another study. One previous study has shown that 180 days of transdermal
testosterone treatment resulted in increase in leg press by 90 days but did not induce further
improvement by 180 days. Another study has shown that major effects of AAS on muscle
strength and lean body mass occurred over the first 12 months of testosterone administration
to older men. In the present study, because the Doped athletes were not in the same AAS
intake “cycle”, the time-dependent effects of AAS on muscles may explain some of the
variations in data [14604].
Density of capillaries in the heart

Changes in the heart compartments that leads to pathological cardiac hypertrophy can be
related to testosterone reduction in aging males since heart cells are susceptible to
androgens. Resistance exercise delays the changes of aging. One study aimed to analyze
alterations of the left ventricle of aged rats subjected to resistance exercise with
administration of testosterone. Wistar rats were divided into five groups: C Group (control), S
Group (sedentary), ST Group (sedentary treated with testosterone), T Group (trained) and
TT Group (trained and treated with testosterone), strength training protocol and testosterone
treatment were 16 weeks long. All groups were sacrificed at 16 months except for C group,
sacrificed at 13 months. There was no change in the weight of the heart or the left ventricle
between the groups. ST group showed increase in Nv [cap] density of capillaries and
collagen, with no differences in interstitial space. Both trained groups (T and TT) showed
increase in the numerical density of capillaries (Nv [cap]) and in the interstitial space, with no
changes in collagen. Resistance exercise combined with testosterone triggered a response
of compensatory adjustment in the increase of Nv [cap], collagen and interstitial space,
increasing perfusion and nutrition to the heart [14638].
Effect on bone tissue
866
The adult skeleton is periodically remodeled by temporary anatomic structures that comprise
juxtaposed osteoclast and osteoblast teams and replace old bone with new. Estrogens and
androgens slow the rate of bone remodeling and protect against bone loss. Conversely, loss
of estrogen leads to increased rate of remodeling and tilts the balance between bone
resorption and formation in favor of the former. Studies from one group during the last 10
years have elucidated that estrogens and androgens decrease the number of remodeling
cycles by attenuating the birth rate of osteoclasts and osteoblasts from their respective
progenitors. These effects result, in part, from the transcriptional regulation of genes
responsible for osteoclastogenesis and mesenchymal cell replication and/or differentiation
and are exerted through interactions of the ligand-activated receptors with other transcription
factors. However, increased remodeling alone cannot explain why loss of sex steroids tilts
the balance of resorption and formation in favor of the former. Estrogens and androgens also
exert effects on the lifespan of mature bone cells: pro-apoptotic effects on osteoclasts but
anti-apoptotic effects on osteoblasts and osteocytes. These latter effects stem from a
heretofore unexpected function of the classical "nuclear" sex steroid receptors outside the
nucleus and result from activation of a Src/Shc/extracellular signal-regulated kinase signal
transduction pathway probably within preassembled scaffolds called caveolae. Strikingly,
estrogen receptor (ER) alpha or beta or the androgen receptor can transmit anti-apoptotic
signals with similar efficiency, irrespective of whether the ligand is an estrogen or an
androgen. More importantly, these nongenotropic, sex-nonspecific actions are mediated by
the ligand-binding domain of the receptor and can be functionally dissociated from
transcriptional activity with synthetic ligands. Taken together, these lines of evidence strongly
suggest that, in sex steroid deficiency, loss of transcriptional effects may be responsible for
the increased osteoclastogenesis and osteoblastogenesis and thereby the increased rate of
bone remodeling. Loss of nongenotropic anti-apoptotic effects on mature osteoblasts and
osteocytes, in combination with an opposite effect on the lifespan of mature osteoclasts, may
be responsible for the imbalance between formation and resorption and the progressive loss
of bone mass and strength. Elucidation of the dual function of sex steroid receptors has
important pathophysiologic and pharmacologic implications. Specifically, synthetic ligands of
the ER that can evoke the nongenotropic but not the genotropic signal may be bone anabolic
agents, as opposed to natural estrogens or selective estrogen receptor modulators that are
antiresorptive agents. The same ligands may also circumvent the side effects associated
with conventional hormone replacement therapy [02025].
Effect on muscle mass, bone mineral density and clinical function after a hip fracture
A total of 63 women who had an operation for a fracture of the hip was randomly allocated to
one year of treatment either with anabolic steroids, vitamin D and calcium (anabolic group) or
with calcium only (control group). The thigh muscle volume was measured by quantitative
CT. The bone mineral density of the hip, femur and tibia was assessed by quantitative CT
and dual-energy x-ray absorptiometry and of the heel by quantitative ultrasound. Quantitative
CT showed that the anabolic group did not lose muscle volume during the first 12 months
whereas the control group did. There was less bone loss in the proximal tibia in the anabolic
group than in the control group. The speed of gait and the Harris hip score were significantly
better in the anabolic group after six and 12 months. Anabolic steroids, even in this moderate
dose, given in combination with vitamin D and calcium had a beneficial effect on muscle
volume, bone mineral density and clinical function in this group of elderly women [02029].
Endogenous steroids
Determining the origin of anabolic androgenic steroids (AAS) that also are produced
endogenously in the human body is a major issue in doping control. In some cases, the
presence of nandrolone and boldenone metabolites might result from endogenous
867
production. The GC-C-IRMS technique (gas chromatography-combustion-isotope ratio mass
spectrometry) enables the carbon isotopic ratio (CIR) to be measured to determine the origin
of these metabolites. The aim of this study was to use GC-C-IRMS to determine the delta-
13
CVPDB values of seized boldenone and nandrolone preparations to decide if the steroids
themselves were depleted in 13C, compared to what is normally seen in endogenously
produced steroids. In addition, several testosterone preparations were analyzed. A total of
69 seized preparations were analyzed. The nandrolone preparations showed delta-13CVPDB
values in the range of -31.5 permille to -26.7 permille. The boldenone preparations showed
delta-13CVPDB values in the range of -32.0 permille to -27.8 permille, and for comparison the
testosterone preparations showed delta-13CVPDB values of -31.0 permille to -24.2 permille.
The results showed that the values measured in the nandrolone and boldenone preparations
were in the same range as those measured in the testosterone preparations. The study also
included measurements of CIR of endogenously produced steroids in a Norwegian/Danish
reference population. The delta-13CVPDB values measured for the endogenous steroids in
this population were in the range of -21.7 to -26.8. In general, most of the preparations
investigated in this study show 13C-depleted delta values compared to endogenously
produced steroids reflecting a northern European diet [14744].
Cholesterol is the starting substance for formation of glucocorticoids, mineralocorticoids and

sex steroids. Precursors of androgens are formed in the adrenals and biotransformed in the
endocrine target organs, the gonads and the prostate gland. Dihydrotestosterone (DHT) is
formed from testosterone in the prostate and is a more potent androgen than testosterone
itself. Some of the testosterone precursors have weak androgenic effects such as
dehydroepiandrosterone (DHEA) and androstenedione.They may be present in dietary
products and abused per se. Most of the end products are eliminated via the kidneys after
conjugation with glucuronic acid or sulphate groups by enzymes in the uridine glucuronosyl
transferase (UGT) and sulphate transferase (SULT) super families, respectively. The UGT
enzymes have different selectivities for the androgens and androgen metabolites. The
specificity, the activity and the genetic variation of the various UGT enzymes is of particular
interest in the conjugation and excretion of testosterone and epitestosterone as these
steroids and their conjugates are quantified in the urine in conventional testosterone doping
tests. Genetic variation in other steroid metabolizing enzymes may also influence the
disposition of testosterone and other AAS [12192].
Androgen disposition and genetic variation
Genetic variation in genes involved in the synthesis,breakdown and elimination of androgens

may affect the bioavailability of administrated AAS, and hence affect the degree of anabolic
and toxic effects of doping. Moreover, genetic variation in the androgen receptor (AR) may
modulate the pharmacodynamic effects of AAS. As a corollary, several of these
polymorphisms may change the serum concentrations of AAS and excretion of AAS
metabolites in the urine. Therefore, genetic variations are an important source of
confounders in doping tests. Several functional polymorphisms in these genes known to alter
the systemic load and excretion rate of androgens are discussed below in relation to doping
and doping control [12192].
The metabolism of testosterone is revisited. Four previously unreported metabolites were

detected in urine after hydrolysis with KOH using a liquid chromatography-tandem mass
spectrometry method and precursor ion scan mode. The metabolites were characterized by a
product ion scan obtained with accurate mass measurements. Androsta-4,6-dien-3,17-dione,
androsta-1,4-dien-3,17-dione, 17-hydroxy-androsta-4,6-dien-3-one and 15-androsten-3,17-
dione were proposed as feasible structures for these metabolites on the basis of the mass
868
spectrometry data. The proposed structures were confirmed by analysis of synthetic
reference compounds. Only 15-androsten-3,17-dione could not be confirmed, owing to the
lack of a commercially available standard. That all four compounds are testosterone
metabolites was confirmed by the qualitative analysis of several urine samples collected
before and after administration of testosterone undecanoate. The metabolite androsta-1,4-
dien-3,17-dione has a structure analogous to that of the exogenous anabolic steroid
boldenone. Specific transitions for boldenone and its metabolite 17β-hydroxy-5β-androst-1-
en-3-one were also monitored. Both compounds were also detected after KOH treatment,
suggesting that this metabolic pathway is involved in the endogenous detection of boldenone
previously reported by several authors [10325].
Metabolism – phase I enzymes

Aldo-keto reductases (AKR1C) are divided into three families of which the AKR1C members
AKR1C1, AKR1C2, AKR1C3,AKR1C4 and AKR1D1 have been shown to play an important
role in steroid metabolism. Genetic variations in AKR1C genes may regulate the local
concentration of steroid hormones. AKR1C3 is involved in the formation and inactivation of
testosterone. Cytochromes P450 (CYPs) CYPs are the most important phase I enzymes in
drug metabolism, accounting for the metabolism of more than 60 percent of all drugs.
Several of the members of the CYP superfamily are also important catalysts in the metabolic
network of steroids, e.g. CYP3A4 catalyzing 4-hydroxylation of testosterone. There is a large
variation in the CYP3A4 activity between individuals, partly explained by polymorphisms in
the CYP3A4 gene and partly by induction or inhibition by exogenous compounds and drugs.
Several polymorphisms in this gene have been associated with altered serum concentrations
of testosterone and oestrogens [12192].
Many anabolic steroids on the market are available as esters in order to achieve a retarded
release. These pro-drugs need to be hydrolyzed prior to activation. It has been shown that
PDE7B is involved in the hydrolysis of testosterone enanthate and nandrolone decanoate
with implications for the serum concentrations and bioavailability of testosterone enanthate. It
is conceivable that higher serum concentrations of testosterone convey an advantage in
terms of strong androgenic influence on the organ receptors, thus being likely to improve the
physical achievements [12192].
Metabolism – phase II enzymes

Uridine glucuronosyl transferases (UGTs) UGT enzymes are considered to be the main
enzymes for inactivation and quantitative metabolic elimination of steroid hormones.
UGT2B17 has been shown to be the main enzyme in testosterone glucuronidation activity in
vitro and in vivo. It has been possible to demonstrate a large variation in the gene deletion
both within, and between ethnic populations with important consequences for the
interpretation of the T : E test. There is a large variation in urinary testosterone
concentrations in UGT2B17 carriers, and this SNP may contribute to the variation in
excretion within each UGT2B17 deletion polymorphism mode. UGT2B7 has also been
identified as the main phase II enzyme involved in epitestosterone glucuronidation. Whether
other SNPs in the UGT2B7 (or in other epitestosterone metabolizing enzyme genes) could
explain the inter-individual variation in epitestosterone excretion needs to be investigated.
Sulphotransferases (SULT) Even though glucuronides are the main conjugated metabolites
of androgens, many steroids are also sulphated to different extent. For some steroids such
as DHEA, sulphation is the major metabolic phase II pathway. As a matter of fact DHEA and
DHEA sulphate are the most abundant androgen precursors in the circulation [12192].
Lately genetic polymorphisms in many transporter proteins have been shown to affect the
outcome of drug treatment. The superfamily of organic anion transporting polypeptides
(OATP), encoded by solute carrier organic anion transporter (SLCO) genes, mediates the
869
uptake of various endogenous compounds including hormones. SLCO1B3 is involved in the
uptake of several hormones including testosterone and SLCO2B1 mediates the transport of
steroid conjugates, such as DHEA sulphate. Both these SLCOB genes are polymorphically
expressed and have been shown to be functional, i.e. there is a correlation with the capacity
to transport hormones [12192].
Steroid hormone binding globulin (SHBG)
Steroid hormone binding globulin (SHBG) Testosterone and DHT bind with high affinity to
SHBG, thereby regulating the concentration of bioactive testosterone. Several genetic
polymorphisms have been characterized in the human SHBG gene. SHBG polymorphisms
are associated with serum concentration of DHT and testosterone. Since genetic variation in
the SHBG gene affects both the binding (and thereby bioavailability) and the urinary
excretion of androgens, polymorphisms in SHBG may modulate both the effects of steroid
doping and the analysis of illicit AAS [12192].
SHBG is the most important carrier protein for androgens. The dimeric protein consists of
two identical peptide chains of 370 amino acids. SHBG synthesis is stimulated by oestrogen
in the liver and decreased by androgens and anabolic steroids. Together with serum albumin
(binding 40–50 % of T), SHBG (binding 50–60 % of T) forms circulating reservoir of T,
balances the concentration of free fraction and decreases the rate of metabolism in the liver.
With respect to genetic variation, studies have revealed SNP which alters SHBG binding
affinity for T. Parallel to carrier proteins, there are transporter proteins which are involved in
the absorption, distribution and elimination of drugs by participating to permeation of the
drugs into cells and access of the drugs to their targets. Genetic polymorphism has also
been shown to occur at this phase of bioprocesses, of which an example is the organic ion
transporter OATP1B3 (encoded by SLCO1B3 gene) and its two polymorphic variants which
transport T with varying efficiencies [14450].
Hydroxysteroid dehydrogenase
The 3beta-hydroxysteroid dehydrogenase/delta(5)-delta(4) isomerase (3beta-HSD) and

1alpha-hydroxylase/17,20-lyase cytochrome P450 (P450c17) enzymes are important in
determining the balance of the synthesis of different steroids such as progesterone (P4),
glucocorticoids, androgens, and estrogens. How this is achieved is not a simple matter
because each of the two enzymes utilizes more than one substrate and some substrates are
shared in common between the two enzymes. The two synthetic pathways, delta(4) and
delta(5), are interlinked such that it is difficult to predict how the synthesis of each steroid
changes when any of the enzyme activities is varied. In addition, the P450c17 enzyme
exhibits different substrate specificities among species, particularly with respect to the 17,20-
lyase activity. The mathematical model developed in this study simulates the network of
reactions catalyzed by 3β-HSD and P450c17 that characterizes steroid synthesis in human,
non-human primate, ovine, and bovine species. In these species, P450c17 has negligible
17,20-lyase activity with the delta(4)-steroid 17alpha-hydroxy-progesterone (17OH-P4);
therefore androstenedione (delta4) is synthesized efficiently only from dehydroepiandro-
sterone (DHEA) through the delta(5) pathway. The model helps to understand the interplay
between fluxes through the delta(4) and delta(5) pathways in this network, and how this
determines the response of steroid synthesis to the variation in 3beta-HSD activity or in the
supply of the precursor substrate, pregnenolone (P5). The model simulations show that A4
synthesis can change paradoxically when 3beta-HSD activity is varied. A decrease in 3beta-
HSD activity to a certain point can increase A4 synthesis by favouring metabolism through
the delta(5) pathway, though further decrease in 3beta-HSD activity beyond that point
870
eventually limits A4 synthesis. The model also showed that due to the competitive inhibition
of the enzymes' activities by substrates and products, increasing the rate of P5 supply above
a certain point can suppress the synthesis of A4, DHEA, and 17OH-P4, and consequently
drive more P5 towards P4 synthesis [11568].
Modulation of follistatin and myostatin propeptide
The purpose of one pilot study was to investigate the impact of training, anabolic steroids
and endogenous hormones on myostatin-interacting proteins in order to identify
manipulations of myostatin signalling. To identify whether analysis of the myostatin
interacting proteins follistatin and myostatin propeptide is suitable to detect the abuse of
anabolic steroids, their serum concentrations were monitored in untrained males,
bodybuilders using anabolic steroids and natural bodybuilders. In addition, we analysed
follistatin and myostatin propeptide serum proteins in females during menstrual cycle. Our
results showed increased follistatin concentrations in response to anabolic steroids.
Furthermore, variations of sex steroid levels during the menstrual cycle had no impact on the
expression of follistatin and myostatin propetide. In addition, we identified gender differences
in the basal expression of the investigated proteins. In general, follistatin and myostatin
propeptide concentrations were relatively stable within the same individual both in males and
females. In conclusion, the current findings provide an insight into gender differences in
myostatin-interacting proteins and their regulation in response to anabolic steroids and
endogenous hormones. Therefore our data provide new aspects for the development of
doping prevention strategies [13162].
Chromosomal damage
The aim of one study was to evaluate DNA damage (micronucleus) and cellular death
(pyknosis, karyolysis and karyorrhexis) in exfoliated buccal mucosa cells from anabolic
steroid users after 2 months of exposure. Two experimental groups consisting of 15 adult
males who practise weight lifting and are anabolic steroid users or 15 adult males who
practise weight lifting, but are non-anabolic steroid users, were recruited. In addition, 20
sedentary males, who do not practise any physical activity regularly, were matched by age
with experimental groups. No significant statistical differences were noticed in individuals
who practise physical activity only. On the other hand, an increase of micronucleated cells
(MNCs) in anabolic steroid (decadurabulin and winstrol) users was observed. Regarding
cytotoxic parameters, the same observation has occurred, that is, significant statistical
differences were noticed in the group exposed to anabolic steroids when compared with
other controls, as depicted by high frequencies of pyknosis, karyolysis and karyorrhexis.
Taken together, the results suggest that genomic instability and cytotoxicity are induced by
anabolic steroid administration in oral mucosa cells as assessed by the micronucleus test
[10446].
Androgen receptors
The androgen receptor (AR) is expressed in many tissues and is activated through binding of
testosterone and DHT. Activation of AR interacts with a broad variety of physiological
processes. Therefore it is likely that genetic variation in AR may correlate with both anabolic
effects and adverse side effects of androgen abuse. It has been speculated that these
polymorphisms may affect the sensitivity to adverse psychic reactions such as aggression.
Several polymorphisms have been described in the AR affecting the receptor affinity. In
particular, a trinucleotide repeat (CAG) has been extensively studied and associated with
disease risks, total fat free mass in men and acne, a common side effect of AAS abuse. In
871
addition to their binding to AR, the testosterone metabolite 3alpha-androstanediol can bind to
GABAA receptors,known to have an important role in the mediation of aggression [12192].
Nearly every cell in the human body has receptors for steroids, so that every organ system is
susceptible to the effects of these molecules. Giving physiologic amounts of testosterone has
no net effect on plasma levels because feedback inhibition shuts down endogenous
production [07031].
The androgen receptor (AR) is a member of the steroid and nuclear receptor superfamily,
and is a soluble protein that functions as an intracellular transcriptional factor. Structurally,
AR contains three major functional domains, N-terminal domain (NTD), DNA-binding domain
(DBD), and ligand binding domain (LBD). AR ligands regulate receptor function through
binding to the LBD, which initiates sequential conformational changes of the receptor. Upon
agonist binding, the receptor then undergoes dissociation from the chaperones, dimerization,
phosphorylation, translocation to the nucleus, and binding to the androgen response
element. Recruitment of other transcription coregulators and transcriptional machinery further
ensures the transactivation of the AR-regulated gene expression upon agonist activation. AR
is mainly expressed in androgen target tissues, such as the prostate, skeletal muscle, liver,
and central nervous system (CNS), with the highest expression level observed in the
prostate, adrenal gland, and epididymis as determined by real-time PCR. AR can be
activated by the binding of endogenous androgens, including testosterone and DHT.
Physiologically, functional AR is responsible for male sexual differentiation in utero and for
male pubertal changes. In adult males, androgen is mainly responsible for maintaining libido,
spermatogenesis, muscle mass and strength, bone mineral density, and erythropoiesis. The
actions of androgen in reproductive tissues, including prostate, seminal vesicle, testis, and
accessory structures, are known as androgenic effects, while the nitrogen-retaining effects of
androgen in muscle and bone are known as anabolic effects. Gonadal production of
testosterone is under the feedback regulation of circulating testosterone through the
hypothalamo-pituitary-gonadal axis [06049].
Androgen receptor (AR) is a member of the nuclear receptor (NR) superfamily of ligand-
dependent transactivation factors. Androgens such as testosterone and 5alpha-
dihydrotestosterone (DHT) act as agonists of AR. AR mediates various biological effects
such as the development of male reproductive tissues, sexual development, and
spermatogenesis. Since androgen declining with age contributes to age-related bone and
muscle loss and increase in fat mass, the anabolic effect of androgen is attractive for the
maintenance of health. Antagonists of AR (flutamide, bicalutamide, and nilutamide) are in
use. AR is structurally characterized by an amino-terminal trans-activation domain
(NTD/activation function 1, AF1), a DNA binding domain (DBD), and a ligand binding domain
(LBD) including a carboxy-terminal transactivation domain (activation function 2, AF2). In the
absence of a ligand, AR is localized in the cytoplasm, where it forms complexes with
chaperones. Upon ligand binding, AR translocates into the nucleus. Following nuclear
translocation, AR binds to androgen responsive elements (ARE) in the promoter regions of
its target genes as a homodimer. Generally, the transcriptional activity of nuclear receptors is
modulated by their interaction with cofactors such as coactivators and corepressors. The
type of ligand that binds to the receptor determines which type of cofactor is chosen. In the
case of agonists, AR interacts with coactivators dominantly over corepressors, and vice
versa in the case of antagonists. Unlike other nuclear receptors, AR AF2 demonstrates weak
transcriptional activity. However, ligand-dependent interaction between NTD and LBD/AF2
(which is termed as the N/C interaction) endows AR with synergistic transactivation potential)
Thus, the N/C interaction is important for the ligand-dependent transactivation potential of AR
[11062].
872
Androgens primarily exert their effects by binding to and activating a specific receptor, AR,
which is expressed in most cells. The AR is a 110 kDa protein to which the natural
androgens, T and DHT, bind with high affinity. In its inactivated state, AR is bound in the
cytoplasm to heat shock proteins. When androgens bind to the AR via the ligand binding
domain (LBD) of the AR, a conformational change is induced that promotes the dissociation
of the heat shock proteins and AR subunits dimerize to form a homodimer. The dimerized AR
complex translocates to the nucleus where it binds to androgen response elements (ARE) in
the regulatory regions of androgen target genes. Binding of AR to the ARE occurs via the
DNA binding domain (DBD). Regulatory cofactors interact with AR to promote DNA binding.
Transcriptional activation by AR also involves cofactors that modify the chromatin structure
and histone complexes in the DNA surrounding the ARE. This is important because the ARE
can be located distantly from the transcription start site of androgen-regulated genes,
therefore, cofactors that alter DNA shape and flexibility are needed for AR to augment
transcription [13084].
The actions of anabolic androgenic steroids are executed via various mechanisms. At
androgen receptor (AR) level, these mechanisms include indirect modulation of expression
by intracellular metabolism and direct effect on the AR topology, which leads to subsequent
interaction with coactivators and transcriptional activity. Human AR is a nuclear transcription
factor, belongs to the nuclear receptor superfamily and mediates male sexual differentiation
as well as the development and maintenance of sexual characteristics. The molecular
structure of AR is well characterised and comprises polymorphic N-terminal domain, a
central well-conserved DNA-binding domain and a C-terminal ligand-binding domain.
According to the literature, more than 300 mutations in the X linked AR gene result in
androgen-insensitivity syndrome, and most of the mutations in the ligand-binding domain
disrupt binding of the natural ligands dihydrotestosterone (DHT) and T [14450].
Different androgens, e.g. virilizing androgens such as testosterone and its precursors as well
as synthetic anabolic steroids, respectively, induce diverse biological effects. The molecular
basis for this variety in biological actions, however, is not well understood. It was
hypothesized that this variability of actions may be due to steroid-specific target gene
expression profiles following androgen receptor (AR)-activation. Therefore, we investigated
androgen receptor dependent transactivation of three structurally different androgen
responsive promoter constructs ((ARE)(2)TATA-luc, MMTV-luc, GRE-OCT-luc) in co-
transfected Chinese hamster ovary (CHO)-cells as an artificial model simulating different
natural target genes. Three virilizing androgens (dihydrotestosterone, testosterone,
methyltrienolone), three anabolic steroids (oxandrolone, stanozolol, nandrolone) and two
testosterone-precursors of gonadal and adrenal origin (dehydroepiandrosterone,
androstenedione) were used as ligands (0.001-100 nM). All steroids proved to be potent
activators of the AR. Remarkably, anabolic steroids and testosterone-precursors showed
characteristic promoter activation profiles distinct from virilizing androgens with significantly
lower (ARE)(2)TATA-luc activation. Hierarchical clustering based on similarity of activation
profiles lead to a dendrogram with two major branches: first virilizing androgens, and second
anabolics/testosterone-precursors. It was concluded that steroid-specific differences in gene
transcription profiles due to androgen receptor activation could contribute to differences in
biological actions of androgens [02023].
Steroid receptors in aging muscle
Skeletal muscle's roles as metabolic sink (i.e. glucose), secreted protein manufacturer (i.e.
lipoprotein lipase), and facilitator of body locomotion make it vital for human health. Muscle
mass loss is linked to frailty, morbidity, and mortality in humans. However, skeletal muscle is
873
composed of highly oxidative, postmitotic fibers, which make it a target for aging sensitivity.
Skeletal muscle mass decreases with advancing age, and this sarcopenia is associated with
decreased muscle protein synthesis, existing fiber atrophy, and muscle fiber loss. Muscle
fiber loss is associated with spinal motoneuron death, which also contributes to motor unit
size expansion and mosaic fiber pattern loss in aged muscle. Another cause of muscle mass
loss with aging is that due to decreased muscle fiber cross-sectional area or atrophy. A
portion of muscle fiber atrophy with advancing age may be related to systemic or intrinsic
biological phenomena related to the aging process and independent of physical activity
levels. It is clear that aging induces changes to skeletal muscle that are independent of an
age-induced loss of muscle mass. These age-related changes manifest themselves long
before any alteration in muscle mass due to age are reported. Skeletal muscle is a dynamic
tissue, and aging decreases its plasticity to many stimuli, such as those requiring
regeneration or remodeling. Deficits in skeletal muscle plasticity can occur at relatively young
ages in the rat. Recovery from toxin-induced injury in skeletal muscle is decreased in both
18- and 32-month-old rats compared with young rats. There is strong evidence that age-
induced decreases in muscle regenerative capacity are not intrinsic to the muscle itself but
rather are dependent on the aging organism as a whole. These facts suggest that signaling
stimuli targeting muscle may be deficient in the aged organism, and, therefore, aged
muscle's plasticity and regenerative capacity could be restored or improved if provided the
appropriate stimulus. Although this theory fits well with skeletal muscle plasticity and/or
adaptability to a regenerative stimuli, it is less certain how this impacts the age-induced loss
of muscle mass. Skeletal muscle is a target of anabolic steroid action; however, anabolic
steroid's affect on aged skeletal muscle is not well understood. The effect of 4 week of
nandrolone decanoate (ND) administration on hindlimb muscles of 5- and 25-month-old
Fischer 344/Brown Norway rats was examined. ND (6 mg/kg body wt) was injected every 7th
day for 4 week. Controls received an oil injection. ND significantly reduced 25-mo-old rat
perirenal fat pad mass by 30 percent. Soleus (Sol) and plantaris (Plan) muscle-to-body
weight ratios were reduced in 25-mo-old rats. ND did not affect Sol or Plan muscle-to-body
weight ratios at either age. Sol DNA concentration was reduced by 25 percent in 25-month-
old rats, and ND increased it to 12 percent greater than 5-month-old rats. ND did not affect
Plan DNA content. Sol androgen receptor (AR) protein in 25-month-old rats was reduced to
35 percent of 5-month-old values. ND increased AR protein by 900 percent in 25-mo-old rat
Sol. Plan AR concentration was not affected by aging but was induced by ND in both age
groups. Aging or ND treatment did not affect glucocorticoid receptor levels in either muscle.
These data demonstrate that fast- and slow-twitch rat hindlimb muscles differ in their
response to aging and ND therapy 02[024].
Testosterone and its pharmaceutical derivatives are potent regulators of skeletal muscle
mass. Anabolic-androgenic steroids are structural derivatives of testosterone manufactured
to maximize anabolic and minimize androgenic effects. Anabolic steroid administration has a
growth effect on female, normal male, and hypogonadal male muscle. Anabolic steroid
therapy for patients with chronic wasting diseases can maintain or increase muscle mass.
Skeletal muscle protein synthesis increases in elderly men administered testosterone. The
rat has proven to be a useful model for studying anabolic steroid's action on skeletal muscle.
The effect of testosterone administration on basal young rat muscle mass is mixed and likely
due to drug type, dosage, and administration schedules. Exogenous testosterone
administration and skeletal muscle loading act synergistically to increase skeletal muscle
mass in healthy and diseased humans. A synergistic relationship between testosterone and
muscle loading is also present in functionally overloaded rat muscle subject to disuse
atrophy. Anabolic steroid administration can reduce rat hindlimb muscle atrophy due to
unloading and abolish unloaded atrophy in the quadriceps muscle. Although testosterone is a
potent skeletal muscle mass effector, little is understood about its molecular regulation and
the effect of aging on this regulation [02024].
874
Androgen receptors (AR) and glucocorticoid receptors (GR) are potential modulators of
skeletal muscle mass. Glucocorticoid- and testosterone-induced cellular regulation involves
binding with its specific cytosolic steroid receptor, translocating to the nucleus, and then
altering gene transcription by binding to its corresponding DNA response element. Signaling
cascades, including RhoA-mediated signaling, can alter AR and GR transcriptional activity.
AR interactions with coactivators and other DNA-binding proteins, including serum response
factor, at the level of DNA binding to alter its transcriptional activity. Circulating testosterone
levels have been well characterized in the Fischer 344/Brown Norway rat, decreasing
dramatically between 4 and 28 months of age. AR expression appears sensitive to circulating
testosterone concentration. However, the influence of circulating testosterone levels on AR
expression appears to be muscle specific. Like other aging phenomena, sarcopenia will likely
be related to both environmental and genetic/biological factors. There has been considerable
focus on the ability of aged skeletal muscle to adapt to exercise training without a clear
understanding of the limitations biological aging places on this response. Anabolic steroids
target skeletal muscle, maintain muscle mass in many wasting disease states, and can act
synergistically with resistance exercise in humans; however, their effect on age-induced
muscle mass loss or sarcopenia is less certain. Additionally, the therapeutic use of anabolic
steroids is limited by their broad spectrum of biological targets. At this time, the anabolic
steroid-induced signaling mechanisms influencing skeletal muscle mass regulation are not
well understood. A better understanding of anabolic steroid action on skeletal muscle will
improve both drug design and the use of combined environmental (i.e. diet, exercise) and
pharmaceutical interventions to offset sarcopenia in the aged individual [02024].
Effect of exercise
Increased serum testosterone (TST) occurs in response to resistance exercise and is
associated with increased muscle mass. However, the effects of elevated TST and
sequential resistance exercise bouts on androgen receptor (AR) expression in humans are
not well known. One study examined three sequential bouts of heavy-resistance exercise on
serum total TST, sex hormone-binding globulin (SHBG) and free androgen index (FAI),
skeletal muscle AR mRNA and protein expression, and myofibrillar protein content. Eighteen
untrained males were randomly assigned to either a resistance-training (RST, n=9) or control
group (CON, n=9). RST performed three lower-body resistance exercise bouts, each
separated by 48 h. At each exercise bout, RST performed three sets of 8-10 repetitions at
75-80 percent one-repetition maximum using the squat, leg press, and leg extension
exercises, respectively, whereas CON performed no resistance exercise. Muscle biopsies
were obtained immediately before the first exercise bout and 48 h after each of the three
bouts, whereas blood samples were obtained immediately before, immediately after, and 30
min after each bout. Data were analyzed with two-way ANOVA and bivariate correlations.
Serum TST and FAI were significantly increased after each exercise bout; however, there
were no significant changes for SHBG. AR mRNA and protein were significantly increased
after the second and third exercise bouts, respectively, and were significantly correlated to
TST and FAI. Myofibrillar protein increased after the third bout. Three sequential bouts of
heavy resistance exercise increases serum TST and are effective at up-regulating AR mRNA
and protein expression that appears to correspond to subsequent increases in myofibrillar
protein [04053].
Androgen receptors
Androgen action is mediated by the androgen receptor, a ligand dependent transcription

factor, belonging to the superfamily of nuclear receptors. The two most important androgens
are testosterone and 5a-dihydrotestosterone and their tissue specific actions are mediated
by the same androgen receptor protein. Binding of androgens by the androgen receptor
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results in two consecutive conformational changes, which are different from those induced by
anti-androgens. The androgen receptor can use different transactivation domains (AF1 and
AF5, respectively, in the NH2-terminal domain and AF2-AD in the COOH-terminal domain)
depending on the "form" of the receptor protein. The AF2 function is strongly dependent on
the presence of nuclear receptor coactivators. Two AF functions are ligand dependent (AF-1
and AF2), whereas AF5 operates in a ligand independent way. The ligand dependency of
AF-1 in the full length androgen receptor and the switch to AF5 in the COOH-terminal
truncated androgen receptor strongly suggests a functional inhibitory action of the ligand-
binding domain on AF-1 in the absence of ligand and on AF5 in the presence of ligand. In
vivo experiments favour a ligand dependent functional interaction between the AF-2 AD core
region in the ligand-binding domain with the NH2-terminal domain. This interaction might be
either direct or indirect, requiring additional factors, and results in androgen receptor driven
transcription activation. The androgen receptor protein can undergo two post-translational
modifications during receptor activation. Firstly, upon synthesis the protein is rapidly
phosphorylated to acquire hormone binding capacities and secondly, upon hormone binding
an additional phosphorylation occurs during transformation to the DNA-binding transcription
activation form [01031].
Canine androgen receptor

Sex steroids, including testosterone, play a major role in determining peak bone mass in
mammals and the subsequent loss of total bone mass with advancing age. Testosterone,
and its active metabolite dihydrotestosterone (DHT), bind with high affinity to the androgen
receptor (AR), a member of the nuclear hormone receptor superfamily. These receptors
function as transcription factors, binding together with accessory proteins to specific DNA
response elements in the promoters of androgen responsive genes. To further characterize
AR function in a model species of relevance to bone and pharmaceutical research, we
cloned a partial canine AR from a canine kidney cDNA library and then cloned the remaining
5' segment by PCR from canine ventral prostate cDNA. The complete sequence obtained
was 3577 bp. This sequence contained a single open reading frame of 2721 bp, potentially
encoding a protein of 907 amino acids with a predicted molecular weight of 98.7 kD.
Sequence analysis of the protein encoded by this open reading frame reveals that the
modular domains providing the DNA binding and ligand binding functions are identical to
those reported for eight other mammalian ARs. Northern analysis of poly-A+ RNA from
ventral prostate revealed three very low abundance transcripts of approximately 9 kb and
RT-PCR analysis showed relatively high expression of AR in canine ventral prostate, testis,
and kidney, with lower levels detectable in spleen, skeletal muscle, heart, and liver.
Competition binding studies using 3H-DHT as ligand demonstrated specific displacement by
DHT, testosterone, and the anabolic steroid stanozolol, with IC50 values of 1.3, 2.5 and 3.8
nM, respectively. Binding of DHT also resulted in the stimulation of an androgen responsive-
luciferase reporter following cotransfection with the canine AR into 293 cells.
Immunohistochemistry using an antibody directed to the C-terminal 19 amino acids of the
human AR showed strong staining of the secretory epithelial cells in canine ventral prostate.
Together, these data indicate that we have cloned the canine AR and that its functional DNA
binding and ligand binding domains are absolutely conserved with those reported for eight
other species [01032].
Expression of androgen receptors in human neck and limb muscles

The purpose of one study was to investigate the immunohistochemical expression of
androgen receptors (AR) in human vastus lateralis and trapezius muscles and to determine
whether long-term strength training and self-administration of androgenic-anabolic steroids
are accompanied by changes in AR content. Biopsy samples were taken from eight high-
level power-lifters (P), nine high-level power-lifters who used anabolic steroids (PAS) and six
untrained subjects (U). Myonuclei and AR were visualised in cross-sections stained with the
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monoclonal antibody against AR and 4',6-diamidino-2-phenylindole. The proportion of AR-
containing myonuclei per fibre cross-section was higher in the trapezius than in the vastus
lateralis. In the trapezius, the proportion of AR-containing myonuclei was higher in P
compared to U and in PAS compared to both P and U. On the contrary, in the vastus
lateralis, there were no differences in AR content between the three groups. Myonuclear
number in both muscles was higher in P compared to U and in PAS compared to both P and
U. In conclusion, AR content differs greatly between human neck and limb muscles.
Moreover, the regulation of AR-containing myonuclei following training and self-
administration of androgenic-anabolic steroids is muscle dependent [00034].
Polymorphisms in androgen and estrogen receptor genes

Besides lifestyle and environmental factors, the life-long exposure to the endocrine milieu of
gonadal steroids is a determining factor to gender specific features of aging. In contrast to
women, men do not experience a sudden cessation of gonadal function comparable to
menopause. However, cross-sectional and longitudinal population studies demonstrate that
the hormones with anabolic actions (e.g. testosterone [T], growth hormone, insulin-like
growth factor [IGF]-1, dehydroepiandrosterone) do decrease progressively with aging in
healthy men, and chronic systemic illnesses accelerate this process. In addition, estrogen
has recently been established to be essential for normal physiology of the male. The slow
progressive decline of the hypothalamic-pituitary-gonadal (HPG) function is thought to be
responsible for many common signs and symptoms of aging men, such as general
weakness, sexual dysfunction, and increased fat mass. There is a large inter-individual
variation in sex hormone levels cross-sectionally within given age groups as well as
longitudinally with aging. A contributing factor to this variability are the numerous functionally
significant polymorphisms that have been detected in the receptors for androgen and
estrogen. In this review, we summarize the recent information on some common
polymorphisms in androgen and estrogen receptor genes and their effect on gender specific
and aging-related symptoms and diseases of men [04054].
Selectivity of ligand action on androgen receptors
Historically, the term androgenic-anabolic steroid (AAS) reflected the view that androgenic
and anabolic effects of androgens could be dissociated, and that in comparison to
testosterone, some androgens were more anabolic than androgenic. However, a large body
of data emerged in the late 1990s that revealed that the selectivity of androgen receptor
signaling could be mediated at multiple levels of the steroid hormone interactome that
encompasses (in addition to the androgen receptor) an interacting web of chaperone
proteins, a repertoire of 300 or so coactivators and corepressors, elements of the chromatin,
effector proteins, and transcription factors that bind specific regions of the androgen
responsive genes. Although the precise molecular mechanisms that mediate tissue selective
actions of selective androgen receptor modulators are not fully characterized, a growing body
of evidence suggests that ligand specificity can be imparted by the recruitment of a specific
repertoire of tissue-specific coactivators and corepressor proteins, the variations in the level
of expression of the coregulator proteins in different tissues, the regulation of chromatin
remodeling, differential activation of signaling pathways in the prostate versus the skeletal
muscle, and by differential susceptibility to the action of the steroid 5 alpha-reductase
enzyme. These landmark discoveries have reinstated the view that multiple levels of the
androgen receptor interact or contribute to tissue specific actions of the AR ligands, and can
be targeted to achieve the desired tissue specificity. Indeed, a number of SARMs have
achieved relative differentiation of androgenic and anabolic activity; being preferentially more
potent in the muscle than in the prostate. Several publications have described the
mechanistic basis of tissue specificity. This growing body of literature suggests that in spite
of the singularity of the AR protein, tissue selectivity of ligand action can be achieved
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[14017].
Androgen receptor polymorphism
Muscle mass and strength, as well as aerobic fitness (VO2max) are related to health and
mortality. Muscle mass and strength is determined by environmental factors, principally
endocrine, nutritional and mechanical loading, and by the genetic background. Gene
polymorphisms, like those encoding for the insulin-like growth factor-1 (IGF-1), type I
collagen (COL1A1), ciliary neurotrophic factor (CNTF), interleukin-6 (IL-6), the vitamin D
receptor (VDR), IGF-2, resistin (RETN) and androgen receptor (AR), have an influence on
either muscle mass or strength. The AR gene is located to the X chromosome (q11.2–q12),
and contains eight exons. The exon 1 contains a polyglutamine tract encoded by CAG
repeats and a polyglycine tract (GGN) encoded by (GGT)3GGG(GGT)2(GGC)n. Polymorphic
tracts are close to the region encoding the transactivation-1 domain of the AR protein. The
CAG and GGN polymorphisms of the androgen receptor (AR) gene are related to incidence
of prostatic cancer, breast cancer, plasma hormone levels and other metabolic,
cardiovascular and even mental diseases. The polyglutamine repeat has an average length
of 22 amino acids (range: 8-35). Short CAG repeats are associated with increased AR
transactivation activity and stronger transcriptional potential. The CAG polymorphisms are
associated with the fat-free mass phenotype in healthy elders. However, it remains to be
established if the AR polymorphism influences muscle mass and fitness in young adults.
The polyglycine repeat length of AR ranges from 10 to 30. Short GGN repeats are
associated with increased AR protein content in cell cultures that may in turn enhance the
response to androgen stimulation. It remains unknown if a short GGN repeat number is
associated to increased muscle mass or strength in humans. The exon-1 of the androgen
receptor gene thus contains two repeat length polymorphisms which modify either the
amount of AR protein inside the cell (GGNn, polyglycine) or its transcriptional activity (CAGn,
polyglutamine). Shorter CAG and/or GGN repeats provide stronger androgen signalling and
vice versa. To test the hypothesis that CAG and GGN repeat AR polymorphisms affect
muscle mass and various variables of muscular strength phenotype traits, the length of CAG
and GGN repeats was determined by PCR and fragment analysis and confirmed by DNA
sequencing of selected samples in 282 men. Individuals were grouped as CAG short (CAGS)
if harbouring repeat lengths of ≤21 and CAG long (CAGL) if CAG >21. GGN was considered
short (GGNS) or long (GGNL) if GGN ≤23 or >23, respectively. No significant differences in
lean body mass or fitness were observed between the CAGS and CAGL groups, or between
GGNS and GGNL groups, but a trend for a correlation was found for the GGN repeat and lean
mass of the extremities. In summary, the length of CAG and GGN repeat of the AR gene do
not appear to influence lean mass or fitness in young men. Additional studies are required to
test if men harbouring the combination CAGS and GGNS have more jumping capacity
[11063].
Antagonists of the androgen receptor
While the androgens of testicular origin (representing about 50 % of total androgens in men
over 50 years) can be completely eliminated by surgical or medical castration with GnRH
(gonadotropin-releasing hormone) agonists or antagonists, the antiandrogens currently
available as blockers of androgen binding to the androgen receptor (AR), namely
bicalutamide (BICA), flutamide (FLU) and nilutamide have too weak affinity to completely
neutralize the other 50 percent of androgens made locally from dehydroepiandrosterone
(DHEA) in the prostate cancer tissue by the mechanisms of intracrinology. Series of steroid
derivatives having pure and potent antagonistic activity on the human and rodent AR were
synthesized. Assays of AR binding and activity in carcinoma mouse Shionogi and human
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LNCaP cells as well as in vivo bioavailability measurements and in vivo prostate weight
assays in the rat were used. The chosen lead steroidal compound, namely EM-5854, has a
3.7-fold higher affinity than BICA for the human AR while EM-5855, an important metabolite
of EM-5854, has a 94-fold higher affinity for the human AR compared to BICA. EM-5854 and
EM-5855 are 14 times more potent than BICA in inhibiting androgen (R1881)-stimulated
prostatic specific antigen (PSA) secretion in human prostatic carcinoma LNCaP cells in vitro.
MDV3100 has a potency comparable to bicalutamide in these assays. Depending upon the
oral formulation, EM-5854 is 5- to 10-times more potent than BICA to inhibit
dihydrotestosterone (DHT)-stimulated ventral prostatic weight in vivo in the rat while
MDV3100 has lower activity than BICA in this in vivo model. These data are supported by
respective 40-fold and 105-fold higher potencies of EM-5854 and EM-5855 compared to
BICA to inhibit cell proliferation in the androgen-sensitive Shionogi carcinoma cell model.
Although the preclinical results data need evaluation in clinical trials in men, combination of
the data obtained in vitro in human LNCaP cells as indicator of potency in the human
prostate and the data on metabolism evaluated in vivo on ventral prostate weight in the rat,
could suggest the possibility of a 70- to 140-fold higher potency of EM-5854 compared to
bicalutamide (Casodex) for the treatment of prostate cancer in men [12115].
Physiological and clinical effects and side effects
The physiological direct effects of testosterone and AASs (AR-mediated) are well known.
They include increases in renal erythropoiesis, lipolysis, protein synthesis, sebaceous
secretion, hair growth and libido. However, the indirect effects should also be considered.
These include antiglucocorticoid effects, which are mediated by testosterone occupation of
cortisol receptors (which have a remarkable affinity with testosterone) and create an anti-
catabolic effect. An increase in muscular activity is certainly the leading result of AAS use. It
constitutes a complex phenomenon involving hypertrophy of skeletal muscle fibres that
contain muscle cells and undifferentiated satellite cells. The latter become myoblasts that are
incorporated into skeletal muscle cells, increasing the number of nuclei, and also the amount
of cytoplasm, actin and myosin, making them larger and more potent. Notably, this
phenomenon does not increase the number of fibres, only their size. Side effects of AASs
are also well known. Their incidence is unclear, as the denominator of AAS use is not clear.
Acne, alopecia and LUTS attributable to prostate enlargement are usually related to the
strong androgenic 5DHT-effect. Erectile dysfunction and libido loss may also occur,
especially after discontinuation, when endogenous testosterone levels are usually low. A
sustained increase in testosterone levels during “cycles” leads to higher aromatizationmrates
of testosterone, which accounts for the gynaecomastia typically found in steroid users.
Hepatic effects are most often related to oral alkylated agents. They include the uncommon
hepatic peliosis, cholestatic jaundice and hepatic neoplasms, such as focal nodular
hyperplasia, which are all closely related to dose and duration of usage. Hepatocellular
carcinoma and Wilm’s tumour are serious and rare side effects that are always related to
long-term and heavy use. Interestingly, there are no reports linking AASs to prostate cancer
or significant increases in PSA levels. The most severe consequences of long-term AAS use
are associated with the cardiovascular system. Hypertension, arrhythmia, erythrocytosis and
ventricular dysfunctions have been reported. Mortality risk among chronic users is estimated
to be 4.6 times higher than among non-users. Cases of renal failure secondary to
rhabdomyolysis and diffuse membranoproliferative glomerulonephritis in heavy users have
been reported. Aggressive behaviour, depression, mood swings, altered libido, euphoria and
even psychosis are some of the psychiatric patterns related to AAS. Overpharmacy may
increase the risk of violent criminality. Withdrawal syndrome and dependency were also
described, and the likelihood of psychiatric effects is greater where there is previous
psychiatric history, or alcohol or drug abuse [11028].
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The adverse endocrine effects of AASs are best understood if one first looks at the native
effects of testosterone. Testosterone is responsible for the in utero masculinization of internal
genitalia, postnatal skeletal muscle development, and the development of male secondary
sexual characteristics. In addition, testosterone is converted in peripheral tissues by 5-alpha-
reductase to dihydrotestosterone (DHT), which contributes to fetal development of external
genitalia, prostate, and seminal vesicles. DHT acts in the cell nucleus of target tissues, such
as skin, male accessory glands, and the prostate, exerting predominantly androgenic, but
also anabolic, effects. Testosterone is converted by the enzyme aromatase to estradiol and
estrone, which are involved in the sexual differentiation of the brain, bone mass accretion,
and fusion of the epiphyses at the conclusion of puberty, in addition to feminizing effects.
Under normal physiologic circumstances, aromatase has a limited role; however, with high-
dose AAS use, this role increases, and, therefore, so does the level of estrogens. An
antiestrogen effect may be present as well with supraphysiologic doses of AASs. Excess
AASs lead to a down-regulation of androgen receptors and AASs then compete with
estrogens for the estrogen receptor. The net outcome of these two different pathways is
difficult to predict. With this information, it is easier to understand the adverse outcomes of
AAS use because many of the effects are amplifications of physiologic effects. Testosterone
acts at the androgen receptor to increase protein synthesis; it also has effects through
conversion to DHT and estrogens. At normal physiologic levels of testosterone, androgen
receptors are saturated, and it is believed that some of the effects of AASs may be through
one or more different mechanisms. Research has shown an antagonist effect at the
glucocorticoid receptor at supraphysiologic levels that leads to an anticatabolic effect..
Glucocorticoids influence glucose synthesis and protein catabolism. The stimulation of
glucocorticoid receptors by glucocorticoids leads to increased protein breakdown in muscle.
High-dose AASs may displace glucocorticoids from the glucocorticoid receptor and inhibit
muscle protein breakdown that leads to an overall anabolic or muscle-building effect. By
competing with glucocorticoids for the glucocorticoid receptor, AASs block the depressed
protein synthesis that usually occurs during stressful training. AASs also exert some effect on
the growth hormone (GH)-insulin-like growth factor (IGF)-1 axis. There seems to be an
androgen-induced stimulation of GH secretion and a direct stimulation of hepatic IGF-1
synthesis. IGF-l stimulates skeletal muscle formation, and GH exhibits anabolic effects.
AASs act on osteoblasts to stimulate proliferation and differentiation that may inhibit bone
breakdown. There also may be some degree of “placebo effect” that allows AAS users to
train harder and increase muscle mass as a result of the increased aggression, euphoria,
and decreased fatigue and recovery time that many AAS users report [07058].
Anticorticoid effects
The supraphysiologic doses of AAS used by athletes reduce the catabolic effects of
endogenous cortisol, which is increased during training, by competitive inhibition of cortisol
binding to the glucocorticoid receptor. This helps to preserve muscle mass by inhibiting
glucocorticoid stimulation of muscle glycogen breakdown and gluconeogenesis. AAS also
increase 2, 3-diphosphoglycerate concentration which decreases hemoglobin-oxygen affinity,
facilitating release of oxygen to the tissues [07002].
Muscle characteristics in bodybuilders
It is well established that skeletal muscle can adapt to the variable functional requirements
through a quantitative mechanism based on changes in muscle mass and fibre size, and a
qualitative mechanism based on a change in fibre type distribution. Human muscles are in
fact mixed muscles expressing three main fibre types, type 1, 2A and 2X in variable
proportions [Harridge et al, 1996]. Type 1, 2A and 2X fibres, in turn, differ in contractile and
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energetic properties that are known to depend on their myosin heavy chain (MHC) isoform
content [Bottinelli & Reggiani, 2000]. Type 1 fibres contain the myosin heavy chain 1 (MHC-
1) isoform and have lower maximum shortening velocity (Vo), maximum power (W max) and
ATPase activity (ATPase), and slower kinetics of stretch activation [Hilber et al, 1999] than
type 2X fibres, which contain MHC-2X [Bottinelli et al, 1996; Stienen et al, 1996]. Type 2A
fibres contain MHC-2A and are intermediate. Moroever, specific force (Po/CSA) is also lower
in type 1 than in type 2A and 2X fibres, whereas no difference is seen between 2A and 2X
fibres. Exercise training is a major factor shaping muscle phenotype. Resistance training has
been studied extensively and it is now well known that it determines both muscle hypertrophy
(quantitative mechanism) and a shift of fibre type distribution (qualitative mechanism)
[Schiaffino & Reggiani, 1996; Bottinelli & Reggiani, 2000; Fluck & Hoppeler, 2003]. However,
several open issues remain. According to a long-lasting [Morpurgo, 1879] and well supported
belief [Gollnick et al, 1981; Gollnick et al, 1983], it is generally assumed that the increase in
muscle mass can be fully accounted for by single muscle fibre hypertrophy. It should be
noted, however, that several findings suggest that hyperplasia can also occur, at least in
some animals (rat, cat, chicken) and in some conditions (compensatory hypertrophy due to
synergist ablation and tenotomy; chronic stretch; resistance training) [Antonio & Gonyea,
1993]. Moreover, the few studies performed on body builders have generally reported a
limited and inconsistent hypertrophy of muscle fibres, failing to account for the obvious and
extreme hypertrophy of the muscles, although a precise quantitative analysis was not
performed [MacDougall et al, 1982; Tesch & Larsson, 1982]. As regards single muscle fibre
properties, it has been recently shown that the myosin isoform-based dependence of
contractile properties of muscle fibres might have relevant exceptions [Bottinelli, 2001]. In
ageing and disuse, in fact, specific tension (Po/CSA) and unloaded shortening velocity (Vo) of
slow (type 1) and fast (type 2A and 2X) fibres have been shown to change [Larsson et al,
1997; D'Antona et al, 2003]. It is unclear whether changes in Po/CSA and Vo of muscle fibres
can also occur in young healthy subjects following training. In two recent studies on
resistance training an increase in CSA and force (Po) of muscle fibres, but no change in
Po/CSA and Vo of slow and fast fibres, was observed, suggesting that the properties of a
given fibre type change mainly by a quantitative mechanism (increase in CSA and absolute
force). However, an increase in Vo of type 1 and 2A fibres has been observed in highly
trained swimmers following a decrease in training intensity [Trappe et al, 2000], and
variations in Po/CSA have been observed in a very recent study on cross-country runners
during a competitive season and changes in endurance training regime [Harber et al, 2004].
Finally, it is still unclear how large and how relevant for muscle function in vivo the adaptation
of fibre type distribution to training can be [Ingalls, 2004]. In longitudinal studies the training-
induced shift in fibre type composition is often small and surprisingly similar for endurance
[Baumann et al, 1987] (duration 8 weeks) and resistance training [Adams et al, 1993;
Andersen et al, 1994; Liu et al, 2003a, b] (duration from 3 weeks to 3 months). A type 2X →
2A shift is mostly observed in spite of the different effects of the two paradigms on muscle
mass and metabolism [Fluck & Hoppeler, 2003]. On the contrary, comparative studies of
different subject populations (cross-sectional studies) have shown a strong bias in fibre type
distribution towards fast fibres in elite sprinters (70 % type 2A and type 2X) and towards slow
fibres in elite marathon runners (60–90 % type 1 fibres) [Sjöstrom et al, 1988; Andersen et al,
2000]. The inconsistency between the former (longitudinal) and the latter (cross-sectional)
studies could be due either to the longer and more intense training of elite athletes or to a
genetically determined bias of fibre type distribution towards fast fibres in sprinters and slow
fibres in marathon runners. To address the above issues, it was reasoned that body builders
(BB) in which muscle adaptations were expected to be particularly evident due to the very
long and intense resistance training could represent a valuable model. BB that performed
hypertrophic heavy resistance exercise (HHRE), a type of resistance training specifically
designed to increase muscle mass, for at least 2 years to compete at national and
international level were enrolled in the study. Muscle phenotype was characterized by
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extreme muscle hypertrophy, that could not be fully accounted for by single muscle fibre
hypertrophy, by a significant expression of MHC-2X, the fastest and most powerful MHC
isoform which is very little present in trained young subjects (Mizuno, 1991), and by a
significantly higher specific force of fast and especially 2X muscle fibres in relation to
controls. Muscle phenotype was therefore clearly adapted to HHRE, but such adaptation was
not fully accounted for by the known qualitative and quantitative mechanisms of muscle
plasticity.mNeedle biopsy samples were taken from vastus lateralis muscle (VL) of five male
body builders (BB, age 27 years), who had being performing hypertrophic heavy resistance
exercise (HHRE) for at least 2 years, and from five male active, but untrained control
subjects (CTRL, age 40 years). The following determinations were performed: anatomical
cross-sectional area and volume of the quadriceps and VL muscles in vivo by magnetic
resonance imaging (MRI); myosin heavy chain isoform (MHC) distribution of the whole
biopsy samples by SDS-PAGE; cross-sectional area (CSA), force (Po), specific force
(Po/CSA) and maximum shortening velocity (Vo) of a large population (n=524) of single
skinned muscle fibres classified on the basis of MHC isoform composition by SDS-PAGE;
actin sliding velocity (Vf) on pure myosin isoforms by in vitro motility assays. In BB a
preferential hypertrophy of fast and especially type 2X fibres was observed. The very large
hypertrophy of VL in vivo could not be fully accounted for by single muscle fibre hypertrophy.
CSA of VL in vivo was, in fact, 54 percent larger in BB than in CTRL, whereas mean fibre
area was only 14 percent larger in BB than in CTRL. MHC isoform distribution was shifted
towards 2X fibres in BB. Po/CSA was significantly lower in type 1 fibres from BB than in type
1 fibres from CTRL whereas both type 2A and type 2X fibres were significantly stronger in
BB than in CTRL. Vo of type 1 fibres and Vf of myosin 1 were significantly lower in BB than in
CTRL, whereas no difference was observed among fast fibres and myosin 2A. The findings
indicate that skeletal muscle of BB was markedly adapted to HHRE through extreme
hypertrophy, a shift towards the stronger and more powerful fibre types and an increase in
specific force of muscle fibres. Such adaptations could not be fully accounted for by well
known mechanisms of muscle plasticity, i.e. by the hypertrophy of single muscle fibre
(quantitative mechanism) and by a regulation of contractile properties of muscle fibres based
on MHC isoform content (qualitative mechanism). Two BB subjects took anabolic steroids
and three BB subjects did not. The former BB differed from the latter BB mostly for the size
of their muscles and muscle fibre [05037].
Long-term effects
The morphological appearance of the vastus lateralis (VL) muscle from high-level power-
lifters on long-term anabolic steroid supplementation (PAS) and power-lifters never taking
anabolic steroids (P) was compared. The effects of long- and short-term supplementation
were compared. Enzyme-immunohistochemical investigations were performed to assess
muscle fiber type composition, fiber area, number of myonuclei per fiber, internal myonuclei,
myonuclear domains and proportion of satellite cells. The PAS group had significantly larger
type I, IIA, IIAB and IIC fiber areas. The number of myonuclei/fiber and the proportion of
central nuclei were significantly higher in the PAS group. Similar results were seen in the
trapezius muscle (T) but additionally, in T the proportion of fibers expressing developmental
myosin isoforms was higher in the PAS group compared to the P group. Further, in VL, the
PAS group had significantly larger nuclear domains in fibers containing > 5 myonuclei. The
results of AS on VL morphology in this study were similar to previously reported short-term
effects of AS on VL. The initial effects from AS appear to be maintained for several years
[05038].
Mesenchymal pluripotent cell as the target of androgen action
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Testosterone supplementation increases muscle mass primarily by inducing muscle fiber
hypertrophy; however, the mechanisms by which testosterone exerts its anabolic effects on
the muscle are poorly understood. The prevalent view is that testosterone improves net
muscle protein balance by stimulating muscle protein synthesis, decreasing muscle protein
degradation, and improving the reutilization of amino acids. However, the muscle protein
synthesis hypothesis does not adequately explain testosterone-induced changes in fat mass,
myonuclear number, and satellite cell number. It was postulated that testosterone promotes
the commitment of pluripotent stem cells into the myogenic lineage and inhibits their
differentiation into the adipogenic lineage. The hypothesis that the primary site of androgen
action is the pluripotent stem cell provides a unifying explanation for the observed reciprocal
effects of testosterone on muscle and fat mass [03037].
Anabolic steroids and antioxidant vitamins on ethanol-induced tissue injury
Various mechanisms are involved in the process of ethanol-induced tissue impairment.

Oxidative stress and its effects are among the most important. It was compared the effects of
antioxidant vitamins (vitamin C and E in combination) and steroids (testosterone and
nandrolone separately) on the toxicity of ethanol in rats. Animals (male Wistar rats, n = 48)
were randomised into following groups-Control, Ethanol, Testosterone, Ethanol +
Testosterone, Ethanol + Nandrolone, Ethanol + Vitamins. Alcohol was given daily by gavage
in a dose of 5 g/kg of body weight. On the 27th day of the study the animals were sacrificed
by decapitation and tissue samples were taken. Metabolic status, parameters of the hepatic
metabolism, hormone levels (testosterone, ACTH, corticosterone), lipoperoxidation markers
(malondialdehyde and conjugated diens in forebrain cortex and in cerebellum) and advanced
glycation end-products were analysed. Tissue samples underwent histological examination.
Histological outcomes showed a protective effect of antioxidants on hepatic and cerebellar
injury caused by chronic ethanol intake. Anabolic steroids protected especially the central
nervous tissue against the toxicity of alcohol. Both, antioxidant vitamins and anabolic steroids
protect against the ethanol-induced toxicity, however, this effect is tissue specific [03038].
Variation in phase II metabolism of sex steroids
Phase II metabolism, usually known as conjugation reactions, generate metabolites that are,
in most cases, biologically inactive and subsequently excreted in bile or urine. Androgenic
and estrogenic sex steroids are mainly inactivated by sulfation or glucuronidation by the
enzyme families’ sulfotransferases (SULTs) or uridine diphosphoglucuronosyltransferases
(UGTs). Genetic variations in UGTs and SULTs have been implicated to play a role in
hormone-dependent diseases and in the outcome for doping test results. Moreover, the use
of drugs may interact with UGTs and SULTs and hence affect the phase II metabolism. The
aim of one research topic forum was to highlight the progress made in this field via review
papers and original articles as well as to promote future research with the aim to further
understand the consequences of inter-individual difference in phase II metabolism and
regulation of sex steroids. In the original article the UGT2B17 deletion polymorphism impact
on testosterone replacement therapy was studied. There was no association with UGT2B17
genotype and testosterone serum peak levels, whereas the increase in testosterone from
week 8 to 18 differed, subjects homozygous for deletion (del/del) had a smaller increase as
compared to UGT2B17 carriers. Moreover, an association between LH levels and UGT2B17
genotype was found; del/del subjects exhibit lower levels of LH during treatment period.
Testosterone is not only being therapeutically used but also a common drug together with
synthetic androgens, i.e. nandrolone to abuse (doping). It was shown that a well
characterized single nucleotide polymorphism in UGT2B15 is associated with the
glucurunidation activity of 19-noradrosterone, the nandrolone metabolite analyzed at the
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WADA accredited doping labs. The significance of different phase II polymorphisms in
female athletes confirmed that UGT2B17 deletion polymorphism is an important determinant
of T/E (a biomarker for testosterone doping) in women. Moreover, the use of hormonal
contraceptives was shown to affect the T/E ratio, which will be important to consider in
doping test programs. The fact that drug use can affect the phase II metabolism and
consequently doping test results were also investigated and it was found that the use of
NSAIDs (diclofenac and ibuprofen) did not interact with the urinary excretion of testosterone-
and epitestosterone-glucuronides, and hence have no impact on T/E ratio. Also, it was noted
that dietary compounds such as white and green tea inhibit testosterone UGT2B17-derived
glucuronidation activity in vitro. Hence, it is possible that in addition to drug use, the diet may
influence the UGTs and alter the risk of hormone-related diseases and impacting doping test
results; however, this needs to be verified in vivo. Even though most androgens are excreted
as glucuronides, some are also subject to sulfate conjugations. It was identified a new copy
number variation polymorphism in the SULT2A1 gene. This CNV alters the capacity to
excrete testosterone and some of its metabolites in the urine. Insertions are associated with
higher excretion rate, both at basal level and after the administration of 500 mg testosterone
enanthate. It is possible that this polymorphism may alter the risk of hormone-related
diseases. Steroid sulfatase (STS), enzymes involved in the de-conjugation of sulfated
compounds, has been highlighted. In addition to phase II enzymes, steroid metabolizing
phase I enzymes may determine the bioavailability of hormones. Mungenast et al. published
an extensive review about the metabolism as well as the receptor effects of estrogens in
relation to ovarian cancer, discussing strategies to target these pathways [150150].
Anabolic steroids effect on other drugs of abuse
Clinical and epidemiological data have reported that the abuse of AAS in humans is often
associated with the abuse of psychotropic drugs, such as cocaine, opiates, alcohol,
cannabis, amphetamine, and 3,4-methylenedioxy-methamphetamine (MDMA). These
surveys have suggested a role of AAS as a gateway to other dependency-inducing drugs.
Based on these findings, different animal paradigms have been used to investigate AAS pre-
exposure effects on neurochemical and behavioral response to other addictive substances.
Consistent with reported higher alcohol intake in AAS abusers, increased voluntary alcohol
consumption after cessation of AAS administration has also been observed in male adult
rats. In line with these findings, corticotropin releasing factor modulation of GABAergic
transmission in the amygdala seems to play a pivotal role in ethanol effects, suggesting that
AAS might alter the sensitivity of these circuits and predispose to alcohol abuse. Chronic
nandrolone decanoate administration has been found to significantly impair CPP induced by
delta9-tetrahydrocannabinol (THC) without affecting CB1 receptor binding. Interestingly,
nandrolone administration increased THC abstinence precipitated by the CB1 cannabinoid
antagonist rimonabant Administration of supra-pharmacological doses of nandrolone
decanoate has been shown to decrease the hyper-locomotion and stereotyped behavior
induced by amphetamine and MDMA, in a dose-dependent manner. Such behavioral
outcomes have been corroborated by microdialysis results. In particular, nandrolone
decanoate attenuated the effect of amphetamine and MDMA on DA baseline and DA
metabolites levels in the NAc. However, the higher dose of nandrolone decanoate has
enhanced the acute effects of MDMA-induced release of 5-HT, followed by exhaustion of
neuronal 5-HT stores. Thus, high-dose nandrolone decanoate treatment might enhance
neuron vulnerability to MDMA, leading to effects resembling MDMA neurotoxicity. In addition,
it has been demonstrated that the effects of amphetamine on the hippocampal and
hypothalamic DOPAC/DA ratio were prevented by nandrolone decanoate, with no changes
to DA baseline levels. Likewise, it has been shown that pretreatment with nandrolone
decanoate attenuates accumbal DA and 5-HT outflow, as well as the consequent
stereotyped behavior induced by cocaine. Nandrolone might decrease neurochemical and
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behavioral effects induced by cocaine via up-regulation of DAT and SERT binding sites. In
these studies, the authors showed that changes in DA and 5-HT systems endure, even after
a long recovery period from the last dose of nandrolone. This confirms the hypothesis that
drug abuse causes long lasting changes in brain dopaminergic and serotonergic pathways.
These data are in line with earlier findings demonstrating that chronic cocaine and
methamphetamine decreased D2-receptor and DAT expression during withdrawal and lasted
up to 11 months after the last drug administration. Collectively, these results demonstrate
that pre-treatment with nandrolone decanoate dose-dependently attenuates neurochemical
and behavioral effects relating to the reward system induced by psychostimulant drugs.
These findings indicate that such reduced dopaminergic and serotonergic activity in brain
regions strictly involved in the reward system might represent the neurochemical substrate
that could underlie a higher prevalence of illicit drug use among AAS abusers. Indeed, to
achieve the desired effect of psychostimulant drugs, AAS users may require increased doses
of these substances [150004].
On the other hand, testosterone has been hypothesized to act as a partial agonist on the
opiod system considering that, depending on type of receptors involved, steroid effects are
brain region specific. As the reinforcing effects of opioids are thought to be mediated
principally by my- and delta-receptors, many data in the literature are available with regard to
altered opioid receptor binding after AAS. In particular, nandrolone has been reported to
increase binding of my-, delta-, and kappa-receptors in the hypothalamus, striatum, and
midbrain periaqueductal gray, while reduced kappa-receptors have been found in NAc. In
addition, increased beta-endorphin levels in the VTA and paraventricular thalamus (PVT)
along with and higher beta-endorphin fiber staining in bed nucleus of the stria terminalis and
PVT have been described. However, the total beta-endorphin immunoreactivity is lower in
arcuate nucleus [150004].
On the other hand, nandrolone has been found to enhance morphine-induced hypothermia
while testosterone increases the antinociceptive effect of a kappa-agonist. However,
contrasting data exist since no effects of AAS on morphine antinociception have been
reported in other animal models. In fact, nandrolone pre-exposure has been shown to inhibit
tolerance to antinociceptive properties of morphine and CPP induced by morphine in mice
and rats. Accordingly, pre-exposure to AAS has been shown to prevent morphine-induced
striatal Fos expression. High variability is present in findings linking AAS to opiate
withdrawal. In monkeys no effect of AAS has been described for naloxone-precipitated
morphine withdrawal paradigm, although it in 2003 was found that nandrolone increased
withdrawal symptoms induced by naloxone in morphine-dependent mice. Moreover, the
dysphoric effect mediated by nandrolone pre-treatment has been correlated to elevated
striatal levels of dynorphin B, which in turn may account for the inhibition of dopaminergic
activity in this brain region. Finally, testosterone has been shown not to increase motivation
for morphine. Such discrepancies may rely on different AAS and schedule of treatment used,
as well as different species or strain of animal used [150004].
Decreased oxygen consumption
It was reported clinical and physical responses to 7 weeks of anabolic-androgenic steroid

(AAS) self-administration in a male recreational bodybuilder. He was self-administrating a
total of 3,250 mg of testosterone when his previous and current clinical and physical trials
records were revisited. Body shape, performance, and biochemistry results were clustered
into three phases labeled PRE (before the self-use), POST I (immediately at the cessation of
the 7-week administration), and POST II (12 weeks after the cessation). Elevated
testosterone and estradiol levels were observed in the POST I phase, while hepatic and
renal functions remained altered in the POST II phase. Body mass and body fat percentages
885
increased throughout the three phases. When adjusted according to body mass, drops in
aerobic and anaerobic power and capacity (2.1 % to 12.9 %) were observed across the
phases. This case report shows that overall performance decreased when a bodybuilding
practitioner self-administered AAS [150151].
Androgen replacement therapy (ART)
Androgen replacement therapy (ART) is usually life-long, and should only be started after
androgen deficiency has been proven by hormone assays. The therapeutic goal is to
maintain physiological testosterone levels. Testosterone rather than synthetic androgens
should be used. Oral 17 alpha-alkylated androgens are hepatotoxic and should not be used
for ART. There is no indication for androgen therapy in male infertility. Although androgen
deficiency is an uncommon cause of erectile dysfunction, all men presenting with erectile
dysfunction should be evaluated for androgen deficiency. If androgen deficiency is
confirmed, investigation for the underlying pathological cause is required. Contraindications
to androgen therapy are prostate and breast cancer. Precautions include using lower starting
doses for older men and induction of puberty. Intramuscular injections should be avoided in
men with bleeding disorders. Androgen-sensitive epilepsy, migraine, sleep apnoea,
polycythaemia or fluid overload need to be considered. Competitive athletes should be
warned about the risks of disqualification. ART should be initiated with intramuscular
injections of testosterone esters, 250 mg every two weeks. Maintenance requires tailoring
treatment modality to the patient's convenience. Modalities currently available include
testosterone injections, implants, or capsules. Choice depends on convenience, cost,
availability and familiarity. There is no convincing evidence that, in the absence of proven
androgen deficiency, androgen therapy is effective and safe for older men per se, in men
with chronic non-gonadal disease, or for treatment of non-specific symptoms. Until further
evidence is available, such treatment cannot be recommended [00035].
Androgen replacement therapy (ART) is usually life-long, and should only be started after
androgen deficiency has been proven by hormone assays. The therapeutic goal is to
maintain physiological testosterone levels. Testosterone rather than synthetic androgens
should be used. Oral 17 alpha-alkylated androgens are hepatotoxic and should not be used
for ART. There is no indication for androgen therapy in male infertility. Although androgen
deficiency is an uncommon cause of erectile dysfunction, all men presenting with erectile
dysfunction should be evaluated for androgen deficiency. If androgen deficiency is
confirmed, investigation for the underlying pathological cause is required. Contraindications
to androgen therapy are prostate and breast cancer. Precautions include using lower starting
doses for older men and induction of puberty. Intramuscular injections should be avoided in
men with bleeding disorders. Androgen-sensitive epilepsy, migraine, sleep apnoea,
polycythaemia or fluid overload need to be considered. Competitive athletes should be
warned about the risks of disqualification. ART should be initiated with intramuscular
injections of testosterone esters, 250 mg every two weeks [corrected]. Maintenance requires
tailoring treatment modality to the patient's convenience. Modalities currently available
include testosterone injections, implants, or capsules. Choice depends on convenience, cost,
availability and familiarity. There is no convincing evidence that, in the absence of proven
androgen deficiency, androgen therapy is effective and safe for older men per se, in men
with chronic non-gonadal disease, or for treatment of non-specific symptoms. Until further
evidence is available, such treatment cannot be recommended [00081].
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Hormonal contraception in the male
The basis of the hormonal approach to male contraception is that spermatogenesis is

dependent on gonadotrophin secretion, directly in the case of follicle stimulating hormone
(FSH) and indirectly, secondary to the production of testosterone, in the case of luteinizing
hormone (LH). Suppression of gonadotrophin secretion, therefore, results in loss of both
endocrine and spermatogenic activity in the testis. This can be achieved by over-riding the
physiological negative feedback control mechanisms at the hypothalamus and pituitary gland
by administration of high doses of an androgen or progestogen, by preventing the stimulatory
effect of gonadotrophin hormone releasing hormone (GnRH) on the gonadotroph, or by a
combination of such agents. Spermatogenic suppression following testosterone
administration has been investigated for over 50 years, but a hormonal male contraceptive
remains stubbornly absent from the pharmacist's shelves. There have, however, been
significant developments in the field in recent years, amongst which is the increasing interest
of the pharmaceutical industry. While this may partly reflect the rapid growth of research into
androgen supplementation in the ageing male, the benefit to contraceptive research will be
improved methods of androgen administration, common to all clinical indications. An ideal
hormonal male contraceptive method might induce universal azoospermia while being devoid
of adverse metabolic effects, in a practical and acceptable formulation. The need for
azoospermia is based on the results of two landmark multicentre studies by the World Health
Organization (WHO) investigating the contraceptive efficacy of sex steroidinduced
azoospermia and oligozoospermia, using a testosterone-only regimen. Incomplete
suppression of spermatogenesis was associated with a significant risk of pregnancy, with a
pregnancy rate of 8.1 per 100 person-years for sperm concentrations between 0.1-3 x
lOVmL, but no pregnancies in 230 person-years with azoospermia1-3. While these results
may not seem surprising, they should be seen as proof of the concept that wide-spread
hormonal male contraception is a real possibility and greatly facilitate present and future
contraceptive efficacy studies. These data also supported the results of laboratory studies
indicating the presence of residual fertilising potential of spermatozoa from men with
testosteroneinduced severe oligozoospermia, negating the possibility of development of a
contraceptive method that did not result in azoospermia. A supraphysiological dose of
testosterone enanthate (TE, 200 mg/week) was used to ensure adequate suppression of
gonadotrophins: this also induced significant effects on lipid metabolism, skin, liver and
haematopoiesis, but not behaviour. While this testosterone-based regimen was a prototype
to investigate contraceptive efficacy, these WHO studies illustrate the major current
problems, i.e. (i) incomplete suppression of spermatogenesis; (ii) lack of a long-duration
androgen preparation; and (iii) the metabolic consequences of androgen administration.
Crucial to development of any new product is that it will be used: surveys of both men and
women indicate firmly positive attitudes towards a “male pill”. There are, therefore, grounds
for cautious optimism that the next decade may see the introduction of the first novel male
contraceptive for several hundred years [00033].
Commonest AASs in use worldwide, according to main effect [11028]
Compound name Brand name
Testosterone-like effect
Testosterone esters: cypionate Deposteron®, Testex Leo®
Testosterone esters: undecanoate Nebido®, Androxon®
Testosterone esters: blends Durateston®, Testoviron®, Sustanon®, Omnadren®
Methyltestosterone Methyltestosterone®, Metandren®
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Methandrostenolone Dianabol®, Anabol®, Naposim®
Chlorodehydromethyltestosterone Turinabol®
Fluoxymesterone Halotestin®
Boldenone Equipoise®, Equilon®
DHT-like effect
Stanozolol Winstrol®, Stromba®
Oxandrolone Anavar®
Oxymetholone Anadrol®, Hemogenin®, Anapolon®
Mesterolone Proviron®
Methenolone Primobolan®
Nandrolone-like effect
Nandrolone decanoate Decadurabolin®
Nandrolone phenylpropionate Durabolin®
Trenbolone Finaplix®, Parabolan®
Nandrolone undecanoate Dynabolon®
Commonly abused anabolic-androgenic steroids [07031]
Oral preparations
Fluoxymesterone (Halotestin)
Mesterolone (Proviron)
Methandienone (Dianabol)
Methyltestosterone (Virilon)
Mibolerone (Cheque)
Oxandrolone (Anavar, Oxandrin)
Oxymetholone (Anadrol)
Stanozolol (Winstrol)
Intramuscular preparations
Boldenone undecylenate (Equipoise)
Methenolone enanthate (Primobolan)
Nandrolone decanoate (Deca Durabolin)
Nandrolone phenpropionate (Durabolin)
Testosterone cypionate (Depotest)
Testosterone enanthate (Andro-Estro)
Testosterone propionate (Testex)
Trenbolone acetate (Finajet)
Molecular function
Anabolic androgenic steroids, a class of steroid hormones related to testosterone, are natural
ligands of androgen receptor (AR), a member of the nuclear receptor superfamily of ligand-
activated transcription factors. AR binds specific DNA elements, known as androgen-
response elements. Testosterone, the main male sexual hormone, binds AR directly and
indirectly, through conversion into dihydrotestosterone (DHT), its more active metabolite. The
effects of androgens can differ depending on the target cells and/or tissues. To gain insight
into transcription activation mechanisms of AR, it was investigated AR protein signaling in
human peripheral blood lymphocytes treated with supraphysiological doses of DHT. It was
performed a comparative proteomic analysis and was identified about 30 differentially
expressed proteins. At least five species contained a consensus androgen-response
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elements sequence in the promoter region of related coding genes. The analysis also
revealed that high doses of DHT activate the drug detoxification process, could stimulate an
increase in cell motility and exert a prosurvival effect rather than an apoptotic one [10324].
Androgen metabolism
Androgens may undergo metabolic reactions prior to their physiological effect, that is, as part
of their biosynthetic pathways. As an example, enzymes CYP11A1 and CYP17 from the
CYP450 family participate in the modification of cholesterol to yield T, which is then
converted to biologically more active DHT by steroid 5alpha-reductase type 2 enzyme
(SRD5A2) in the prostate. For rational targeting of analysis and appropriate result
interpretation in doping control and for ABP purposes, however, the bioprocesses concerning
metabolism and urinary excretion, and interindividual variability within these processes, are
of major importance. Owing to highly non-polar nature of anabolic steroids, the parent
compounds are often converted by metabolising reactions prior to their elimination and
excretion in urine. A rough division into two main categories can be made, namely phase I
and phase II metabolic reactions. These processes typically aim at termination of
pharmacological activity, modification of steroid structure into less potent, more polar and
better water-soluble form, and thus an enhanced excretion of steroids into urine. In human
body, several organs are involved in metabolic processes, the liver being the main site of the
reactions [14450].
Phase I reactions (i.e. functionalisation) of androgens include hydroxylation, oxidation and

reduction, and involve CYP450 enzymes, dehydrogenases (e.g. type 5 17beta-
hydroxysteroid dehydrogenase (AKR1C3)) and 5alpha-reductases and 5beta-reductases,
which catalyse the reactions. In general, CYP450 family plays a significant role in
metabolism and genetic variability in humans, as 70–80 percent of all drugs are metabolised
via isoenzymes of families CYP1, 2 and 3, and expression of each CYP is influenced by a
unique combination of factors including genetic polymorphisms. From the putative 57
functional isoenzymes, the highest expressed forms in the liver are 3A4, 2C9, 2C8, 2E1 and
1A2, from which 3A4 contributes to 6beta-hydroxylation of T and shows ethnicity-related
polymorphism. Furthermore, in the metabolism of T, the CYP17 gene promoter
polymorphism has been suggested to explain naturally elevated T/E ratios due to
involvement in catalysis of 5-androstene-3beta,17alpa-diol, an important precursor of E.
Phase II reactions, conjugations, play a remarkable role in the metabolism of androgens, as
in an average, the unconjugated fraction represents only less than 3 percent of the total
amount of urinary excreted compounds. Glucuronidation, that is, conjugation with glucuronic
acid, is the main conjugation reaction of androgens in humans. Reaction is catalysed by
UGTs, which are a family of membrane-bound enzymes in the endoplasmic reticulum.
Human genome contains four UGT families, from which UGT1 (9 members) and UGT2 (10
members), especially the members of subfamily UGT2B, are the most significant genomes in
glucuronidation of androgens. With regard to UGT isoenzymes, polymorphism has been
reported for several genes, but in doping control context, a deletion polymorphism in the
gene coding UGT2B17 is of profound significance. It is strongly associated with urinary levels
of T glucuronide and thus with T/E ratio, and interethnic variation has been observed in the
prevalence of gene deletion [14450].
Exogenous factors
Human metabolism is subjected to significant variations caused by multiple external factors.

With regard to the urinary steroid profiling, environmental conditions, drug administration and
889
diet have been identified as sources of alteration of steroids metabolism and excretion from
the body [14450].
Drugs and medication
From the athlete and doping control perspective, all personal properties and genetic
polymorphism involved at each level have an influence on the formation of ‘normal profile’ of
an individual and justify the shift from population-based reference values to the direction of
the ABP. However, the phenotype of an individual is also regulated strongly by the
exogenous factors, which may temporarily interfere with the homeostasis and the metabolic
routes of endogenous steroids. The effect of various pharmaceutical preparations (e.g.
endogenous and exogenous steroids, oral contraceptives, human chorionic gonadotropin, LH
and glucocorticosteroids) on T/E and steroid profile is extensively summarised earlier,
emphasising the alterations in androgen synthesis arising from the feedback received via
hypothalamic–pituitary axis [14450].
Another category of exogenous factors that define the steroid profile includes compounds
affecting the metabolism and elimination of androgens. In general, the endogenous
compounds, drugs and other xenobiotics undergo the same metabolic pathways, and thus
compete and interfere (either by enzyme inhibition or induction) with each other. Taking into
account the reactions connected to androgen metabolism, the most significant ones are
those involving 5α-reductases. Consequently, 5α-reductase inhibitors, such as finasteride,
which are aimed at the treatment of prostatic hyperplasia and which influence mainly the type
2 5α-reductase present in prostate, suppress the formation of DHT from T, and thus interfere
with the interpretation of the ABP profile. Analogous to this mechanism of effects, type 5 17β-
hydroxysteroid dehydrogenase (AKR1C3) catalyses the reduction of 4-androstene-3,17-
dione to T, and the inhibition of this pathway would be desired, for example, for the treatment
of hormone-dependent and hormone-independent cancers. Several compounds, such as
non-steroidal anti-inflammatory drugs (NSAIDs), steroid hormone analogues and
benzodiazepines, have been explored as inhibitors of AKR1C3 and could impact the
measured T concentration [14450].
For conjugation reactions, inhibition properties of NSAIDs have been demonstrated for
steroid glucuronidation in an in vitro assay, but the observations were not confirmed by in
vivo experiments. One particular therapeutic drug, ketoconazole, should be mentioned due to
its unique property to inhibit T synthesis, as well as the binding of DHT to SHBG, and to
exhibit inhibition of CYP3A4 system. As all these features may have an implication to steroid
profile, anti-doping laboratories report the presence of ketoconazole as part of confirmation
analysis [14450].
Ethanol and tea
Aside the investigated physiological effects of alcohol on physical performance skills and the
widespread habit among top level athletes, ethanol could have an effect on metabolic
pathways linked to steroids biotransformation, and this may be mainly due to a competitive
inhibition of oxidative enzymes such as 17beta-hydroxysteroid dehydrogenases (17HSD)
and UGTs (i.e. UGT2B17) involved in alcohol and steroid metabolisms. The main observed
effects of ethanol on steroid profile are the decrease in androsterone and etiocholanolone
concentrations up to 10 percent of the basal levels and less significant increase in T
excretion resulting in a slight rise of T/E ratio. Urinary steroid concentrations in women are
more sensitive to these modifications caused by ethanol consumption, and obviously the
dose and the frequency of alcohol abuse are key factors that determine the amplitude of
alterations in metabolism [14450].
890
Since quantification of urinary steroids is influenced by the presence of alcohol in the body,
monitoring of alcohol markers is necessary for anti-doping laboratories. Ethylglucuronide
(EtG) and ethylsulfate are widely used parameters in clinical and forensic toxicology to
control ethanol consumption or abstinence. In 2011, it was published a study showing that
EtG is the most suitable quantitative marker of ethanol consumption, allowing the evaluation
of steroid profiling alteration. The question of the threshold is still remaining and various
studies are in progress to establish a shared EtG concentration level at which alcohol could
impact significantly the steroid concentrations. In case of abnormally high T/E ratio due to
ethanol drinking, additional analysis with GC-C-IRMS would prove that no exogenous T was
misused by the athlete, showing the usefulness of this technique as described further in this
review. However, some precautions need to be taken in the interpretation of GC-C-IRMS
results as few studies provided evidence that diet components and geographical origin may
affect delta values of the investigated steroid compounds [14450].
It has also been reported that in vitro green and white teas suppress UGT2B17, a key
enzyme for the glucuronidation of T. The inhibition of this pathway would increase free T
level in human tissues and a potential doping in optimising free T. As for the NSAIDs, the
influence of green tea on T metabolism, as shown in in vitro experiments, has most probably
no effect on the urinary steroid profile. Furthermore, the publication of these results were
appeased by anti-doping experts saying that the required amount of administered tea for a
significant change in steroids concentrations is considerable and that a human interpretation
of steroid profiles is always performed in any suspicious case [14450].
Environment and bacterial contamination
Although it is known for many years that bacteria and microorganisms also alter steroid
profiles, peer-reviewed papers investigating the ability of microbiological contamination to
modify the urinary steroid profile were published only recently. During the diuresis and
storage in the bladder, urine is germ free, but when leaving the human body or subjected to
bacterial exposure, enzyme activity linked to microorganisms may lead to a rise or a drop of
endogenous steroid concentrations or even to the hydrolysis of conjugated T metabolites.
5alpha-androstanedione and 5beta-androstanedione, originating from a bacterial
transformation of androsterone glucuronide and etiocholanolone glucuronide, respectively,
are markers that WADA-accredited laboratories screen and quantify in urine to reveal an
adulteration of the biological samples with microorganisms. Another marker of bacterial
contamination is an increase of free T concentrations, which may lead to elevated T/E ratio.
Besides modifications of endogenous steroid profiles, other anabolic androgenic steroids
such as 19-nortestosterone (nandrolone) and boldenone could be produced by
microorganisms in urine matrix. Identification of microorganisms that could be found in
contaminated urine among the huge diversity of bacteria is possible through a variety of
accurate methods such as sensory observations, assessing turbidity, presence of precipitate
and smell and measurement of pH. In 2010, it was published an approach based on PCR
and 16S rRNA gene sequencing for microbes identification, and thus potential steroid profiles
adulteration. Methods using matrix-assisted laser desorption ionisation-time of flight MS has
recently been developed as a very effective tools to identify bacteria in biological fluids. This
approach is of valuable interest in clinical microbiology but is not easily adapted to the
prevailing technologies in the anti-doping laboratories. Despite the efficiency of these
techniques, identification and quantification of microbial degradation products such as 5alpa-
androstanedione and 5beta-androstanedione is still the preferred approach advocated by
WADA in TD2014EAAS [14450].
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Physiological cellular effects of androgens
Androgens promote anabolism in the musculoskeletal system while generally repressing

adiposity, leading to lean body composition. Circulating androgens decline with age,
contributing to frailty, osteoporosis, and obesity, however the mechanisms by which
androgens modulate body composition are largely unknown. Here we demonstrate that aged
castrated rats develop increased fat mass, reduced muscle mass and strength, and lower
bone mass. Treatment with testosterone or 5alpha-dihydrotestosterone (DHT) reverses the
effects on muscle and adipose tissues while only aromatizable T increased bone mass.
During the first week, DHT transiently increased soleus muscle nuclear density and induced
expression of insulin-like growth factor-1 (IGF-1) and its splice variant mechano growth factor
(MGF) without early regulation of the myogenic factors MyoD, myogenin, MNF, or myostatin.
A genome-wide microarray screen was also performed to identify potential pro-myogenic
genes that respond to androgen receptor activation in vivo within 24 hours. Of 24,000 genes
examined, 70 candidate genes were identified whose functions suggest initiation of
remodeling and regeneration, including the type II muscle genes for myosin heavy chain II
and parvalbumin and the chemokine MCP-1. Interestingly, Axin and Axin2, negative
regulators of beta-catenin, were repressed, indicating modulation of the beta-catenin
pathway. DHT increased total levels of beta-catenin protein, which accumulated in nuclei in
vivo. Likewise, treatment of C2C12 myoblasts with both IGF-1Ea and MGF c-terminal
peptide increased nuclear beta-catenin in vitro. Thus it was propose that androgenic
anabolism involves downregulation of Axin, and induction of IGF-1, leading to nuclear
accumulation of beta-catenin, a pro-myogenic, anti-adipogenic stem cell regulatory factor
[09053].
Role of satellite cells in anabolic steroid-induced muscle growth
Both androgenic and estrogenic steroids are widely used as growth promoters in feedlot
steers because they significantly enhance feed efficiency, rate of gain, and muscle growth.
However, despite their widespread use relatively little is known about the biological
mechanism by which androgenic and estrogenic steroids enhance rate and efficiency of
muscle growth in cattle. Treatment of feedlot steers with a combined estradiol (E2) and
trenbolone acetate (TBA) implant results in an increased number of muscle satellite cells,
increased expression of IGF-1 mRNA in muscle tissue, and increased levels of circulating
IGF-1. Similarly, treatment of bovine satellite cell (BSC) cultures with either TBA or E2 results
in increased expression of IGF-1 mRNA, increased rates of proliferation and protein
synthesis, and decreased rates of protein degradation. Effects of E2 on BSC are mediated at
least in part through the classical E2 receptor, estrogen receptor-alpha (ESR1), the IGF-1
receptor (IGFR1), and the G protein-coupled estrogen receptor-1 (GPER-1), formerly known
as G protein-coupled receptor-30 (GPR30). The effects of TBA appear to be primarily
mediated through the androgen receptor. Based on current research results, it is becoming
clear that anabolic steroid-enhanced bovine muscle growth involves a complex interaction of
numerous pathways and receptors. Consequently, additional in vivo and in vitro studies are
necessary to understand the mechanisms involved in this complex process. The fundamental
information generated by this research will help in developing future, safe, and effective
strategies to increase rate and efficiency of muscle growth in beef cattle [14041].
Episodical secretion
Testosterone and cortisol respond to exercise stimuli and modulate adaptation. Episodic
basal secretion of these hormones may modify the responsiveness of these hormones. It
892
was attempted to identify episodic steroid secretion via frequent salivary sampling and
investigate any interaction between ultradian rhythmicity and induced changes in
testosterone. Salivary testosterone and cortisol concentrations of seven males (age 20-40
years) were measured every 10 min between 0800 and 1600 h on three consecutive days.
On either the second or third day, three interventions designed to elicit a hormonal response
were randomly assigned: sprint exercise (two 30-s maximal efforts on a cycle ergometer);
boxing (two 30-s maximal punching efforts); and a violent video game (10 min of player vs.
player combat). On the other days subjects were inactive. Testosterone data on non-
intervention days suggested pulsatile secretion with a pulse interval of 47 + 9 min (mean +
SD). The sprint intervention substantially affected hormones: it elicited a small transient
elevation in testosterone (by a factor of 1.21; factor 90 % confidence limits x/ divided by 1.21)
10 min after exercise, and a moderate elevation in cortisol peaking 50 min post-exercise
(factor 2.3; x/ divided by 2.6). The testosterone response correlated significantly with the
change in testosterone concentration in the 10 min prior to the sprint and with a measure of
randomness in testosterone fluctuations. Thus, the salivary testosterone response to
exercise may be dependent on the underlying ultradian rhythm and aspects of its regulation.
This interaction may have important implications for adaptation to exercise [10326].
Circadian rhythm
Diurnal variation of sports performance usually peaks in the late afternoon, coinciding with
increased body temperature. This circadian pattern of performance may be explained by the
effect of increased core temperature on peripheral mechanisms, as neural drive does not
appear to exhibit nycthemeral variation. This typical diurnal regularity has been reported in a
variety of physical activities spanning the energy systems, from Adenosine triphosphate-
phosphocreatine (ATP-PC) to anaerobic and aerobic metabolism, and is evident across all
muscle contractions (eccentric, isometric, concentric) in a large number of muscle groups.
Increased nerve conduction velocity, joint suppleness, increased muscular blood flow,
improvements of glycogenolysis and glycolysis, increased environmental temperature, and
preferential meteorological conditions may all contribute to diurnal variation in physical
performance. However, the diurnal variation in strength performance can be blunted by a
repeated-morning resistance training protocol. Optimal adaptations to resistance training
(muscle hypertrophy and strength increases) also seem to occur in the late afternoon, which
is interesting, since cortisol and, particularly, testosterone (T) concentrations are higher in the
morning. T has repeatedly been linked with resistance training adaptation, and higher
concentrations appear preferential. This has been determined by suppression of endogenous
production and exogenous supplementation. However, the cortisol (C)/T ratio may indicate
the catabolic/anabolic environment of an organism due to their roles in protein degradation
and protein synthesis, respectively. The morning elevated T level (seen as beneficial to
achieve muscle hypertrophy) may be counteracted by the morning elevated C level and,
therefore, protein degradation. Although T levels are higher in the morning, an increased
resistance exercise-induced T response has been found in the late afternoon, suggesting
greater responsiveness of the hypothalamo-pituitary-testicular axis then. Individual
responsiveness has also been observed, with some participants experiencing greater
hypertrophy and strength increases in response to strength protocols, whereas others
respond preferentially to power, hypertrophy, or strength endurance protocols dependent on
which protocol elicited the greatest T response. It appears that physical performance is
dependent on a number of endogenous time-dependent factors, which may be masked or
confounded by exogenous circadian factors. Strength performance without time-of-day-
specific training seems to elicit the typical diurnal pattern, as does resistance training
adaptations. The implications for this are athletes are advised to coincide training times with
performance times, and individuals may experience greater hypertrophy and strength gains
when resistance training protocols are designed dependent on individual T response [10054].
893
Testosterone and cortisol respond to exercise stimuli and modulate adaptation. Episodic
basal secretion of these hormones may modify the responsiveness of these hormones. We
sought to identify episodic steroid secretion via frequent salivary sampling and investigate
any interaction between ultradian rhythmicity and induced changes in testosterone. Salivary
testosterone and cortisol concentrations of seven males (age 20-40 years) were measured
every 10 min between 0800 and 1600 h on three consecutive days. On either the second or
third day, three interventions designed to elicit a hormonal response were randomly
assigned: sprint exercise (two 30-s maximal efforts on a cycle ergometer); boxing (two 30-s
maximal punching efforts); and a violent video game (10 min of player vs player combat). On
the other days subjects were inactive. Testosterone data on non-intervention days suggested
pulsatile secretion with a pulse interval of mean 47 + 9 min. The sprint intervention
substantially affected hormones: it elicited a small transient elevation in testosterone (by a
factor of 1.21) 10 min after exercise, and a moderate elevation in cortisol peaking 50 min
post-exercise (factor 2.3). The testosterone response correlated with the change in
testosterone concentration in the 10 min prior to the sprint and with a measure of
randomness in testosterone fluctuations. Thus, the salivary testosterone response to
exercise may be dependent on the underlying ultradian rhythm and aspects of its regulation.
This interaction may have important implications for adaptation to exercise [10055].
Testosterone is the principal androgenic steroid produced by the testes. Testosterone is also
a precursor to estrogen synthesis by the ovary in women. Steroids are hormones derived
from cholesterol, and androgens promote the development and maintenance of male
characteristics.. Many androgen actions in the body are mediated by binding to the androgen
receptor, a nuclear receptor that modulates transcription of responsive genes. Whether of
endogenous or exogenous origin in males and in females, excess testosterone creates an
advantage in sports. While the anabolic effects of testosterone in hypogonadal males were
well-accepted, early studies testing the effects of testosterone supplementation to eugonadal
men were not well-controlled. More recent studies have shown that testosterone stimulates
muscle mass and reduces body fat. Androgens likely also act on specific substrates in the
brain to increase aggression and motivation for competition. Exogenous testosterone has
been banned from Olympic competition since 1976, and was classified in the United States
as a controlled substance by the Anabolic Steroid Control Act of 1990. Defining the upper
limit for endogenous testosterone is complicated by the dynamic changes in testosterone
across a number of temporal scales. On the shortest time-scale, testosterone production in
the gonads follows the pulsatile release of luteinizing hormone. This introduces a level of
unpredictability for estimating circulating androgen concentrations in any single biologic
sample. Secondly, in both sexes, testosterone follows a diurnal rhythm with peak
concentrations in the morning followed by progressive decline over the course of the day,
rising again at night during sleep. In women, there is evidence that testosterone
concentrations also vary as a function of the menstrual cycle, with peak testosterone
concentrations in the peri-ovulatory window, and lower values in the early follicular and late
luteal phases. On a somewhat longer time-scale, testosterone concentrations exhibit
circannual variation and peak in the fall. Lastly, men's testosterone concentrations slowly
decline over the lifespan while women face an abrupt decline in testosterone at menopause.
Due to the dynamic regulation of endogenous testosterone production, including the acute
effects of competition and exercise, testosterone concentrations may vary considerably
within and among individuals. Accordingly, it has been difficult to establish a threshold
separating endogenous testosterone from exogenous sources. Furthermore, disorders of
sexual differentiation (DSD) can produce elevated concentrations of endogenous androgens,
potentially creating a competitive advantage for female athletes with DSD. Due to variability
in endogenous secretion, and similarities with exogenous testosterone, it has been
challenging to establish allowable limits for testosterone in competition. Endogenous
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androgen production is dynamically regulated by both exercise and winning in competition.
Furthermore, testosterone may promote athletic performance, not only through its long-term
anabolic actions, but also through rapid effects on behavior [12100].

Interindividual variation in genetics, in enzyme distribution and, consequently, in drug
metabolism are discussed later in this review in detail. Briefly, two main families of enzymes
contributing the drug metabolism in humans are cytochrome P450 (CYP450), which is
responsible for phase I reactions, and uridine diphosphate glucuronosyltransferase (UGT)
enzymes, which catalyse the phase II conjugation reaction with glucuronic acid. Gender-
dependent differences in enzyme activity have been demonstrated for several CYP
isoenzymes and for UGTs, supporting the possibility of quantitative differences between
female and male athletes. However, the genes for CYP and UGT proteins are not linked to
X-chromosome, and, thus, the prevalence of poor metabolisers should not be expected to be
different between genders. In fact, reference concentration ranges of urinary T and excreted
metabolites have been published previously with lower levels in female participants than in
male participants. Periodical variations in hormones concentrations are well established in
different species and matrices. In humans, T is also subjected to these fluctuations, as is
previously shown in serum, saliva and urine. This daily, monthly and even yearly based
variability of steroid hormones concentrations should not significantly impact the longitudinal
follow-up of participants, and is included within the normal intraindividual variation of the
steroid profile components [14450].
Regarding the urinary steroid profile and physical exercise there are studies concluding
differences between sedentary and exercising individuals, and that the physical activity may
influence the elimination of androgens due to changes in sex hormone binding globulin
(SHBG). A group of trained female athletes was investigated by Bricout et al with respect to
urinary steroid profiles during menstrual cycle and compared with non-athlete (sedentary)
group. T and E were measured from glucuronide-conjugated fraction by radioimmunoassay
(RIA), and based on this study, the T/E remained stable between the follicular phase and
luteal phase of menstrual cycle within athlete (0.66 ± 0.05 vs 0.69 ± 0.33) and non-athlete
(0.72 ± 0.26 vs 0.67 ± 0.31) groups. As a conclusion, it was stated that although physical
training may have an effect on androgen metabolism, active sportswomen can be considered
as members of normal population as long as there are no signs of secondary amenorrhoea
induced by physical activity. It was shown that high workload during the Tour de France does
not influence the T/E ratio in top-level athletes [14450].
AAS suppress HPT function. When individuals stop taking AAS after a lengthy course of use
(i.e. several months or longer), HPT activity may be suppressed for months, or years and
some individuals may never regain normal testosterone levels. Further, AAS may also
produce direct toxic effects on the testis, which may be irreversible, so that some AAS users
will continue to display primary hydoi: pogonadism even after hypothalamic and pituitary
functions have returned to normal. Several case reports have described successful treatment
of AAS-induced hypogonadism with clomiphene, human chorionic gonadotropin, and/or
human menopausal gonadotropin. However, case reports also have described failure with
these interventions. To date, there are no systematic treatment studies in AAS-induced
hypogonadism. The suppression of pituitary luteinizing hormone (LH) and follicle-stimulating
hormone (FSH) secretion by AAS can be associated with suppression of spermatogenesis
and infertility in men, and menstrual irregularity and infertility in women [14017].
895
AASs suppress HPT function. When individuals stop taking AASs after a lengthy course of
use (i.e. several months or longer), HPT activity may be suppressed for months, or years;
and some individuals may never regain normal testosterone levels. Furthermore, AAS may
also produce direct toxic effects on the testis, which may be irreversible, so that some AAS
users will continue to display primary hypogonadism even after hypothalamic and pituitary
functions have returned to normal. Several case reports have described successful treatment
of AAS-induced hypogonadism with clomiphene, human chorionic gonadotropin, and/or
human menopausal gonadotropin. However, case reports also have described failure with
these interventions. To date, we are not aware of any systematic treatment studies in AAS-
induced hypogonadism. The suppression of pituitary LH and FSH secretion by AAS can be
associated with suppression of spermatogenesis and infertility in men and menstrual
irregularity and infertility in women [14426].
Effect on older men
Although testosterone levels and muscle mass decline with age, many older men have
serum testosterone level in the normal range, leading to speculation about whether older
men are less sensitive to testosterone. It was determined the responsiveness of androgen-
dependent outcomes to graded testosterone doses in older men and compared it to that in
young men. The participants in this randomized, double-blind trial were 60 ambulatory,
healthy, older men, 60-75 years of age, who had normal serum testosterone levels. Their
responses to graded doses of testosterone were compared with previous data in 61 men, 19-
35 yr old. The participants received a long-acting GnRH agonist to suppress endogenous
testosterone production and 25, 50, 125, 300, or 600 mg testosterone enanthate weekly for
20 wk. Fat-free mass, fat mass, muscle strength, sexual function, mood, visuospatial
cognition, hormone levels, and safety measures were evaluated before, during, and after
treatment. Of 60 older men who were randomized, 52 completed the study. After adjusting
for testosterone dose, changes in serum total testosterone (change, -6.8, -1.9, +16.1, +49.5,
and +101.9 nmol/liter at 25, 50, 125, 300, and 600 mg/wk, respectively) and hemoglobin
(change, -3.6, +9.9, +20.9, +12.6, and +29.4 g/liter at 25, 50, 125, 300, and 600 mg/wk,
respectively) levels were dose-related in older men and significantly greater in older men
than young men. The changes in FFM (-0.3, +1.7, +4.2, +5.6, and +7.3 kg, respectively, in
five ascending dose groups) and muscle strength in older men were correlated with
testosterone dose and concentrations and were not significantly different in young and older
men. Changes in fat mass correlated inversely with testosterone dose and were significantly
different in young vs. older men; young men receiving 25- and 50-mg doses gained more fat
mass than older men. Mood and visuospatial cognition did not change significantly in either
group. Frequency of hematocrit greater than 54 percent, leg edema, and prostate events
were numerically higher in older men than in young men. Older men are as responsive as
young men to testosterone's anabolic effects; however, older men have lower testosterone
clearance rates, higher increments in hemoglobin, and a higher frequency of adverse effects.
Although substantial gains in muscle mass and strength can be realized in older men with
supraphysiological testosterone doses, these high doses are associated with a high
frequency of adverse effects. The best trade-off was achieved with a testosterone dose (125
mg) that was associated with high normal testosterone levels, low frequency of adverse
events, and significant gains in fat-free mass and muscle strength [04070].
Effects on andropause
Andropause is the gradual reduction of the male sex hormone (testosterone) with increasing
896
age. Its symptoms are sexual dysfunction, weakness, fatigue, insomnia, loss of motivation,
mood disorders and reduction of bone density. Treatment of andropause with testosterone
has been recently considered. The aim of one study was to evaluate the effect of
testosterone in the treatment of andropause in men. For men who met the inclusion criteria
(50 years of age and older) laboratory tests and clinical examinations were conducted by an
urologist in order to diagnose prostate cancer, prostate disease, urinary tract infection and
active urinary retention. After obtaining consent, the patients were enrolled in the study. Data
were analyzed using SPSS version 20. Descriptive statistics (frequency and percentage,
mean, standard deviation) and the paired t-test were used to compare levels of testosterone.
To determine the correlation between age and testosterone levels, the Pearson correlation
was used. Finally, to compare the treatment processes during the treatment period the
repeated measures ANOVA was used. The mean age of patients was 57 ± 3 years. A total of
31 patients (39 %) were smokers, among them 30 percent smoked daily, 3 percent weekly
and 6 percent smoked for fun. The mean testosterone level before treatment was 240.6 ±
125.4 and at 1, 3 and 6 months after treatment the level was raised, so that at the end of the
sixth months it was 578.7 ± 141.7. The level of increase was statistically significant [150152].
Treatment with testosterone in men over 50 years with andropause will increase testosterone
levels and also quality of life, sexual desire, erection, energy levels, ability to exercise and
feel the joy of life more than before. Depression was decreased and they had sleepy feelings
after dinner. Testosterone is responsible for secondary sex characteristics, sexual desire and
erection. Also, it increases metabolic processes in the muscles, bones, bone marrow,
immune system and brain. Therefore, a reduction in the level of testosterone leads to
symptoms that are caused by the decrease of these processes. Total testosterone levels in
men consist of three parts:
- testosterone that binds strongly to SHBG (sex hormone binding globulin); this
accounts for almost 80 percent of total testosterone and serves as a reserve
source
- testosterone also binds to albumin, but this binding is weak and easily available
- free testosterone
Free testosterone and testosterone bound to albumin are biologically active and are 20
percent of total testosterone. Testosterone levels in men aged 40 is reduced, and the total
testosterone level is reduced 0.3 percent annually. The average amount of testosterone in
men of 75 years of age is 66 percent of men aged 25. The decline of testosterone levels with
aging, on one hand, is related to testis dysfunction (Leydig cell mass reduction, a decrease in
performance of the remaining cells, as well as reduced testis circulation), and on the other
hand, to reduced testosterone-stimulating hormones, including GnRH from the hypothalamus
and gonadotropin from the pituitary gland [150152].
Andropause is the gradual reduction of the male sex hormone (testosterone) with increasing
age, and its symptoms are sexual dysfunction, weakness, fatigue, insomnia, loss of
motivation, mood disorders and reduction of bone density. Recently, andropause treatment
with testosterone has been considered. The serum level of testosterone is different in
individuals, however, in most experiments 10 nmol/L to 35 nmol/L has been found to be
within the normal range. One of the issues facing older people is finding the lower level of
testosterone that causes andropause. So, to diagnosis andropause, in addition to
testosterone serum levels, clinical symptoms should be considered. Besides considering the
possible benefits of testosterone therapy in older men for andropause treatment, possible
complications including benign prostatic hyperplasia, prostate cancer, exacerbation of sleep
apnea, gynecomastia, polycythemia and liver toxicity should be considered. Andropause has
been widely considered in recent years by the medical societies. Some studies of
897
testosterone therapy have mentioned significant progress in reducing the symptoms.
However, the benefit of testosterone therapy in this age group still faces uncertainty
[150152].
Andropause and aging are associated with neuroendocrine dysfunctions. Growth hormone
and testosterone play a significant role in several processes affecting adaptation and thereby
also everyday functioning. The aim of research project was now to evaluate the effects of
recombinant human growth hormone and testosterone enanthate injections on body mass
and body composition, aerobic and anaerobic fitness and lipid profile in middle-aged men.
The research group was comprised of 14 men aged 45-60 years. Two series of laboratory
analyses were performed. Independent tests were carried out at baseline and after 12 weeks
of the experiment. The data were analyzed using Statistica 9.1 software. A two-way repeated
measures ANOVA revealed a statistically significant effect of the intervention programme on
fat-free mass, total body fat, total cholesterol, high-density lipoprotein cholesterol, low-density
lipoprotein cholesterol, triglyceride, testosterone, insulin-like growth factor 1, and growth
hormone. Furthermore, ANOVA revealed a statistically significant effect of the rhGH and T
treatment on maximal oxygen uptake, anaerobic threshold and maximal work rate. It should
be emphasized that the lipid profile was affected not only by rhGH+T replacement therapy,
but also by the prescribed physical activity programme. The strength and endurance fitness
programme alone did not cause significant changes in body mass and composition, nor the
anaerobic and aerobic capacity. On the other hand, the rhGH and T treatment stimulated
these changes significantly [14040].
Muscle atrophy after androgen deprivation

The molecular factors targeted by androgens and estrogens on muscle mass are not fully
understood. One study aimed to explore gene and protein expression of Atrogin-1, MuRF1,
and myostatin in an androgen deprivation-induced muscle atrophy model. It was examined
the effects of Orx either with or without testosterone (T) or estradiol (E2) administration on
Atrogin-1 gene expression, and MuRF1 and myostatin gene and protein expression.
Measurements were made in soleus (SOL), extensor digitorum longus (EDL) and levator
ani/bulbocavernosus (LA/BC) of male C57BL/6 mice. Thirty days of Orx resulted in a
reduction in weight gain and muscle mass. These effects were prevented by T. In LA/BC,
Atrogin-1 and MuRF1 mRNA was increased throughout 30 days of Orx, which was fully
reversed by T and partially by E2 administration. In EDL and SOL, a less pronounced
upregulation of both genes was only detectable at the early stages of Orx. Myostatin mRNA
levels were downregulated in LA/BC and upregulated in EDL following Orx. T, but not E2,
reversed these effects. No changes in protein levels of MuRF1 and myostatin were found in
EDL at any time point following Orx. It was concluded that the atrophy in SOL and EDL in
response to androgen deprivation, and its restoration by T, is accompanied by only minimal
changes in atrogenes and myostatin gene expression. The marked differences in muscle
atrophy and atrogene and myostatin mRNA between LA/BC and the locomotor muscles
suggest that the murine LA/BC is not an optimal model to study Orx-induced muscle atrophy
[14042].
Erythropoesis
AAS use is associated with dose-related increases in hemoglobin and hematocrit, and
polycythemia is a frequent adverse event of AAS use. Androgens stimulate erythropoiesis by
increasing sensitivity to erythropoietin, suppressing hepcidin transcription, and increasing
iron availability for erythropoiesis [14017].
898
Exercise-induced low testosterone levels
During the last 30 years a large number of research studies have been conducted examining
reproductive endocrine dysfunction in exercising women. The number of similar studies
examining men is still relatively small. Nevertheless, an increasing amount of research
studies in men indicate endurance exercise training has significant effects upon the major
male reproductive hormone, testosterone, and the hypothalamic-pituitary-testicular axis that
regulates reproductive hormones. This review article addresses one reproductive endocrine
dysfunction found in exercising men, what has been deemed the "exercise-hypogonadal
male condition". Specifically, men with this condition exhibit basal (resting-state) free and
total testosterone levels that are significantly and persistently reduced. The exact
physiological mechanism inducing the reduction of testosterone is currently unclear, but is
postulated to be a dysfunction (or perhaps a readjustment) within the hypothalamic-pituitary-
testicular regulatory axis. The time course for the development of the "exercise-hypogonadal
condition" or the threshold of exercise training necessary to induce the condition remains
unresolved. The potential exists for these reduced testosterone levels within the exercise-
hypogonadal male to disrupt and be detrimental to some anabolic or androgenic
testosterone-dependent physiological processes. Unfortunately, extremely few research
studies have addressed whether such processes are affected, and thus findings are
inconclusive. Conversely, the alterations in testosterone levels brought about by endurance
exercise training have the potential for cardiovascular protective effects and thus could be
beneficial to the health of these men. Current evidence suggests this condition is limited to
men who have been persistently involved in chronic endurance exercise training for
extended periods of time (i.e. years). Many questions, however, regarding the male
reproductive endocrine adaptive process to exercise and exercise training remain
unanswered, necessitating the need for further research on this topic [05033].
Seven resistance-trained men performed six bouts of resistance exercise, each separated by
at least 1 week, in a crossover design. High, moderate and low volumes of exercise were
used, each performed twice and followed immediately post-exercise by either a placebo or
carbohydrate-protein supplementation. All bouts of resistance exercise were performed using
a load equal to 100 percent of each subject's ten-repetition maximum (10-RM), and all rest
periods between sets of exercise were 1 min. Blood was obtained before and at intervals
after exercise until 120 min post-exercise. Lactate levels were significantly elevated
immediately post-exercise, and to a significantly greater extent after the greatest volume of
exercise. Levels of growth hormone rose significantly after the greatest volume of exercise
only. Those of insulin and glucose rose significantly after supplementation only. Cortisol
levels tended to be higher after the greatest volume of exercise, but the differences were not
significant. Supplementation had no effect on the lactate, growth hormone or cortisol
responses to resistance exercise. The data indicate that volume of exercise and protein-
carbohydrate supplementation can alter the metabolic and hormonal responses to resistance
exercise independently. However, cortisol levels remain high after a high volume of
resistance exercise, irrespective of whether a post-exercise carbohydrate-protein supplement
is used [02026].
Androgen receptors after exercise
The purpose of one investigation was to examine androgen receptor (AR) content in the
vastus lateralis following two resistance exercise protocols of different volume. Nine
resistance-trained men performed the squat exercise for 1 (SS) and 6 sets (MS) of 10
899
repetitions in a random, counter-balanced order. Muscle biopsies were performed at
baseline, and 1h following each protocol. Blood was collected prior to, immediately following
(IP), and every 15 min after each protocol for 1h. No acute elevations in serum total
testosterone were observed following SS, whereas significant 16-23 percent elevations were
observed at IP, 15, and 30 min post-exercise following MS. No acute elevations in plasma
cortisol were observed following SS, whereas significant 31-49 percent elevations were
observed for MS at IP, 15, and 30 min post-exercise. Androgen receptor content did not
change 1h following SS but significantly decreased by 46 percent following MS. These
results demonstrated that a higher volume of resistance exercise resulted in down-regulation
of AR content 1h post-exercise. This may have been due to greater protein catabolism
associated with the higher level of stress following higher-volume resistance exercise
[05034].
Modulation of training on anabolic steroids values
Relationship between dietary intake and serum anabolic hormone concentrations of

testosterone (T), free testosterone (FT), and growth hormone were examined at rest as well
as after the heavy-resistance exercise (HRE) in 8 strength athletes (SA) and 10 physically
active non-athletes (NA). In the first part of the study serum basal anabolic hormone
concentrations and dietary intake were examined in the total group of subjects. In the second
part of the study a subgroup of 5 SA and 5 NA performed the high volume and high intensity
HRE. Dietary intake was registered by dietary diaries for 4 days preceding the loading day.
Significant correlations were observed between serum basal T and fat and protein intake in
the total group of subjects. However, when the two groups were examined separately the
significant relationships between serum basal T and dietary fat and protein could be noticed
in SA only. Both serum T and FT responses to HRE were correlated with fat and protein. The
results suggest the possible role of diet leading to alterations in serum T and FT during
prolonged strength training, and that diets with insufficient fat and/or excessive protein may
compromise the anabolic hormonal environment over a training program [04055].
Endogenous steroids
The detection of misuse with naturally occurring steroids is a great challenge for doping
control laboratories. Intake of natural anabolic steroids alters the steroid profile. Thus,
screening for exogenous use of these steroids can be established by monitoring a range of
endogenous steroids, which constitute the steroid profile, and evaluate their concentrations
and ratios against reference ranges. Elevated values of the steroid profile constitute an
atypical finding after which a confirmatory IRMS procedure is needed to unequivocally
establish the exogenous origin of a natural steroid. However, the large inter-individual
differences in urinary steroid concentrations and the recent availability of a whole range of
natural steroids (e.g. dehydroepiandrosterone and androstenedione) which each exert a
different effect on the monitored parameters in doping control complicate the interpretation of
the current steroid profile. The screening of an extended steroid profile can provide additional
parameters to support the atypical findings and can give specific information upon the
steroids which have been administered. The natural concentrations of 29 endogenous
steroids and 11 ratios in a predominantly Caucasian population of athletes were determined.
The upper reference values at 97.5 percent, 99 percent and 99.9 percent levels were
assessed for male (n=2027) and female (n=1004) populations. Monitoring minor metabolites
and evaluation of concentration ratios with respect to their natural abundances could improve
the interpretation of the steroid profile in doping analysis [09052].
900
The use of gas chromatography (GC)-combustion (C)-isotope ratio mass spectrometry
(IRMS) demonstrates that a single oral administration of dehydroepiandrosterone (DHEA,
100 mg) to a male subject significantly lowers the 13C content of etiocholanolone (Et) and
androsterone (A) in the subject's urine. The difference in carbon isotope ratio (delta13C per
thousand) values between Et and A increases from 1.6 per thousand at the time of
administration to 5.1 per thousand at 26 h post-administration, indicating preferential
metabolism of administered DHEA to form Et in relation to A. Multiple oral administrations of
DHEA to a male subject reveals lower d13C values during the excretion period of Et (-31.7
per thousand to -34.6 per thousand) and A (-31.4 per thousand to -33.0 per thousand) to that
of the delta13C value of the administered DHEA (-31.3 per thousand). Reference distributions
of delta13C Et and delta13C. A constructed from normal athlete populations within Australia
and New Zealand show a small natural discrimination against 13C in the formation of Et
relative to A (mean=0.3 per thousand, n=167). Amplified differences between delta13C Et and
delta13C A, and in vivo 13C depletion measured by GC-C-IRMS are shown to be potentially
useful for doping control [05063].
The detection of the administration of an androgen such as testosterone that could be

present normally in human bodily fluids is based upon the methodical evaluation of key
parameters of the urinary profile of steroids, precisely measured by GC/MS. Over the years,
the markers of utilization were identified, the reference ranges of diagnostic metabolites and
ratios were established in volunteers and in populations of athletes, and their stability in
individual subjects were studied. The direct confirmation comes from the measurement of
delta 13C values reflecting their synthetic origin, ruling out a potential physiological anomaly.
Several factors may alter the individual GC/MS steroid profile besides the administration of a
testosterone-related steroid, the nonexhaustive list ranging from the microbial degradation of
the specimen, the utilization of inhibitors of 5alpha-reductase or other anabolic steroids,
masking agents such as probenecid, to inebriating alcohol drinking. The limitation of the
testing strategy comes from the potentially elevated rate of false negatives, since only the
values exceeding those of the reference populations are picked up by the GC/MS screening
analyses performed by the laboratories on blind samples, excluding individual particularities
and subtle doping. Since the ranges of normal values are often described from samples
collected in Western countries, extrapolating data to all athletes appears inefficient.
Furthermore, with short half-life and topical formulations, the alterations of the steroid profile
are less pronounced and disappear rapidly. GC/C/IRMS analyses are too delicate and
fastidious to be considered for screening routine samples. An approach based upon the
individual athlete's steroid profiling is necessary to pick up variations that would trigger
further IRMS analysis and investigations [10090].
One review attempted to give a synopsis of the major aspects concerning the biochemistry of
endogenous androgens, supplemented with several facets of physiology, particularly with
respect to testosterone. Knowledge regarding the precursors and metabolism of endogenous
testosterone is therefore fundamental to understanding many of the issues concerning
doping with testosterone and its prohormones, including the detection of their administration.
Further, adverse findings for nandrolone are frequent, but this steroid and 19-
norandrostenedione are also produced endogenously, an appealing hypothesis being that
they are minor by-products of the aromatization of androgens. At sports tribunals pertaining
to adverse analytical findings of natural androgen administration, experts often raise issues
that concern some aspect of steroid biochemistry and physiology. Salient topics included
within this review are the origins and interconversion of endogenous androgens, the
biosynthesis of testosterone and epitestosterone, the mechanism of aromatization, the
molecular biology of the androgen receptor, the hypothalamic-pituitary-testicular axis,
disturbances to this axis by anabolic steroid administration, the transport (binding) of
androgens in blood, and briefly the metabolism and excretion of androgens [10091].
901
Intake of natural anabolic steroids alters the steroid profile. Thus, screening for exogenous
use of these steroids can be established by monitoring a range of endogenous steroids,
which constitute the steroid profile, and evaluate their concentrations and ratios against
reference ranges. Elevated values of the steroid profile constitute an atypical finding after
which a confirmatory IRMS procedure is needed to unequivocally establish the exogenous
origin of a natural steroid. However, the large inter-individual differences in urinary steroid
concentrations and the recent availability of a whole range of natural steroids (e.g.
dehydroepiandrosterone and androstenedione) which each exert a different effect on the
monitored parameters in doping control complicate the interpretation of the current steroid
profile. The screening of an extended steroid profile can provide additional parameters to
support the atypical findings and can give specific information upon the steroids which have
been administered. The natural concentrations of 29 endogenous steroids and 11 ratios in a
predominantly Caucasian population of athletes were determined. The upper reference
values at 97.5 percent, 99 percent and 99.9 percent levels were assessed for male (n=2027)
and female (n=1004) populations. Monitoring minor metabolites and evaluation of
concentration ratios with respect to their natural abundances could improve the interpretation
of the steroid profile in doping analysis [10092].
By means of gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS)

urinary steroids obtained from a reference population of 56 subjects were analyzed for their
13
C/12C-ratios. The analytes encompassed androsterone (A), etiocholanolone (E), 11beta-
hydroxyetiocholanolone (OHE), 11beta-hydroxyandrosterone (OHA), and 5beta-pregnane-
3alpha,20alpha-diol (PD). A and E represent androgen metabolites (AM). PD, OHE, and
OHA have sources independent from androgen metabolism. The delta(13)C-values of the
latter compounds may be compared to those of AM in order to detect doping with synthetic
androgens and thus may serve as endogenous reference compounds (ERC). In order to
allow for classification of conspicuous samples, reference ranges and limits were calculated
for delta13C-values of selected steroids and differences hereof (Delta13C-values). When A is
compared to ERCs, Delta13C-values larger than 3 per thousand are very unlikely. A set of
additional parameters was surveyed by a questionnaire. Several factors turned out to exert
significant influence on the delta13C-values of urinary steroids. These encompass the identity
of the steroid itself, gender, oral contraception, travels, and physical activity [07059].
It was carried out a study of 141 normal male participants (aged 8-26 years), categorised the
population into five groups based on the development stages according to Tanner's scale
and compared the excretion profiles of T and E between different age groups. According to
their report, excretion of both markers increased significantly during development and
correlated significantly with age. However, a significant difference was observed between the
increase of T and E relative to age, T excretion increasing much faster than E and indicating
the potential instability of T/E during puberty. In another study, originating from approximately
same time, it was studied a population of 140 male participants (aged 13-20 years) with
respect to urinary excretion of several endogenous steroids and luteinising hormone (LH).
Although they concluded that the increase in excretion rates of glucuronide-conjugated T and
E correlated with pubertal development, the result was somewhat contrary to earlier one with
respect to T/E, where the observed differences were not significant. In this study, ratio of T-
glucuronide to LH, which has been proposed as additional information on T misuse,
increased throughout puberty. An independent study fro with 100 male participants (aged 10-
17) showed insignificant change in T/E between different stages, although higher instability
of the ratio was associated to prepubertal stages. In a group of adolescent girls (aged 6-17,
n=256), the same research group observed a decreasing T/E ratio during development, most
902
obviously due to larger relative increase in E excretion. The results were similar between
exercising and control group of participants [14450].
Effect of smoking
Cigarette tobacco smoke is a potent environmental contaminant known to adversely affect

health including fertility and pregnancy. To examine the associations between second-hand
cigarette tobacco-smoke exposure, or active smoking and serum concentrations of steroid
hormones using tandem mass spectrometry. Healthy women (18-45 years) from the general
community in the Metropolitan Washington, DC were recruited at the follicular stage of their
menstrual cycle. Participants were assigned to one of three study groups: active smokers
(n=107), passive smokers (n=86), or non-smokers (n=100). Classifications were based on a
combination of self-reporting and serum cotinine concentrations. Serum androgens,
estrogens, progestins, androstenedione, aldosterone, cortisol, corticosterone, dehydroepi-
androsterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), 11-deoxycortisol and 25-
hydroxy-vitamin D3 (25-OHVitD3) and cotinine were measured by isotope dilution tandem
mass spectrometry (LC/MS/MS) (API-5000). Serum estrone, estradiol, and estriol
concentrations were lower in active and passive smokers than in non-smokers. The three
study groups differed significantly in serum concentrations of 16-OHE1, aldosterone and 25-
OHVitD3, as well as in the ratios of many of the steroids. Pair-wise comparison of the groups
demonstrated significant differences in hormone concentrations between smokers and non-
smokers for aldosterone; passive smokers and non-smokers for aldosterone, progesterone
and estriol. Moreover, for smokers and passive smokers, there were no significant
differences in these hormone concentrations. It was concluded that smoke exposure was
associated with lower than normal median steroid hormone concentrations. These processes
may be instrumental in explaining some adverse effects of tobacco smoke on female health
and fertility [11070].
Effects of dietary components on testosterone metabolism
The potential interference in testosterone metabolism through ingested substances has

ramifications for:
- a range of pathologies such as prostate cancer

- medication contra-indications
- disruption to the endocrine system
- potential confounding effects on doping tests
Conjugation of anabolic steroids during phase II metabolism, mainly driven by UDP-

glucuronosyltransferase (UGT) 2B7, 2B15, and 2B17, has been shown to be impaired in vitro
by a range of compounds including xenobiotics and pharmaceuticals. Following early reports
on the effects of a range of xenobiotics on UGT activity in vitro, the work was extended to
reveal similar effects with common non-steroidal anti-inflammatory drugs. Notably, recent
studies have evidenced inhibitory effects of the common foodstuffs green tea and red wine,
along with their constituent flavonoids and catechins. This review amalgamates the existing
evidence for the inhibitory effects of various pharmaceutical and dietary substances on the
rate of UGT glucuronidation of testosterone; and evaluates the potential consequences for
health linked to steroid levels, interaction with treatment drugs metabolized by the UGT
enzyme and steroid abuse in sport [13209].
903
As a major route for excretion of exogenous and endogenous compounds, there is
considerable interest in the roles of the UDP-glucuronosyltransferase (UGT) family, which
has led to widespread investigations of their potential effects in health and disease. In
particular, genetic and chemical modification of UGT activity relating to steroid metabolism
has ramifications for a range of pathologies such as prostate cancer, medication contra-
indications, disruption to the endocrine system, and potential confounding effects on doping
tests in sport. Therefore, it is timely to review lifestyle factors that affect UGT activity.
Variations in the activity of UGT isozymes occur as a result of gender and ethnic origins
giving different levels of expression of UGT forms and altered ratios of testosterone/
epitestosterone excreted in urine). In addition to genetic variations, from a steroid
metabolism viewpoint, one current focus of investigation is on the regulation of specific UGT
activity via induction or inhibition by exogenous compounds such as pharmaceuticals and
dietary components. Several reports show induction of UGT activity by a range of
compounds including phytochemicals and pharmaceuticals. Early studies reported the
effects of drugs and dietary compounds on UGT activity in isolated microsomes or in rats
without detailing the specific UGT isozymes involved. Liver microsomal glucuronidation of
estradiol and estrone was inhibited by green and black teas, along with a constituent
catechin [(-)-epigallocatechin gallate] and several flavonoids (kaempferol, quercetin, rutin,
flavone, naringenin, hesperetin). Green tea polyphenols had a strong inhibitory effect of
glucuronidation in vitro and showed a small increase in liver glucuronidation activity against
estrone and estradiol was observed in vitro in rats with green tea as the sole fluid source.
Consequent alterations in steroid metabolism have been debated to have a range of putative
effects including varying responses to doping tests, inter-medication interactions, and
susceptibility to developing cancer. From a treatment perspective, the roles of common
compounds, including dietary components have been investigated as UGT inhibitors with a
view to enhancing bioavailability of drugs. This approach to impairing metabolism and thus
increasing the half-lives of drugs has been the subject of patent protection for a wide range
of drugs (raloxifene, 2-methoxyestradiol, irinotecan, estradiol, labetalol, dilevalol, zidovudine,
and morphine) using numerous inhibitors from plant origin (epicatechin gallate,
epigallocatechin gallate, octyl gallate, propyl gallate, quercetin, tannic acid, benzoin gum,
capsaicin, dihydrocapsaicin, eugenol, gallocatechin gallate, geraniol, menthol, menthyl
acetate, naringenin, allspice berry oil, N-vanillylnonanamide, clovebud oil, peppermint oil,
silibinin, and silymarin) [13210].
Early reports demonstrated that a number of compounds interfere with the activity of
UGT2B17 which is the major isozyme for clearance of anabolic steroids, having greater than
double the activity of the next most active form UGT2A1. It has been reported that
epitestosterone and two non-steroidal anti-inflammatory drugs (NSAID) act as competitive
inhibitors against UGT2B17. Using human microsomes and recombinant enzymes they
demonstrated that diclofenac and ibuprofen inhibited testosterone glucuronidation without
having significant effects on epitestosterone glucuronidation. Similar inhibitory effects on
testosterone glucuronidation were reported for both UGT2B15 and UGT2B17 isozymes in in
vitro studies. The authors measured IC50 values for diclofenac inhibition of testosterone
glucuronidation by UGT2B15 and UGT2B17 of 25 microM and 65 microM respectively, at
testosterone concentrations of 10 μM. The corresponding IC50 values for ibuprofen were 121
μM and 1340 microM against UGT2B15 and UGT2B17 respectively. To date, no
commensurate studies have been reported demonstrating an effect of pharmaceuticals on
testosterone glucuronidation in vivo. A recent report showed only a slight modification but no
significant effects of concomitant use of maximum recommended doses of ibuprofen or
diclofenac with testosterone on the urinary ratios of testosterone/epitestosterone in
individuals with either two, one, or no allele of the UGT2B17, and no effect when
ibuprofen/diclofenac was administered prior to single dose of testosterone. Given the
competitive nature of the inhibition, at least for diclofenac, the experiment was limited by
904
restriction to maximum doses of the NSAID. Thus, doses of 50 mg × 3 per day of the single
competitive inhibitor, although well reasoned, may not elicit an inhibitory effect given that
ibuprofen can also elevate UGT enzyme activity in vivo. Although reports of in vivo studies
are lacking to date, the potential effects of inhibiting major testosterone-metabolizing
enzymes warrants further exploration, especially if common substances are considered
where maximum dosage effects do not limit intake. From one standpoint, this effect could
alter the results of a doping test which is based on the ratio of the glucuronidated
testosterone and epitestosterone. Following these advances, researchers have recently
explored the effects of dietary components on steroid metabolism [13210].
Tea and cacao

It was first reported the effects of dietary green and white teas on the activity of UGT2B17
toward testosterone glucuronidation. Using an high performance liquid chromatography
(HPLC) assay, testosterone glucuronidation was monitored in the presence of tea extracts
using human UGT2B17 supersomes. Under the conditions studied, green and white tea
preparations inhibited the reaction by about 20 percent with a white tea powder inhibiting
glucuronidation by 30 percent. HPLC analysis of the teas revealed key constituents such as
epicatechin (EC) and epigallocatechin gallate (EGCG). Analysis via a Dixon plot revealed
that EGCG was acting as a competitive inhibitor with an IC50 value of 64 microM which
equaled that found previously for diclofenac. At a concentration of 1 mM, EC inhibited
testosterone (at 10 microM) glucuronidation by some 55 percent [13210].
Cacao also inhibits UGT2B17 but to a lesser extent (15 %). Under these conditions, at
testosterone concentrations of 12 microg/mL, white and green tea preparations inhibited over
70 percent of activity with a white tea powder form showing inhibition of some 90 percent.
For the individual phenolics, inhibition was insignificant for gallocatechin and caffeine but
ranged up to 22 and 42 percent for (−)epicatechin and (+)epicatechin respectively. Extensive
inhibition of testosterone glucuronidation was observed for epicatechin gallate (70 %),
epigallocatechin gallate (78 %), and catechin gallate (90 %). Analysis of the tea and cacao
samples by HPLC revealed catechins were present in these samples at lower levels in
comparison to the tea samples. The cacao samples, whilst inhibiting testosterone
glucuronidation, did so at a much lesser rate than the tea samples which could be linked with
having lower levels of inhibiting catechin compounds at the same concentrations of the tea
samples [13210].
Red wine
Red wine and its constituents were shown to inhibit testosterone glucuronidation by human
UGT2B17 supersomes. Under the conditions studied, red wine inhibited glucuronidation by
up to 70 percent over a 2-h period, with little effect arising from the alcohol content. Phenolic
components were selected following HPLC analysis of the selected red wine and quercetin,
caffeic acid, and gallic acid inhibited UGT2B17 testosterone glucuronidation by 72, 22, and 9
percent respectively, with concentrations of phenolic : testosterone of 100 : 250 microM. For
the most active phenolic, reducing the quercetin concentration to 2 microM, maintained
inhibition of 20 percent in spite of the 10-fold excess of testosterone [13201].
Testosterone plus an ornithine decarboxylase inhibitor
Because of its anabolic effects on muscle, testosterone is being explored as a function-

promoting anabolic therapy for functional limitations associated with aging; however,
concerns about testosterone's adverse effects on prostate have inspired efforts to develop
strategies that selectively increase muscle mass while sparing the prostate. Testosterone's
promyogenic effects are mediated through upregulation of follistatin. It was shown that the
905
administration of recombinant follistatin (rFst) increased muscle mass in mice, but had no
effect on prostate mass. Consistent with the results of rFst administration, follistatin
transgenic mice with constitutively elevated follistatin levels displayed greater muscle mass
than controls, but had similar prostate weights. To elucidate signaling pathways regulated
differentially by testosterone and rFst in prostate and muscle, we performed microarray
analysis of mRNAs from prostate and levator ani of castrated male mice treated with vehicle,
testosterone, or rFst. Testosterone and rFst shared the regulation of many transcripts in
levator ani; however, in prostate, 593 transcripts in several growth-promoting pathways were
differentially expressed after testosterone treatment, while rFst showed a negligible effect
with only 9 transcripts differentially expressed. Among pathways that were differentially
responsive to testosterone in prostate, we identified ornithine decarboxylase (Odc1), an
enzyme in polyamine biosynthesis, as a testosterone-responsive gene that is unresponsive
to rFst. Accordingly, we administered testosterone with and without α-difluoromethylornithine
(DFMO), an Odc1 inhibitor, to castrated mice. DFMO selectively blocked testosterone's
effects on prostate, but did not affect testosterone's anabolic effects on muscle. Co-
administration of testosterone and Odc1 inhibitor presents a novel therapeutic strategy for
prostate-sparing anabolic therapy [13211].
Influence of alcohol on steroid metabolism
Besides the (mis)use of natural steroids, the impact of ethanol consumption on steroid
profiles was subjected to further investigations. In a comprehensive study with 21 male and
15 female volunteers, alterations in steroid profile parameters were correlated with urinary
ethanol-glucuronide and ethanol-sulfate concentrations, and threshold values of 48 microg/ml
and 15.5 microg/ml for men and women, respectively, were suggested. When exceeded, an
influence on urinary steroid profiles due to ethanol-induced suppression of steroid
biotransformation processes should be considered during data interpretation [13012].
Salivary hormones
Saliva contains cells and compounds, of local and non-local oral origin, namely inorganic,
organic non-protein, protein/polypeptide, and lipid molecules. Moreover, some hormones,
commonly assayed in plasma, such as steroids, are detectable in oral fluid and
peptide/protein, and non-steroid hormones have been investigated. The sports practice
environment and athletes' availability, together with hormone molecule characteristics in
saliva and physical exercise behavior effects, confirm this body fluid as an alternative to
serum. One review focused on the relation between salivary steroids and psycho-
physiological stress and underlines how the measurement of salivary cortisol provides an
approach of self-report psychological indicator and anxiety change in relation to exercise
performance. The correlation between salivary and plasma steroid hormone (cortisol,
testosterone, and dehydroepiandrosterone (DHEA)) levels, observed during exercise, has
been considered, underlining how the type, duration, and intensity of the exercise influence
the salivary steroid concentrations in the same way as serum-level variations. Training
conditions have been considered in relation to the salivary hormonal response. One review
focuses on studies related to salivary hormone measurements, mainly steroids, in physical
exercise. Saliva use in physical disciplines, as a real alternative to serum, could be a future
perspective [11068]
The combination of resistance and plyometric training, or complex training, may yield greater
functional gains than either method alone. As steroid hormones respond to exercise stimuli
906
and modulate the functional outcomes, it is possible that complex training creates an
enhanced anabolic physiological milieu for adaptation. It was investigated acute responses of
salivary testosterone and cortisol to complex exercise bouts. After a standardized warm-up,
16 semiprofessional rugby players performed 1 of 4 exercise bouts in a cross-over manner:
power-power; power-strength; strength-power; or strength-strength. Each player completed
each of the 4 bouts twice over a 4-week period in a balanced random order such that each
player performed a total of 8 bouts. The power block consisted of 3 sets of 3 repetitions of
jump squat exercise at 50 percent of 1-repetition maximum load. The strength block
consisted of three sets of three repetitions of box squat exercise at a 3-repetition maximum
load. There were 3-minute rest periods between sets and 4-minute rest periods between
exercise blocks. Saliva was sampled before, during, and immediately after the exercise bout.
The greatest overall hormonal responses were a small increase in testosterone (13 %; 90 %
confidence limits ± 7 %) and a trivial increase in cortisol (27 %; ± 30 %) after the strength-
power bout. A clear difference was observed between the strength-power and the power-
power bouts immediately after exercise for testosterone (10 %; ± 8 %) and cortisol (29 %; ±
17 %). The preceding exercise block had little effect on subsequent strength and power
performance. The hormonal response after the strength-power bout suggests that this
exercise sequence provides an enhanced anabolic milieu for adaptation [11069].
The aim of one study was to examine the acute response to plasma and salivary cortisol and
testosterone to three training protocols. Ten trained endurance athletes participated in three
experimental trials, such as interval training (INT), tempo run (TEMP) and bodyweight-only
circuit training (CIR), on separate days. Blood and saliva samples were collected pre- and 0,
15, 30 and 60 min post-exercise. Peak post-exercise salivary cortisol was higher than pre-
exercise in all trials. After INT, salivary cortisol remained elevated above pre-exercise than
60 min post-exercise. Salivary testosterone also increased post-exercise in all trials. Plasma
and salivary cortisol were correlated between individuals and within individuals. Plasma and
salivary testosterone was also correlated between and within individuals. Peak cortisol and
testosterone levels occurred simultaneously in plasma and saliva, but timing of post-exercise
hormone peaks differed between trials and individuals. Further investigation is required to
identify the mechanisms eliciting an increase in hormones in response to CIR. Furthermore,
saliva is a valid alternative sampling technique for measurement of cortisol, although the
complex, individual and situation dependent nature of the hormone response to acute
exercise should be considered [13212].
Salivary testosterone (T) and cortisol (C) concentrations were monitored across a sports
competition. Data were compared using two enzyme-immunoassay (EIA) methods and two
sample preparations to determine their influence on hormone concentrations. A group of
male athletes (n=19) provided a saliva sample the morning before and one day after (24h
post) an international rugby union match. Following an extraction procedure, the samples
were analysed for T and C concentrations using a commercial kit (CM(E)) and an in-house
method (IH(E)). Raw samples (no extraction procedure) were also tested using the
commercial kit (CM(R)). There were no significant changes in T and C levels from pre to post
competition with each EIA method and sample preparation, but significant differences in T
(IH(E)>CM(E)>CM(R)) and C (CM(R)>IH(E) and CM(E)) concentrations were seen when
both samples were pooled. Bland-Altman analyses confirmed the presence of fixed and
proportional bias. Strong and significant correlations were demonstrated between the IH(E)
and CM(E) measures of salivary T and C. The T and C values from the raw and extracted
samples were also strongly correlated. The measurement of salivary T and C concentrations
across an international sports event was influenced by different EIA methods and sample
preparations, but all measures were strongly correlated with some bias. Both T and C were
907
unresponsive to the sports event, but within the group results large individual variation was
seen [13213].
One study examined salivary cortisol and testosterone responses to two, different high-
intensity, about 30-min cycles separated by 2 h rest before and after an 11-day intensified
training period. Twelve recreationally active, healthy males completed the study. Saliva
samples were collected before, immediately after and 30 min after both bouts with salivary
cortisol and testosterone concentrations assessed. Compared with pre-training blunted
exercise-induced salivary cortisol, testosterone and cortisol/testosterone responses to both
bouts post-training were observed. Comparing pre- with post-training the absolute exercise-
induced salivary cortisol, testosterone and cortisol/testosterone decreased from 11.1 to 3.1
and 7.0 to 4.4 nmol/L (cortisol), from 407 to 258 and from 473 to 274 pmol/L (testosterone)
and from 12 to 4 and 7 to 5 (cortisol/testosterone) for the first and second bouts, respectively.
No differences in the pre- and post-training rating of perceived exertion (RPE) and heart rate
(HR) responses during the cycles or times to fatigue were found. Fatigue and Burnout scores
were higher post- compared with pre-training. These high-intensity exercise bouts can detect
altered hormonal responses following intensified training. This test could assess an athlete's
current hormonal status, reductions in salivary cortisol and testosterone responses
suggestive of increased fatigue [13214].
In soccer
One study investigated the contribution of salivary testosterone concentration, years from
peak height velocity (YPHV) and height by body mass interaction on jumping performance
(counter movement jump; CMJ) and aerobic fitness (Yo-Yo intermittent endurance test, level
1) in young elite soccer players. Forty-five participants (age: 13 years; body mass: 49 kg,
height: 156 cm) belonging to a top level Brazilian soccer club were evaluated at four time
points across a single semester. None of the assessed players had reached PHV. The data
from the four evaluations were averaged and multiple linear regression analysis conducted.
For CMJ, the model explained 43 percent of the variance; salivary testosterone concentration
was the primary contributor and the YPHV contributed 10 percent of the variance. The model
explained 29 percent of the variance in Yo-Yo. The salivary testosterone was the primary
and single significant contributor. A significant difference was noted between high and low
testosterone groups divided a posteriori to CMJ performance. These results suggest an
important role for hormonal status in interpreting physical performance in preadolescent
soccer players [13215].
Effects on cognitive functions
The illicit use of anabolic androgenic steroids (AAS) has gained popularity among
adolescents in the last decade. However, although it is known that exposure to AAS impairs
cognition in adult animal models, the cognitive effects during adolescence remain
undetermined. An inhibitory avoidance task (IAT) was used to assess the effect of AAS
(17alpha-methyltestosterone; 17alpha-meT, 7.5 mg/kg) in male and female periadolescent
rats. A single injection of 17α-meT immediately before the footshock produced significant
impairment of inhibitory avoidance learning in males but not females. Generalized anxiety,
locomotion, and risk assessment behaviors (RAB) were not affected. The results show that
exposure to a single pharmacological dose of 17alpha-meT during periadolescence exerts
sex-specific cognitive effects without affecting anxiety. Thus, disruption of the hormonal
milieu during this early developmental period might have negative impact on learning and
memory [13114].
908
Effects of training
Performing strength exercise, whether acutely or in a training programme, leads to

alterations at the hypothalamic-pituitary-testicular and hypothalamic-pituitary-adrenal axes.
One way to evaluate these changes is by analysis of the excretion of steroid hormones in the
urine. One study determined the variations in the urine profile of glucuroconjugated steroids
after a single session of strength exercise and after a 4-week programme of strength training.
The subjects were a group (n=20) of non-sportsman male university students who worked
out 3 days a week, performing the exercises at 70-75 percent of one repetition maximum
strength (1-RM). Four urine samples were collected per subject: (A) before and (B) after a
standard session prior to initiating the training programme, and (C) before and (D) after the
same standard session at the end of the study, and they were assayed by gas
chromatography coupled to mass spectrometry. The concentrations of the different
hormones were determined relatively to the urine creatinine level (ng steroid/mg creatinine)
to correct for diuresis. After the exercise sessions, both before and after the training
programme, there was a fall in the urine excretion of androgens and estrogens, but no
statistically significant changes in the excretion of tetrahydrocortisol (THF) and
tetrahydrocortisone (THE). The anabolic/catabolic hormones ratio also decreased after the
acute session, although only androstenodione + dehydroepiandrosterone (DHEA)/THE +
THF ratio had a significant decrease. After the training programme, there was a significant
improvement in the strength of the muscle groups studied, and an increased urinary
excretion of all the androgens with respect to the initial state of repose, with the difference
being significant in the case of epitestosterone. The androsterone (A) + etiocholanolone
(E)/THE + THF ratio increased significantly concerning the initial state. It was therefore
concluded that subjects suffer variations of the urine profile with regard to the steroid
hormones before and after the acute strength sessions and after the training period. The
alteration after the training programme seems to be due to the subjects' hypothalamic-
hypophysis-testicular and hypothalamic-pituitary-adrenal axes adaptations, which enable
them to increase physical strength [07060].
The purpose of one study was to examine the effects of an 8-week basic training with added
strength training or endurance training on both the performance of a 3K-combat loaded run
test and the acute neuromuscular and hormonal responses. All training groups significantly
improved their run-test times: strength training by 12 percent, endurance training by 12
percent, and normal training by 10 percent. Significant acute decreases were observed in
maximal isometric force of leg extensors in all subject groups following the run. Increases
were observed in acute testosterone responses after the test in all groups both at pre- and
post-training. However, endurance training and normal training demonstrated significantly
lower acute post-training serum cortisol responses than strength training. In conclusion, the
present results indicate that within a demanding basic training, the added training for
endurance training and especially strength training may be compromised in their adaptation
potential due to interference from the demands of basic training [10044].
Age-related skeletal muscle loss is thought to stem from suboptimal nutrition and resistance
to anabolic stimuli. Impaired microcirculatory (nutritive) blood flow may contribute to anabolic
resistance by reducing delivery of amino acids to skeletal muscle. In one study, it was
employed contrast-enhanced ultrasound, microdialysis sampling of skeletal muscle
interstitium, and stable isotope methodology, to assess hemodynamic and metabolic
responses of older individuals to endurance type (walking) exercise during controlled amino
acid provision. It was hypothesized that older individuals would exhibit reduced
microcirculatory blood flow, interstitial amino acid concentrations, and amino acid transport
909
when compared with younger controls. It was reported that aging induces anabolic
resistance following endurance exercise, manifested as reduced (by 40 %) efficiency of
muscle protein synthesis. Despite lower (by 40-45 %) microcirculatory flow in the older than
in the younger participants, circulating and interstitial amino acid concentrations and
phenylalanine transport into skeletal muscle were all equal or higher in older individuals than
in the young, comprehensively refuting our hypothesis that amino acid availability limits
postexercise anabolism in older individuals. The data point to alternative mediators of age-
related anabolic resistance and importantly suggest correction of these impairments may
reduce requirements for, and increase the efficacy of, dietary protein in older individuals
[10447].
The objectives of one investigation were to study the inflammatory and performance
responses after an acute bout of intense plyometric exercise during a prolonged recovery
period. Participants were randomly assigned to either an experimental group (P, n=12) that
performed intense plyometric exercises or a control group (C, n=12) that rested. The delayed
onset of muscle soreness (DOMS), knee range of motion (KROM), creatine kinase (CK) and
lactate dehydrogenase (LDH) activities, white blood cell count, C reactive protein (CRP), uric
acid (UA), cortisol, testosterone, IL-6, IL-1b strength (isometric and isokinetic), and counter-
movement (CMJ) and static (SJ) jumping performance were measured at rest, immediately
postexercise and at 24, 48, 72, 96, and 120 hours of recovery. Lactate was measured at rest
and postexercise. Strength remained unchanged throughout recovery, but CMJ and SJ
declined significantly by 8-20 percent. The experimental group induced a marked rise in
DOMS, CK, and LDH (peaked 24-48 hours postexercise) and a KROM decline. An acute-
phase inflammatory response consisting of leukocytosis (postexercise and at 24 hours), an
IL-6, IL-1b, CRP, and cortisol elevation (during the first 24 hours of recovery) and a delayed
increase of UA (peaked at 48 hours) and testosterone (peaked at 72 hours) was observed in
P. The results of this investigation indicate that performing an acute bout of intense
plyometric exercise may induce a short-term muscle damage and marked but transient
inflammatory responses. Jumping performance seems to deteriorate for as long as 72 hours
postexercise, whereas strength appears to remain unchanged. The acute-phase
inflammatory response after a plyometric exercise protocol appears to follow the same
pattern as in other exercise models. These results clearly indicate the need of sufficient
recovery between successive plyometric exercise training sessions [10045].
One study aimed to develop a quantitative and in vivo magnetic resonance imaging (MRI)
approach to investigate the muscle growth effects of anabolic steroids. A protocol of MRI
acquisition on a standard clinical 1.5 T scanner and quantitative image analysis was
established and employed to measure the individual muscle and organ volumes in the intact
and castrated guinea pigs undergoing a 16-week treatment protocol by two well-documented
anabolic steroids, testosterone and nandrolone, via implanted silastic capsules. High
correlations between the in vivo MRI and postmortem dissection measurements were
observed for shoulder muscle complex, masseter, temporalis, neck muscle complex,
prostate gland and seminal vesicles, and testis. Furthermore, the longitudinal MRI
measurements yielded adequate sensitivity to detect the restoration of growth to or towards
normal in castrated guinea pigs by replacing circulating steroid levels to physiological or
slightly higher levels, as expected. These results demonstrated that quantitative MRI using a
standard clinical scanner provides accurate and sensitive measurement of individual muscles
and organs, and this in vivo MRI protocol in conjunction with the castrated guinea pig model
constitutes an effective platform to investigate the longitudinal and cross-sectional growth
effects of other potential anabolic steroids. The quantitative MRI protocol developed can also
be readily adapted for human studies on most clinical MRI scanner to investigate the
anabolic steroid growth effects, or monitor the changes in individual muscle and organ
volume and geometry following injury, strength training, neuromuscular disorders, and
910
pharmacological or surgical interventions [08151].
The purpose of one study was to examine the changes in neuromuscular, perceptual and
hormonal measures following professional rugby league matches during different length
between-match microcycles. Twelve professional rugby league players from the same team
were assessed for changes in countermovement jump (CMJ) performance (flight time and
relative power), perceptual responses (fatigue, well-being and muscle soreness) and salivary
hormone (testosterone and cortisol) levels during 5, 7 and 9 d between-match training
microcycles. All training was prescribed by the club coaches and was monitored using the
session-RPE method. Lower mean daily training load was completed on the 5 d compared
with the 7 and 9 d microcycles. CMJ flight time and relative power, perception of fatigue,
overall well-being and muscle soreness were significantly reduced in the 48 h following the
match in each microcycle. Most CMJ variables returned to near baseline values following 4 d
in each microcycle. Countermovement jump relative power was lower in the 7 d microcycle in
comparison with the 9 d microcycle. There was significantly increased fatigue at 48 h in the 7
and 9 d microcycles but had returned to baseline in the 5 d microcycle. Salivary testosterone
and cortisol did not change in response to the match. Neuromuscular performance and
perception of fatigue are reduced for at least 48 h following a rugby league match but can be
recovered to baseline levels within 4 d. These findings show that with appropriate training, it
is possible to recover neuromuscular and perceptual measures within 4 d after a rugby
league match [10330].
The combination of resistance and plyometric training, or complex training, may yield greater
functional gains than either method alone. As steroid hormones respond to exercise stimuli
and modulate the functional outcomes, it is possible that complex training creates an
enhanced anabolic physiological milieu for adaptation. It was investigated acute responses of
salivary testosterone and cortisol to complex exercise bouts. After a standardized warm-up,
16 semiprofessional rugby players performed 1 of 4 exercise bouts in a cross-over manner:
power-power; power-strength; strength-power; or strength-strength. Each player completed
each of the 4 bouts twice over a 4-week period in a balanced random order such that each
player performed a total of 8 bouts. The power block consisted of 3 sets of 3 repetitions of
jump squat exercise at 50 percent of 1-repetition maximum load. The strength block
consisted of three sets of three repetitions of box squat exercise at a 3-repetition maximum
load. There were 3-minute rest periods between sets and 4-minute rest periods between
exercise blocks. Saliva was sampled before, during, and immediately after the exercise bout.
The greatest overall hormonal responses were a small increase in testosterone (13 %; 90 %
confidence limits + 7 %) and a trivial increase in cortisol (27 %; + 30 %) after the strength-
power bout. A clear difference was observed between the strength-power and the power-
power bouts immediately after exercise for testosterone (10 %; + 8%) and cortisol (29 %; +/-
17%). The preceding exercise block had little effect on subsequent strength and power
performance. The hormonal response after the strength-power bout suggests that this
exercise sequence provides an enhanced anabolic milieu for adaptation [10331].
Appropriate physical activity is one of the bases of healthy lifestyle. In fact, physical exercise
and playing sport may be associated with both improvements and injury to both general and
reproductive health. A biologically normal testosterone secretion appears fundamental in
males to guarantee both a physiological exercise adaptation and safe sport participation. The
reproductive system is highly sensitive to the effects of exercise-related stress and the
reproductive hormones may both increase and decrease after different acute or chronic
exercises. Exercise and sport participation may positively or negatively influence andrological
health status depending on the type, intensity and duration of performed physical activity and
on individual health status. In addition, prohibited substances administration (e.g.
androgenic-anabolic steroids, and so forth) in competitive and non-competitive athletes
911
represents the main cause of iatrogenic andrological diseases. Preventing and treating
andrological problems in active healthy and unhealthy individuals is as important as
promoting a correct lifestyle. Physicians need to be educated on the relationships between
the male reproductive system and sport participation and on the great role of the pre-
participation physical examination in the prevention of andrological diseases [12121].
Effects according to MRI techniques
MRI and image quantitation play an expanding role in modern drug research, because MRI
offers high resolution and non-invasive ability, and provides excellent soft tissue contrast.
Moreover, with development of effective image segmentation and analysis methods, in-vivo
and serial tissue growth measurements could be assessed. In the study, MR image
acquisition and analysis protocol were established and validated for investigating the effects
of anabolic steroids and precursors on muscle growth and body composition in a guinea pig
model. Semi-automatic and interactive segmentation methods were developed to accurately
label the tissue of interest for tissue volume estimation. In addition, a longitudinal tissue area
outlining procedure was proposed for study of tissue geometric features in relation to tissue
growth. Finally, a fully automatic data retrieval and analysis scheme was implemented to
facilitate the overall huge amount of image quantitation, statistical analysis, as well as study
group comparisons. As a result, highly significant differences in muscle and organ growth
were detected between intact and castrated guinea pigs using the selected anabolic steroids,
indicating the viability of employing such protocol to assess other anabolic steroids.
Furthermore, the anabolic potential of selected steroid precursors and their effects on muscle
growth, in comparison with that in respective positive control groups of castrated guinea pigs,
were evaluated with the proposed protocol [05062].
Morphology versus performance after use of anabolic steroids
It was hypothesized that treatment with testosterone (T) and recombinant human growth
hormone (rhGH) would increase lean mass (LM) and muscle strength proportionally and an
in a linear manner over 16 weeks. This was a multicenter, randomized, controlled, double-
masked investigation of T and rhGH supplementation in older (71 ± 4 years) community-
dwelling men. Participants received transdermal T at either 5 or 10 g/day as well as rhGH at
0, 3.0 or 5.0 μg/kg/day for 16 weeks. Body composition was determined by dual-energy X-
ray absorptiometry (DEXA) and muscle performance by composite one-repetition maximum
(1-RM) strength and strength per unit of lean mass (muscle quality, MQ) for five major
muscle groups (upper and lower body) at baseline, week 8 and 17. The average change in
total LM at study week 8 compared with baseline was 1.50 ± 1.54 kg in the T only group and
2.64 ± 1.7 in the T + rhGH group and at week 17 was 1 in the T only group and 2.14 ± 1.96
kg in the T + rhGH group. 1-RM strength improved modestly in both groups combined (12.0 ±
23.9 %) at week 8 but at week 17 these changes were twofold greater. MQ did not
significantly change from baseline to week 8 but increased for the entire cohort, T only, and T
+ rhGH groups by week 17. Despite sizeable increases in LM measurements at week 8, tests
of muscle performance did not show substantive improvements at this time point [12195].
Effect of training status and exercise mode on endogenous steroid hormones in men
From the perspective of an athlete, an increase in anabolic-androgenic hormones can

improve performance by decreasing body fat and increasing lean body mass and muscular
strength. During aging, there is a decline in anabolic-androgenic hormones, namely
testosterone and dehydroepiandrosterone sulfate (DHEAS), that may have significant
negative effects on body composition, physical function, and libido in men. Increased
912
anabolic hormone concentrations could slow the aging process and maintain quality of life in
older individuals. Certain age-related disease states that are catabolic in nature, such as
osteoporosis or sarcopenia, could be attenuated by an increase in anabolic hormone
concentrations. The purpose of one study was to determine the acute anabolic and catabolic
hormone response to endurance and resistance exercise bouts of equal volume in subjects
with differing training status. Twenty-two healthy men were recruited who were either
resistance trained (n=7), endurance trained (n=8), or sedentary (n=7). Three sessions were
completed: a resting session, a 40-min run at 50-55 percent maximal oxygen consumption,
and a resistance exercise session. Expired gases were monitored continuously during
exercise, and the endurance and resistance exercise sessions were individually matched for
caloric expenditure. Blood samples were drawn before exercise and 1, 2, 3, and 4 h after the
start of the exercise. Plasma was analyzed for luteinizing hormone, dehydroepiandrosterone
sulfate, cortisol, and free and total testosterone. Androgens increased in response to
exercise, particularly resistance exercise, whereas cortisol only increased after resistance
exercise. Dehydroepiandrosterone sulfate levels increased during the resistance exercise
session and remained elevated during recovery in the resistance-trained subjects.
Endurance-trained subjects displayed less pronounced changes in hormone concentrations
in response to exercise than resistance-trained subjects. After an initial postexercise
increase, there was a significant decline in free and total testosterone during recovery from
resistance exercise, particularly in resistance-trained subjects. On the basis of the results of
this study, it appears that the endogenous hormone profile of men is more dependent on
exercise mode or intensity than exercise volume as measured by caloric expenditure. The
relatively catabolic environment observed during the resistance session may indicate an
intensity-rather than a mode-dependent response [03039].
Sedentary, resistance-trained, and endurance-trained subjects were used in this study to

identify differences in testosterone, LH, DHEAS, and cortisol, as well as the ratios of
testosterone and DHEAS to cortisol. These hormones were evaluated both at rest and during
recovery from a 40-min run and a resistance exercise session. Resistance-trained subjects
tended to have higher androgen levels and slightly higher LH levels. Androgens increased in
response to exercise, particularly resistance exercise, whereas cortisol only increased after
resistance exercise. DHEAS levels increased during the resistance exercise session and
remained elevated during recovery in the resistance-trained subjects. LH showed a delayed
increase during recovery from the run in resistance trained subjects. Endurance-trained
subjects displayed less pronounced changes in hormone concentrations in response to
exercise. There was a significant decline in free and total testosterone during recovery from
resistance exercise, and the ratios of anabolic hormones (free and total testosterone and
DHEAS) to cortisol were lower during resistance exercise, which, paradoxically, suggests a
less anabolic environment [03039].
Endurance-trained men tend to have lower levels of testosterone compared with sedentary
controls, whereas resistance-trained subjects have been shown to have higher basal
testosterone levels. Cross-sectional work found that both endurance- and resistance-trained
subjects had lower testosterone levels than sedentary control subjects. The main signaling
peptide of testosterone, luteinizing hormone (LH), may also be altered by training, with
significantly elevated levels reported in endurance-trained men. Other research has focused
on the ratio of testosterone to cortisol, which has been reported to both increase and
decrease during resistance training. Testosterone concentrations have been shown to
increase after an acute bout of resistance or endurance exercise. In response to prolonged
endurance exercise (e.g. a marathon), testosterone levels will typically decline. Others have
reported no change in testosterone after resistance exercise. There has been considerably
less research looking at adrenal androgens; however, DHEAS has been shown to increase
in response to endurance exercise. In contrast, it was found no change in
913
dehydroepiandrosterone after resistance exercise in men. In many cases, conflicting results
can be attributed to differences in the mode or volume of exercise or in the training status of
the subjects. There are few studies that have looked at the interaction between training
status and mode of exercise in determining the hormonal response to exercise. Previous
work comparing hormone responses between endurance and resistance exercise in men
and women used exercise bouts of equal duration, which does not necessarily reflect equal
energy expenditure or work output, and the total volume of exercise may contribute to
differences observed in the hormone response to various exercise protocols. It was also
compared the hormone response between bouts of continuous and interval cycling exercise
of equal work outputs, but they used only endurance-trained subjects. It is known that
training status can influence the hormone response to exercise, but it is not clear whether the
mode of training can affect the hormone response to different modes of exercise. This
information could be useful in designing training programs that will result in the most
favorable ratio of anabolic and catabolic hormones. Therefore, the objective of this study was
to determine the acute steroid hormone response to endurance and resistance exercise
bouts of equitable volume in subjects with differing training status. It was hypothesized that
androgens would increase with exercise and that resistance exercise would result in a more
anabolic hormonal environment than endurance exercise. It was also expected that
sedentary subjects would have a greater hormone response than either resistance- or
endurance-trained subjects in response to both modes of exercise. On the basis of the
results of this study, it appears that the circulating endogenous hormone profile is more
dependent on exercise mode or intensity than exercise volume as measured by caloric
expenditure. The relatively catabolic environment observed during the resistance session
may indicate an intensity rather than mode dependent response. This study also provides
evidence that hormone levels and exercise-induced hormone changes are different in
subjects of different training status. Endurance-trained subjects displayed a less pronounced
hormone response to exercise, whereas resistance-trained subjects demonstrated a more
pronounced hormone response to exercise. These differences may be related to specific
exercise intensity characteristics or training adaptations. Of course, it is not known whether
training per se alters hormone profiles or whether athletes are self-selected into certain
activities because their physical or physiological characteristics predispose them to success
[03039].
Effects on training in males
The acute response of free salivary testosterone and cortisol concentrations to four
resistance exercise (RE) protocols in 23 elite men rugby players was investigated. It was
hypothesized that hormonal responses would differ among individuals after four distinct RE
protocols: four sets of 10 repetitions (reps) at 70 percent of 1 repetition maximum (1RM) with
2 minutes' rest between sets (4 x 10-70 %); three sets of five reps at 85 percent 1RM with 3
minutes' rest (3 x 5-85 %); five sets of 15 reps at 55 percent 1RM with 1 minute's rest (5 x
15-55 %); and three sets of five reps at 40 percent 1RM with 3 minutes' rest (3 x 5-40 %).
Each athlete completed each of the four resistance exercise protocols in a random order on
separate days. Testosterone and cortisol concentrations were measured before exercise,
immediately after exercise, and 30 minutes post exercise. Each protocol consisted of four
exercises: bench press, leg press, seated row, and squats. Pooled testosterone data did not
change as a result of resistance exercise, whereas cortisol declined significantly. Individual
athletes differed in their testosterone response to each of the protocols, a difference that was
masked when examining the pooled group data. When individual data were retrospectively
tabulated according to the protocol in which each athlete showed the highest testosterone
response, a significant protocol-dependent testosterone increase for all individuals was
revealed. Therefore, resistance exercise induced significant individual, protocol-dependent
hormonal changes lasting up to 30 minutes after exercise. These individual responses may
914
have important ramifications for modulating adaptation to resistance exercise and could
explain the variability often observed in studies of hormonal response to resistance exercise
[08152].
In aging men
Worldwide many aging males practice sports. A high prevalence of late-onset male
hypogonadism has been observed in general population. Sport-participation influences the
neuroendocrine system and may decrease serum testosterone. One preliminary study was
designed to estimate the prevalence and the symptoms of undiagnosed testosterone
deficiency in aging athletes. This observational survey was performed in 183 caucasian male
athletes >50 years, in the setting of pre-participation screening. Pituitary-gonadal hormones
and symptoms of hypogonadism were investigated. Serum total testosterone (TT), sex
hormone binding globulin, luteinizing hormone (LH), follicle stimulating hormone (FSH),
prolactin (PRL), free-T4, and thyroid stimulation hormone (TSH) were assayed, and free
testosterone, bioactive testosterone, and the LH/TT ratio were calculated. The International
Index of Erectile Dysfunction (IIEF-15) and the Center for Epidemiological Studies
Depression Scale (CES-D) were administered. Hypogonadal athletes were compared with
eugonadal athletes as controls. Prevalence and clinical symptoms of severe (TT < 8 nmol/L)
or mild (TT between 8 and 12 nmol/L) testosterone deficiency were investigated. The mean
sample age was 62 + 8 years (range 50-75). Severe or mild testosterone deficiency was
observed in 12 percent and 18 percent respectively, of overall athletes, with the highest
prevalence in athletes >70 years (28 % and 25 %, respectively). Serum total testosterone did
not correlate with age, training duration, or questionnaire scores. No differences were
observed for nonspecific symptoms of hypogonadism, IIEF-15 and CES-D scores between
eugonadal and severe hypogonadal athletes. It was concluded that independently of its
etiology, a significant percentage of aging athletes had undiagnosed testosterone deficiency.
In a relevant number of these cases, testosterone deficiency was not overtly symptomatic.
Our results suggest that sport-participation per se can influence the symptoms of
hypogonadism [10334].
Effects on training in females
The purpose of one investigation was to study the effects of an 11-week training period
performed by female weightlifters. Two weeks before this investigation, baseline measures
for total testosterone, cortisol, and testosterone:cortisol ratio were collected. The 11-week
training program consisted of the core exercises (i.e. clean, clean and jerk, and snatch) and
other supplemental exercises (i.e. clean pull, snatch pull, squat, and front squat). Hormonal,
isometric, and dynamic middle thigh pull force-time curve characteristics were assessed
biweekly throughout the duration of the investigation, whereas volume load and training
intensity were assessed weekly throughout the investigation. The testosterone:cortisol ratio
of the baseline was significantly different from the ratio of weeks 1 and 9. When the week-to-
week values were compared, week 1 was significantly different from week 3. A very strong
correlation was found between the percentage change of the testosterone:cortisol ratio and
volume load from weeks 1 to 11. Moderate to very strong correlations were noted between
the percentage change in volume load and isometric peak force, peak force during the 30
percent isometric peak force trial, and peak force during the 100-kg trial during the 11 weeks
of training. The primary finding of this study was that alterations in training volume load can
result in concomitant changes in the anabolic-to-catabolic balance, as indicated by the
testosterone:cortisol ratio, and the ability to generate maximal forces [08153].
Although androgens are produced in small amounts in women, androgens have direct and
significant effects on many aspects of female physiology. Moreover, androgens are
915
precursors to estrogens, which are the predominant female sex hormones. The
measurement of androgens in blood is important in the diagnosis of both gonadal and
adrenal functional disturbances, as well as monitoring subsequent treatments. The accuracy
of such measurements is crucial in sports medicine and doping control. Therefore, the
concentration of androgens in female subjects is frequently measured. Analysing such
compounds with accuracy is especially difficult, costly and time consuming. Therefore,
laboratories widely use direct radioimmunoassay kits, which are often insensitive and
inaccurate. It is especially complicated to determine the level of androgens in women, as the
concentration is much lower compared to the concentration found in males. Additionally, the
amount of androgens in fluids tends to decrease with aging. Analyses of hormone
concentrations are influenced by a myriad of factors. The factors influencing the outcome of
these tests can be divided into in vivo preanalytical factors (e.g, aging, chronobiological
rhythms, diet, menstrual cycle, physical exercise, etc.), in vitro preanalytical factors (e.g.
specimen collection, equipment, transport, storage, etc.) and as mentioned before, analytical
factors. To improve the value of these tests, the strongly influencing factors must be
controlled. This can be accomplished using standardised assays and specimen collection
procedures. In general, sufficient attention is not given to the preanalytical (biological)
factors, especially in the measurement of androgens in females. Biological factors (non-
pathological factors) that may influence the outcome of these tests in female subjects have
so far received little attention [08154].
The aim of one study was to examine the effect of supra-maximal exercise on circulating
concentrations of salivary testosterone, salivary cortisol, and salivary immunoglobulin A in
female adolescents. Nineteen apparently healthy females aged 15-16 years participated in
this study. All participants completed 6 × 8 s sprints, interspersed with 30 s recovery intervals
on a cycle ergometer. Salivary testosterone, cortisol, and immunoglobulin A samples were
taken before and 5 min after exercise. Experimental procedures continued over two
mornings, at least 3 h after a light breakfast. Participants refrained from performing any
strenuous physical activity for at least 24 h prior to the exercise test. None of the participants
were engaged in a structured training programme. The group mean for peak power output
was 562 ± 113 W. Female adolescents recruited for this study showed no changes in
salivary testosterone, cortisol or immunoglobulin A following repeated bouts of supra-
maximal cycling. To date, there has been a paucity of information concerning adolescents'
hormonal and mucosal immune function responses to supra-maximal exercise. The data
provide further guidance with regard to physical activities and sports prescription for female
adolescents. Further research, on a larger sample of females, is required to elucidate the
physiological significance of these findings [10335].
In virtually all sports, participants "warm-up" prior to formal competition. Women athletes
from a highly ranked varsity college volleyball team and, in a second study, a highly ranked
varsity college tennis team gave saliva samples before warm-up, at mid-warm-up (volleyball)
or after warm-up (tennis), and immediately after intercollegiate competition. For volleyball
and tennis, warm-up was associated with a substantial elevation in saliva levels of
testosterone which was carried over through the period of actual competition. Cortisol levels
were relatively unchanged during warm-up, but typically rose during competition. Thus, as
women prepare for athletic competition by warming up, testosterone levels rise in apparent
anticipation of the coming contest and then remain high through the period of play. In
volleyball and tennis, after-practice testosterone level was significantly higher than before-
practice level, and practice session increases in testosterone (but not cortisol) were positively
correlated with increases in testosterone during intercollegiate competition. When practice
and competitive play share as yet undetermined key elements, individual differences in this
endocrine response to "competition" appear stable across practice and intercollegiate
competition [10336].
916
Effects of resistance exercise
Even though the side effects of AAS intake have been widely studied, yet little is known
about how resistance exercise can alter these side effects. This study aimed to determine
the effects of one session resistance exercise and the use of AAS on hemodynamic
characteristics and muscle damage markers in professional bodybuilders. Sixteen
bodybuilders were divided into two groups: bodybuilders using AAS for at least 5 years
(users; n=8) and AAS-free bodybuilders (non-users; n=8). The exercise protocol was a circuit
strength training session involved three sets of 8-9 repetitions at 80-85 percent of 1-RM.
Heart rate (HR), blood pressure (BP) and concentrations of serum creatine kinase (CK) and
lactate dehydrogenase (LDH) were measured at three different time points, immediately
before and after the exercise session and 24-h following the exercise session. The users
group showed significantly greater basal levels of hemodynamic characteristics (i.e. HR and
BP) and cell damage markers (i.e. CK and LDH) compared to those in the non-users group.
Furthermore, the exercise session significantly increased the levels of HR and CK in the
users group compared to those in the non-users group immediately after the exercise. No
significant differences were observed in BP and LDH responses to exercise between the
users and the non-users groups. These findings indicate that the use of AAS could be
potentially harmful as it enhances the levels of the hemodynamic characteristics and the
muscle enzymes. These harmful effects of AAS intake could be more evident in response to
resistance exercise [150153].
Intramuscular anabolic signaling following resistance exercise

Maintaining skeletal muscle mass and function is critical for disease prevention, mobility and
quality of life, and whole-body metabolism. Resistance exercise is known to be a major
regulator for promoting muscle protein synthesis and muscle mass accretion. Manipulation of
exercise intensity, volume, and rest elicit specific muscular adaptations that can maximize
the magnitude of muscle growth. The stimulus of muscle contraction that occurs during
differing intensities of resistance exercise results in varying biochemical responses regulating
the rate of protein synthesis, known as mechanotransduction. At the cellular level, skeletal
muscle adaptation appears to be the result of the cumulative effects of transient changes in
gene expression following acute bouts of exercise. Thus, maximizing the resistance
exercise-induced anabolic response produces the greatest potential for hypertrophic
adaptation with training. The mechanisms involved in converting mechanical signals into the
molecular events that control muscle growth are not completely understood; however,
skeletal muscle protein synthesis appears to be regulated by the multi-protein
phosphorylation cascade, mTORC1 (mammalian/mechanistic target of rapamycin
complex 1). The purpose of this review is to examine the physiological response to
resistance exercise, with particular emphasis on the endocrine response and intramuscular
anabolic signaling through mTORC1. It appears that resistance exercise protocols that
maximize muscle fiber recruitment, time-under-tension, and metabolic stress will contribute
to maximizing intramuscular anabolic signaling; however, the resistance exercise parameters
for maximizing the anabolic response remain unclear [150154].
Effects on stress
After dominance-related encounters, testosterone levels increase in winners and decrease in

losers. In humans, many exceptions have been described. It is possible that the complicated
patterns in humans result from the methods limitations – measurement of hormone
concentrations in simulated competitive events or sport instead in real-life situations. It was
studied changes in hormonal levels and self-estimated attractivity in real situations, namely in
917
students after written exams. It was observed that the testosterone and cortisol increased or
decreased in relation to the number of wrong answers on the exam. The number of wrong
answers was a better predictor of the hormonal changes (increase of both testosterone and
cortisol in successful, decrease in unsuccessful students) than the self-estimated number of
wrong answers or a subjectively opinionated impression from the exam. On the contrary, the
concentration of hormones before the exam and self-estimated attractivity were better
predictors of the subjective impression from the exam than the number of wrong answers.
The results suggest that the students' subconsciousness, which directly influences the
concentration of hormones, is able to objectively estimate results of an exam better than their
consciousness [10337].
Effect of anabolic steroids on endurance capacity
The majority of studies demonstrated that AAS were not able to increase endurance
performance in athletes. However, in two studies, an improvement of aerobic capacity was
noticed. Remarkably, the volunteers in both studies were strength athletes who did not train
for endurance performance [04002].
The administration of anabolic androgenic steroids (AAS) increases skeletal muscle mass
(hypertrophy) and protein synthesis, and these responses are enhanced when AAS is given
in combination with resistance exercise. Skeletal muscle fibers are multinucleated, and
hypertrophy is accompanied by an increase in the number of myonuclei, thereby maintaining
a relatively constant myonuclear domain, i.e. myonuclei per cytoplasmic volume. A primary
source of new myonuclei appears to be from the activation, proliferation, and incorporation of
satellite cells, in that inactivation of satellite cells via irradiation prevents hypertrophy in
functionally overloaded muscles. However, there are no reports examining the relationship
between protein synthesis and mitotic activity in extensor and flexor muscles after AAS
treatment with and without exercise. In addition, a “membrane stabilizing effect” of AAS
agents that diminishes the rise in serum creatine kinase (CK) efflux after muscle damage has
been suggested. Resistance exercise, such as weight lifting, appropriately induces muscle
hypertrophy and is commonly associated with muscle damage and increased levels of serum
CK in humans. It was reported a morphological and biochemical myogenic response
associated with muscle damage and regeneration in the plantarflexor muscles after a single
exhaustive session of weight lifting in previously nontrained adult rats. The severity of weight
lifting-induced muscle damage was associated with the level of increase in serum CK activity
after the exercise bout. In addition, it was that found [3H]thymidine and [14C]leucine labeling
in vivo to be useful methods to detect the mitotic activity of proliferating cells and amino acid
uptake in the muscles after the exercise session. For example, after activation of satellite
cells, other stem cells, and/or fibroblasts, thymidine uptake in the nuclei of these cells is
essential for DNA duplication and cell proliferation, and amino acid uptake is necessary for
the differentiation of these cells. Similarly, elevations in amino acid uptake and protein
synthesis are necessary for increasing the cytoplasm in hypertrophying muscle fibers. There
also is evidence that AAS treatment may directly improve the endurance capacity of skeletal
muscles. For example, improved submaximal running capacity of rats and an improved
fatigue resistance of rat skeletal muscles tested via electrical stimulation have been reported
after AAS treatment. The influence of an anabolic androgenic steroid (AAS) on thymidine and
amino acid uptake in rat hindlimb skeletal muscles during 14 days after a single exhaustive
bout of weight lifting therefore was determined. Adult male rats were divided randomly into
Control or Steroid groups. Nandrolone decanoate was administered to the Steroid group 1
week before the exercise bout. [3H]thymidine and [14C]leucine labeling were used to
determine the serial changes in cellular mitotic activity, amino acid uptake, and myosin
synthesis. Serum creatine kinase (CK) activity, used as a measure of muscle damage,
918
increased 30 and 60 min after exercise in both groups. The total amount of weight lifted was
higher, whereas CK levels were lower in Steroid than in control rats. [3H]thymidine uptake
peaked 2 days after exercise in both groups and was 90 percent higher in Control than in
Steroid rats, reflecting a higher level of muscle damage. [ 14C]leucine uptake was
approximately 80 percent higher at rest and recovered 33 percent faster postexercise in
Steroid than in Control rats. In a separate group of rats, the in situ isometric mechanical
properties of the plantaris muscle were determined. The only significant difference was a
higher fatigue resistance in the Steroid compared with the Control group. Combined, these
results indicate that AAS treatment ameliorates CK efflux and the uptake of [ 3H]thymidine
and enhances the rate of protein synthesis during recovery after a bout of weight lifting, all
being consistent with there being less muscle damage, and enhances in vivo work capacity
and the in situ fatigue resistance of a primary plantarflexor muscle [01036].
Effect of anabolic steroids on muscle growth
In spite of the widespread abuse of androgenic steroids by athletes and recreational body-
builders, the effects of these agents on athletic performance and physical function remain
poorly understood. Experimentally induced androgen deficiency is associated with a loss of
fat-free mass; conversely, physiologic testosterone replacement of healthy, androgen-
deficient men increases fat-free mass and muscle protein synthesis. Testosterone
supplementation of HIV-infected men with low testosterone levels and of older men with
normally low testosterone concentrations also increases muscle mass. However, we do not
know whether physiologic testosterone replacement can improve physical function and
health-related quality of life, and reduce the risk of falls and disability in older men or those
with chronic illness. Testosterone increases maximal voluntary strength in a dose-dependent
manner and thus might improve performance in power-lifting events. However, testosterone
has not been shown to improve performance in endurance events. The mechanisms by
which testosterone increases muscle mass are not known, but probably involve alterations in
the expression of multiple muscle growth regulators [01034].
The abuse of androgenic steroids by athletes and the proposed anabolic applications of
these agents in sarcopenia (loss of muscle mass and strength) associated with aging or
chronic illness is based on the premise that these agents increase muscle mass and improve
measures of skeletal muscle performance, and that androgen-induced changes in skeletal
muscle performance translate into improvements in athletic performance and health-related
outcomes. The premise remains unsubstantiated. There is agreement that testosterone
supplementation increases muscle mass and maximal voluntary strength in a variety of
clinical and experimental paradigms, but we do not know whether testosterone improves
athletic performance or health-related outcomes, and whether beneficial effects of androgens
can be achieved without significant long-term adverse effects. Opinion as to the effects of
testosterone on the muscle in healthy eugonadal men has been enormously controversial for
more than five decades. The athletes who abuse androgenic steroids believe fervently that
these drugs increase muscle mass and strength; however, the academic community decried
their use, citing lack of verifiable evidence. The historical aspects of the use of androgenic/
anabolic steroids have been extensively reviewed. Although their use is most common
among weight-lifters and heavy throwers, almost all types of athletes whose event requires
explosive strength, including football players, swimmers and track and field athletes, have
been known to use steroids. Their use has spread to high-school athletes and to amateur
bodybuilders. Disqualification of highly celebrated athletes in recent years has focused
substantial media attention on this issue [01034].
919
Some strength athletes use androgenic-anabolic steroids (AAS) to improve body dimensions,
though the drugs' long- and short-term effects have not been definitively established. One
study sought to investigate the short-and long-term effects of AAS self-administration on
body dimensions and total and regional body composition. One prospective, unblinded study
involved 35 experienced male strength athletes: 19 AAS users (drugs were self-
administered) and 16 nonuser controls engaged in their usual training regimens. At baseline,
8 weeks, and 6 weeks after AAS withdrawal (for AAS users) circumferences were measured
at 10 sites, and skinfolds measured at 8 sites. To assess differences in AAS regimens, 9
subjects took AAS for 8 weeks (short-AAS) and 10 athletes took AAS for 12 to 16 weeks
(long-AAS). Body composition and anthropometry were assessed at baseline, at the end of
AAS use, and 6 weeks later. Lean body mass (LBM) was calculated from body weight and
percentage fat. Total and regional body composition was measured by dual-energy x-ray
absorptiometry. AAS use increased users' body weight by 4.4 kg and LBM by 4.5 kg, and
produced increases in several circumferences. Percentage of fat decreased (17.0 % to 16.0
%), but fat mass remained unchanged. Changes persisted 6 weeks after drug withdrawal but
were not less than those taken at 8 weeks. Bone-free lean mass of all regional body parts
increased in subjects taking AAS, but fat mass was unaffected. Short- and long-term AAS
users did not differ in any parameter measured at 8 weeks or after drug withdrawal. It was
concluded that in AAS users, 8 weeks of self-administered AAS increased body weight, lean
body mass, and limb circumferences, but decreased percentage fat compared with controls.
Changes remained 6 weeks after drug withdrawal, though for some measurements only
partially. AAS stimulated the bone-free lean mass of all body parts, but it did not affect fat
mass. Short-term and long-term AAS administration produced comparable effects [01035].
The AAS-induced increase in muscle mass can be attributed to both muscle hypertrophy and
the formation of new muscle fibres. It has also been also hypothesised that the fundamental
process for muscle fibre growth appeared to be the incorporation of the satellite cells into
pre-existing fibres to maintain a constant nucleus to cytoplasm ratio. Moreover, key roles
seem to be played by satellite cells, that is, they are enhanced by AAS administration, and
androgen receptors. Androgen receptors are expressed in myonuclei of muscle fibres and in
capillaries and are more present in neck and shoulder muscles than in limb muscles. AAS
administration induces an increase in androgen receptor-containing myonuclei in the
shoulder girdle muscles but not in the vastus lateralis, although it was demonstrated that high
doses of testosterone were also able to increase the myonuclear number per fibre in the
vastus lateralis muscle. It has also been observed that muscle hypertrophy induced by
exogenous testosterone administration was associated with an increase in satellite cell
number, changes in satellite cell ultrastructure and a proportionate increase in myonuclear
number. These observations may, at least partially, explain the regional differences in body
changes and muscle fibre adaptation. Moreover, it has been hypothesised that alterations in
muscle mass may be explained by action of AAS on the mesenchymal pluripotent cell
[04002].
The most prevalent reason for athletes initiating AAS use is to promote muscle mass and
strength. A series of well designed studies investigated the effects of exogenous
testosterone administration, with and without an accompanying strength training programme,
on muscle tissue in eugonadal males. Through use of magnetic resonance imaging
measurements they observed that 10 weeks of testosterone administration (600 mg/week)
may lead to increments of the area of the triceps brachii and quadriceps muscles. The gains
in muscle mass were larger when testosterone administration was combined with a strength
training programme, and may lead to increments of approximately 15 percent of the area of
the triceps brachii and quadriceps muscles. In another study, different dosages of
testosterone enantate (25, 50, 125, 300 and 600 mg/week) for 10 weeks were administered
to non-exercising volunteers. It was found that the effects on thigh muscle volume and
920
quadriceps muscle volume were highly dose dependent. In another study it was registered
gains in muscle mass of rectus femoris muscle and the triceps brachii muscle by means of
ultrasonography. In yet another study it was demonstrated that the effects of AAS on lean
body mass could be attributed to real muscle growth, since no alterations of hydrational
status of lean body mass could be observed [04002].
Because of the relationship between strength and fast twitch muscle fibres it has been
supposed that AAS would affect type II (fast twitch) muscle fibres more than type I (slow
twitch) fibres. In two cross-sectional studies investigating muscular adaptation at the cellular
level in strength athletes, the largest difference in muscle fibre size between AAS users and
non-users was observed in type I muscle fibres of the vastus lateralis and the trapezius
muscle as a result of long-term AAS self-administration. On the other hand, prospective
studies presented equivocal results. In athletes self-administering high doses of several AAS
simultaneously, studies found increments of type I muscle fibres of the vastus lateralis
muscle, whereas the type II fibres remained unaltered. It has been demonstrated that
polydrug regimens for 8 weeks increased muscle fibre size of the deltoid muscle in strength
athletes, with the most profound effect on the type II fibres reflected by a growth of nearly 15
percent. Administration of a single anabolic steroid (i.e. intramuscular nandrolone decanoate
200 mg/week) for 8 weeks, however, had no effect on the size of deltoid muscle fibres.In
muscle biopsy samples from the vastus lateralis, it was found that administration of
testosterone 300 and 600mg increased the cross-sectional areas of type I muscle fibres and
the myonuclear number per fibre, while type II muscle fibres were only enlarged after the
600mg administration regimen in eugonadal males.Lower doses of testosterone 25, 50 and
125 mg/week had no effects on muscle fibre cross-sectional areas [04002].
From these studies it can be concluded that AAS administration may increase muscle mass
in a dose-dependent relationship, but independent of the regimen used (single drug vs
polydrug regimen). Whether type I or type II muscle fibres are more profoundly affected is not
clear yet, but that might be related to the substance(s) and/or dose administered. The
underlying mechanism has been the subject of a study and it was found that the increase in
muscle mass can be attributed to muscle hypertrophy and also the formation of new muscle
fibres. It was also hypothesised that the fundamental process for muscle fibre growth
seemed to be the incorporation of the satellite cells into pre-existing fibres to maintain a
constant nucleus to cytoplasm ratio. Moreover, key roles seem to be played by satellite cells
(i.e. they are enhanced by AAS administration) and androgen receptors. Androgen receptors
are expressed in myonuclei of muscle fibres and in capillaries and are more present in neck
and shoulder muscles than in limb muscles. AAS administration induces an increase in
androgen receptor-containing myonuclei in the shoulder girdle muscles but not in the vastus
lateralis, although it was also demonstrated that high doses of testosterone were also able to
increase the myonuclear number per fibre in the vastus lateralis muscle. It has also been
observed that muscle hypertrophy induced by exogenous testosterone administration was
associated with an increase in satellite cell number, changes in satellite cell ultrastructure
and a proportionate increase in myonuclear number. These observations may, at least
partially, explain the regional differences in body changes and muscle fibre adaptation
[04002].
Problems with studies presented

Considerable debate has raged in the academic community for five decades on whether
androgenic steroids had anabolic effects on the muscle, due in part to the shortcomings of
previous studies; several reviews have discussed these study design issues. For instance,
many of the studies that examined the effect of androgenic steroids were neither blinded nor
randomized. Some studies included competitive athletes, whose desire to win at any cost
prevent them from complying with a standardized regimen of diet and exercise. Nutritional
921
intake was not controlled in many of the studies; changes in energy and protein intake might
have had independent effects on nitrogen balance. Exercise stimulus was not standardized
and, in some studies, the participants were allowed to exercise ad libitum. Therefore, the
effects of androgen administration could not be separated from the effects of resistance
exercise training. Most of the studies performed before the 1980s used relatively small doses
of androgenic steroids, equivalent to or less than the replacement dose of testosterone used
for the treatment of androgen-deficient men. In contrast, athletes use supraphysiological
doses of androgenic steroids. Because of these problems of study design, the results of
these previous studies were inconclusive. With the advent of magnetic resonance imaging
and more refined methods for the assessment of body composition, it has become possible
to detect small changes in muscle volume and fat-free mass with a greater degree of
precision and accuracy than was feasible before. Consequently, studies published in the past
6 years by a number of groups have now established that testosterone supplementation
does increase muscle mass and strength [01034].
A reduction in serum testosterone is associated with decreased fat-free mass

Healthy, hypogonadal men have lower fat-free mass and higher fat mass compared with
those of age-matched eugonadal men. It has been reported that experimental suppression of
serum testosterone by administration of a gonadotropinreleasing hormone (GnRH) agonist
analog in healthy young men is associated with a significant reduction in fat-free mass, an
increase in fat mass, and a decrease in fractional muscle protein synthesis. An age-
associated decline in serum testosterone concentrations correlates with decreased
appendicular muscle mass and reduced lower extremity strength in white and in African-
American men. Effects of physiologic testosterone replacement in healthy, young
hypogonadal men. Testosterone replacement increases nitrogen retention in castrated males
of several animal species, eunuchoidal men, boys before puberty, and women. Several
studies have reexamined the effects of testosterone on body composition and muscle mass
in hypogonadal men in more detail. It was administered 100 mg testosterone enanthate
intramuscularly weekly for 10 weeks to seven hypogonadal men after a 10-12 week period of
androgen withdrawal. Testosterone replacement was associated with a 4.5 + 0.6 kg increase
in body weight because of a 5.0 + 0.8 kg increase in fat-free mass, estimated from
underwater weight, whereas body fat did not change. Similar increases in fat-free mass were
observed using the deuterium water dilution method. Arm and leg muscle cross-sectional
areas, assessed by magnetic resonance imaging, increased significantly. Substantial
increases in muscle strength were also noted after treatment. It was also reported a 15
percent increase in fat-free mass and an 11 percent decrease in fat mass in hypogonadal
men treated with a replacement dose of testosterone enanthate. Their muscle mass
increased by 20 percent and accounted for 65 percent of the increase in fat-free mass. The
muscle accretion during testosterone treatment was associated with a 56 percent increase in
fractional muscle protein synthesis. In another study, a cyclodextrin-complexed testosterone
formulation produced a modest increase in fat-free mass (+0.9 kg) and muscle strength (+8.7
kg) in hypogonadalmen; however, the testosterone dose used in that study was smaller than
the doses used in previous studies. Taken together, these studies provide convincing
evidence that physiologic androgen replacement in healthy, young hypogonadal men is
associated with significant gains in fat-free mass, muscle size and maximal voluntary
strength [01034].
Effect of supraphysiologic doses of testosterone on body composition and muscle strength

Intense controversy persisted until recently with respect to the effects of supraphysiologic
doses of androgenic steroids on body composition and muscle strength. It was conducted a
placebo-controlled, double-blind, randomized clinical trial to assess separately the effects of
supraphysiologic doses testosterone and resistance exercise on fat-free mass, muscle size
and strength. Healthy eugonadal men, 19-40 years of age, who were within 15 percent of
922
their ideal body weight, were randomly assigned to one of four groups: placebo but no
exercise; testosterone but no exercise; placebo plus exercise; testosterone plus exercise.
The men received 600 mg testosterone enanthate or placebo weekly for 10 weeks. Serum
total and free testosterone concentrations, measured 7 days after each injection, increased
fivefold; these were nadir values and serum testosterone concentrations at other times must
have been greater. Serum concentrations of luteinizing hormone (LH) were markedly
suppressed in the two testosterone-treated groups, but not the placebo-treated groups,
providing additional evidence of compliance. Men in the exercise groups underwent weight-
lifting exercises three times weekly; the training stimulus was standardized on the basis of
the participants’ initial one-repetition maximum (1RM) and the sessions were well
supervised. Fat-free mass by underwater weighing, muscle size by magnetic resonance
imaging, and muscle strength of the arms and legs in bench-press and squat exercises were
measured before and after 10 weeks of treatment. The eugonadal men given testosterone
alone had greater gains in muscle size in the arm change in triceps area compared with and
leg than those given placebo injections. Testosterone treatment was also associated with
greater gains in strength in the bench-press and squat exercise capacity than were placebo-
injections. Testosterone and exercise, given together, produced greater increase in fat-free
mass and muscle size than either placebo or exercise alone, and greater gains in muscle
strength than were achieved in either non-exercising group. Serum concentrations of
prostate-specific antigen (PSA) did not change during treatment and no abnormalities were
detected in the prostate on digital rectal examination during the 10-week treatment period.
These results demonstrate that supraphysiologic doses of testosterone, especially when
combined with strength training, increase fat-free mass, muscle size and strength in healthy
eugonadal men. In another study it was administered testosterone enanthate at a dose of 3
mg/kg per week to healthy, eugonadal men, 19-40 years of age. This was an open-label
study that was not placebo-controlled. Muscle mass, estimated from creatinine excretion,
increased by a mean of 20 percent and total body potassium mass increased 12 percent
after 12 weeks of testosterone treatment. In a separate study, a similar dose of testosterone
enanthate given for 12 months to men with muscular dystrophy was associated with a 5 kg
increase in lean body mass (approximately 10 %) at 3 months; these gains were maintained
for 12 months. It was also examined fat-free mass by dualenergy X-ray absorptiometry
(DEXA) scan in 13 nonathletic, eugonadal men treated with 200 mg testosterone enanthate
weekly for 6 months during the course of a male contraceptive study. This was an open-label
study that included untreated men as controls. Testosterone treatment increased serum
testosterone percent and was associated with a 10 percent increase in fat-free mass and a
16 percent decrease in fat mass. Collectively, these data demonstrate that, when dietary
intake and exercise stimulus are controlled, supraphysiologic doses of testosterone produce
further increases in fat-free mass and strength in eugonadal men. Thus it is likely that
strength training may augment the effects of androgen on the muscle [01034].
Improvement of muscle mass and strength but not muscle quality

In populations prone to muscle wasting different anabolic strategies have been investigated
to augment total lean tissue and skeletal muscle mass. These anabolic interventions have
included androgen therapies (testosterone and semisynthetic derivatives of testosterone))
and resistance training. There is compelling evidence that both types of interventions
increase myofibrillar protein synthesis. Because maximum voluntary strength is proportional
to skeletal muscle mass, it is not surprising that these strategies also augment skeletal
muscle strength. Moreover, recent data suggest that treatment with testosterone increases
lean tissues and maximum voluntary strength in a dose-related manner in healthy volunteers,
suggesting that the gains in strength may be directly proportional to change in skeletal
muscle mass with this form of anabolic stimulus. However, with resistance training, there are
theoretical reasons to expect that the relative gains in strength may be greater than the gains
in muscle mass, possibly due to neuromuscular adaptations or other factors. Muscle quality
923
is a quantitative concept to assess the relationship of skeletal muscle strength to muscle
mass. Muscle quality is determined by calculating the ratio of skeletal muscle strength per
unit of skeletal muscle. Maximum voluntary strength is frequently measured using the one-
repetition maximum (1-RM) method. Skeletal muscle mass may be quantified by determining
cross-sectional area of muscle groups by using imaging procedures such as magnetic
resonance imaging (MRI) or computed tomography, of appendicular lean tissue by dual-
energy X-ray absorptiometry (DEXA), or of muscle fiber area by histological staining of
muscle tissue. As such, muscle quality may be used to determine whether increases in
strength are proportional to increases in muscle mass (i.e, no change in muscle quality) or
whether increases in strength are of greater magnitude than the relative increases in muscle
mass (namely, improvements in muscle quality). Improvements in muscle quality suggest
that the muscle is capable of developing greater force relative to the size of the muscle.
Enhancements in muscle quality indicate that there are factors contributing to the
augmentation in strength that are beyond the gross increases in muscle size. Understanding
the mechanisms whereby muscle quality is improved will be important in determining the
most efficient anabolic interventions to improve physical function. In fact, an expert panel
meeting at the National Institutes of Aging Workshop (Sarcopenia and Physical Performance
in Old Age) in 1996 concluded that comprehensive evaluations of age-related changes in
muscle quality should be a top priority. Therefore, enhancing muscle quality should be of
particular importance in populations (e.g. older persons or those with disabilities) in which the
loss of muscle mass may result in decrements of physical function, frailty, risk of falls and
bone fractures, and immobility and risk for pulmonary embolism, loss of independence, and
thus declining overall health [03040].
The relationship of strength to muscle area was used to assess change in muscle quality
after anabolic interventions. Study 1: asymptomatic human immunodeficiency virus-positive
men (39 years) were randomized to nandrolone (600 mg/wk) +/- resistance training (RT).
Study 2: older healthy men (72 years) were randomized to oxandrolone (20 mg/day) or
placebo. Maximum voluntary strength was determined by the 1-repetition maximum (1-RM)
method for leg press, flexion and extension, and cross-sectional area of leg muscles by MRI.
From study week 0 to study week 12, muscle quality was unchanged with nandrolone,
oxandrolone, or oxandrolone placebo, respectively, for total thigh muscles. Lower-extremity
1-RM strength increased several times greater with RT+nandrolone (51-63 % increases)
than with nandrolone alone (5-16 %), despite similar increases in muscle area; therefore,
muscle quality increased for total thigh muscle, for hamstrings, and for quadriceps. Thus
androgen therapy alone did not improve muscle quality, but the addition of RT to nandrolone
produced substantive improvements [03040].
Age dependence of androgens on muscles

Skeletal muscle is primarily composed of highly oxidative, postmitotic fibers, which undergo
constant remodeling, and aging can induce an imbalance within this remodeling process.
Age-induced muscle mass loss is associated with decreased muscle protein synthesis,
muscle fiber loss, alpha-motoneuron loss, and a decline in fiber cross-sectional area.
Although skeletal muscle is a dynamic tissue, aging decreases its plasticity to many stimuli,
and this is especially true of stimuli requiring muscle regeneration or remodeling. There is
strong evidence that age-induced decreases in muscle regenerative capacity are not intrinsic
to the muscle itself but rather are dependent on the aging organism as a whole. Systemic-
related changes with aging include alteration in cardiovascular, endocrine, and immune
systems. Signaling stimuli targeting muscle may be deficient in the aged organism; therefore,
aged muscle's regenerative capacity could possibly be restored if provided the appropriate
stimuli. Functional work overload of rat hindlimb muscle induces a rapid remodeling
response, which includes structural damage, fiber growth, satellite cell activation, and
macrophage infiltration. Each of these responses has been hypothesized to be an important
924
component for the large increases in muscle mass and protein content induced by functional
overload. Aging alters rat hindlimb muscle growth induced by functional overload. Plantaris
(Plan) muscle from 36-mo-old rats does not adapt to 8 week of functional overload in the
same manner as muscles from young rats. However, the response of aged rat hindlimb
muscle during the onset of functional overload has not been determined [03041].
Anabolic steroids are structural derivatives of testosterone, which can increase skeletal
muscle mass and protein synthesis in adult and aged individuals. Exogenous testosterone
administration with muscle loading can synergistically increase skeletal muscle mass in
healthy and diseased humans. A synergistic relationship between testosterone and muscle
loading is present in functionally overloaded rat muscle subject to disuse atrophy. Anabolic
steroid administration can also abolish unloaded atrophy in the rat quadriceps muscle.
Although anabolic steroids are a potent skeletal muscle mass effector, the intracellular
signaling mechanisms induced by the interaction of overload and anabolic steroids have not
been demonstrated. Steroid receptors, a subgroup of the nuclear receptor superfamily, are
transcription factors showing high homology in their DNA-binding domain. The androgen
receptor and glucocorticoid receptor are both potential modulators of skeletal muscle mass.
These receptors can alter cellular gene expression by binding ligand in the cytosol,
translocating to the nucleus, and binding to their corresponding DNA response element to
alter gene transcription. Steroid receptors have also been shown to have ligand-independent
effects on cellular gene expression, which can be modulated by several signaling pathways.
Targets of androgen and glucocorticoid receptor-induced transcriptional activity related to
skeletal muscle mass regulation have not been well characterized. The expression of the
skeletal muscle androgen receptor is sensitive to circulating hormone levels, age of the
animal, and muscle loading conditions. In humans, the skeletal muscle androgen receptor is
downregulated after puberty, and androgen receptor levels are also decreased in young rat
soleus (Sol) muscles compared with older animals. Skeletal muscle androgen receptor
protein expression increases in aged men administered a physiological dose of testosterone
for 1 month. Rat Sol and Plan androgen receptor concentrations are induced by anabolic
steroid administration in both young and old rats. Androgen receptor ligand binding capacity
in rat skeletal muscle increases in functionally overloaded muscle [03041].
One study's purpose was to examine whether functional overload with nandrolone decanoate
(ND) administration increased muscle mass and steroid receptor concentration in aged rat
soleus (Sol) and plantaris (Plan) muscle. ND (6 mg/kg body wt) was administered once a
week for 4 wk, whereas control rats received sesame seed oil injections. Functional overload
of the hindlimb Sol and Plan was induced by synergistic gastrocnemius muscle ablation at
the beginning of the fourth week. Adult (5 mo of age) and aged rats (25 mo of age) were
randomly assigned to four groups: control, overload, control-ND, and overload-ND. Seven
days of functional overload increased adult Sol muscle mass 27 percent, whereas the aged
Sol muscle mass did not change. The aged overloaded Sol muscle receiving ND significantly
increased muscle weight by 35 percent and total muscle protein by 24 percent. Aged Plan
muscle did not increase muscle weight with overload or ND treatment. Androgen receptor
protein was induced by ND treatment and functional Ov, and combining the two treatments
induced Sol androgen receptor protein concentration above either alone. Sol glucocorticoid
receptor protein concentration increased in overload groups of both ages. ND administration
can increase aged Sol muscle mass and protein content after 7 days of functional overload,
and the cooperative induction of androgen receptor may be important for this response
[03041].
Effects on muscles and tendons
925
Combined androgenic-anabolic steroids (AAS) and overloading affects tendon collagen
metabolism and ultrastructure and is often associated with a higher risk of injury. The aim of
this prospective study was to investigate whether such effects would be reflected in the
patellar tendon properties of individuals with a history of long-term resistance training and
AAS abuse (RTS group), compared with trained (RT) and untrained (CTRL) nonsteroids
users. Tendon cross-sectional area (CSA), stiffness, Young's modulus, and toe limit strain
were measured in vivo, from synchronized ultrasonography and dynamometry data. The
patellar tendon of RT and RTS subjects was much stiffer and larger than in the CTRL group.
However, stiffness and modulus were higher in the RTS group (26 % and 30 %, respectively)
than in the RT group. Conversely, tendon CSA was 15% (P < 0.05) larger in the RT group
than in RTS, although differences disappeared when this variable was normalized to
quadriceps maximal isometric torque. Yet maximal tendon stress was higher in RTS than in
RT (15 %), without any statistical difference in maximal strain and toe limit strain between
groups. The present lack of difference in toe limit strain does not substantiate the hypothesis
of changes in collagen crimp pattern associated with AAS abuse. However, these findings
indicate that tendon adaptations from years of heavy resistance training are different in AAS
users, suggesting differences in collagen remodeling. Some of these adaptations (e.g. higher
stress) could be linked to a higher risk of tendon injury [13116].
Skeletal muscle regeneration efficiency declines with age for both men and women. This
decline impacts on functional capabilities in the elderly and limits their ability to engage in
regular physical activity and to maintain independence. Aging is associated with a decline in
sex hormone production. Therefore, elucidating the effects of sex hormone substitution on
skeletal muscle homeostasis and regeneration after injury or disuse is highly relevant for the
aging population, where sarcopenia affects more than 30 percent of individuals over 60 years
of age. While the anabolic effects of androgens are well known, the effects of estrogens on
skeletal muscle anabolism have only been uncovered in recent times. Hence, the purpose of
this review is to provide a mechanistic insight into the regulation of skeletal muscle
regenerative processes by both androgens and estrogens. Animal studies using estrogen
receptor (ER) antagonists and receptor subtype selective agonists have revealed that
estrogens act through both genomic and non-genomic pathways to reduce leukocyte
invasion and increase satellite cell numbers in regenerating skeletal muscle tissue. Although
animal studies have been more conclusive than human studies in establishing a role for sex
hormones in the attenuation of muscle damage, data from a number of recent well controlled
human studies is presented to support the notion that hormonal therapies and exercise
induce added positive effects on functional measures and lean tissue mass. Based on the
fact that aging human skeletal muscle retains the ability to adapt to exercise with enhanced
satellite cell activation, combining sex hormone therapies with exercise may induce additive
effects on satellite cell accretion. There is evidence to suggest that there is a “window of
opportunity” after the onset of a hypogonadal state such as menopause, to initiate a
hormonal therapy in order to achieve maximal benefits for skeletal muscle health. Novel
receptor subtype selective ligands and selective estrogen and androgen receptor modulators
(SERMs, SARMs) promise to reduce health risks associated with classical hormonal
therapies, whilst maintaining the positive effects on muscle repair. Dietary supplements
containing compounds with structural similarity to estrogens (phytoestrogens) are
increasingly used as alternatives to classical hormone-replacement therapies (HRT), but the
effects on skeletal muscle are currently largely unknown. Research has started to investigate
the combined effects of exercise and alternative HRTs, such as soy isoflavones, on skeletal
muscle regenerative processes to provide safer and more efficient therapies to promote
muscle regeneration and maintenance of muscle mass and strength in the aging population
[13117].
Androgen-dependent impairment of myogenesis in muscular atrophy

926
Spinal and bulbar muscular atrophy (SBMA) is an inherited neuromuscular disease caused
by expansion of a polyglutamine (polyQ) tract in the androgen receptor (AR). SBMA is
triggered by the interaction between polyQ-AR and its natural ligands, testosterone and
dihydrotestosterone (DHT). SBMA is characterized by the loss of lower motor neurons and
skeletal muscle fasciculations, weakness, and atrophy. To test the hypothesis that the
interaction between polyQ-AR and androgens exerts cell-autonomous toxicity in skeletal
muscle, we characterized the process of myogenesis and polyQ-AR expression in DHT-
treated satellite cells obtained from SBMA patients and age-matched healthy control
subjects. Treatment with androgens increased the size and number of myonuclei in
myotubes from control subjects, but not from SBMA patients. Myotubes from SBMA patients
had a reduced number of nuclei, suggesting impaired myotube fusion and altered contractile
structures. The lack of anabolic effects of androgens on myotubes from SBMA patients was
not due to defects in myoblast proliferation, differentiation or apoptosis. DHT treatment of
myotubes from SBMA patients increased nuclear accumulation of polyQ-AR and decreased
the expression of interleukin-4 (IL-4) when compared to myotubes from control subjects.
Following DHT treatment, exposure of myotubes from SBMA patients with IL-4 treatment
rescued myonuclear number and size to control levels. This supports the hypothesis that
androgens alter the fusion process in SBMA myogenesis. In conclusion, these results
provide evidence of an androgen-dependent impairment of myogenesis in SBMA that could
contribute to disease pathogenesis [13118].
Long-term effects on muscles

Previous strength training with or without the use of anabolic steroids facilitates subsequent
re-acquisition of muscle mass even after long intervening periods of inactivity. Based on in
vivo and ex vivo microscopy we here propose a cellular memory mechanism residing in the
muscle cells. Female mice were treated with testosterone propionate for 14 days, inducing a
66% increase in the number of myonuclei and a 77 percent increase in fibre cross-sectional
area. Three weeks after removing the drug, fibre size was decreased to the same level as in
sham treated animals, but the number of nuclei remained elevated for at least 3 months (>10
% of the mouse lifespan). At this time, when the myonuclei-rich muscles were exposed to
overload-exercise for 6 days, the fibre cross-sectional area increased by 31 percent while
control muscles did not grow significantly. It was suggested that the lasting, elevated number
of myonuclei constitutes a cellular memory facilitating subsequent muscle overload
hypertrophy. The findings might have consequences for the exclusion time of doping
offenders. Since the ability to generate new myonuclei is impaired in the elderly our data also
invites speculation that it might be beneficial to perform strength training when young in order
to benefit in senescence [13119].
Inhibitory actions on tendons

One study investigated the structural changes in the rat calcaneal tendon (CT), superficial
flexor tendon (SFT), and deep flexor tendon (DFT) in response to jump exercises and
anabolic androgenic steroids (AAS). Animals were divided into four groups: sedentary,
trained, AAS-treated sedentary rats, and AAS-treated trained animals. Training increased the
volume density (Vv%) of blood vessels in all regions of the CT and DFT, cell Vv% in the
peritendinous sheath of the proximal and distal regions of the SFT and proximal region of
DFT, and cell Vv% in the tendon proper of the proximal and distal regions of the SFT and
DFT. The combination of AAS and load exercises showed little increased blood vessel Vv%
at the proximal region of the CT, intermediate region of the SFT, and all regions of the DFT
as opposed to an increase in adipose cell Vv% in the CT proximal region. The AAS reduced
the levels of hydroxyproline in the proximal region of the DFT and in the distal region of the
STF. In conclusion, exercise promoted benefits to the adaptation of the tendons to overload.
These effects were absent when load exercise was combined with AAS. The abusive
927
consumption of AAS contributes to tendon inertness and rigidity, and increases the potential
risk of injury [13120].
Effect of exercise of serum sex steroid levels
Acute and chronic exercise-induced changes in serum sex steroid hormone levels have so
far been reported in many studies. For instance, serum level of testosterone were reduced
during marathon running and treadmill running using the Bruce protocol decreased serum
testosterone levels in physically active men. On the contrary, repeat sprint exercise
(consisting of 10 repetitions of 30-s sprinting at a target load of 150 % of the work capacity)
increased serum total testosterone, free testosterone and DHT levels in healthy active young
men. Furthermore, progressive maximal intensity exercise on a cycle ergometer increased
serum testosterone levels after 20 min from the beginning of exercise and returned to
baseline within 10 min after exercise termination. A 2 h subsequent prolonged exercise
increased by 18-25 percent the concentration of serum testosterone. Concerning the
response of serum sex steroid hormone levels to chronic exercise reported in recent studies,
it was previously demonstrated that in high-sucrose induced obesity in rats and Zucker fatty
rats, lower serum sex steroid hormone levels, such as DHEA and DHT. However, 6-week
aerobic exercise training greatly increased these serum sex steroid hormone levels. Thus,
the response to acute exercise of serum sex steroid hormone levels is inconsistent. This
difference may be affected by characteristics of subjects, exercise design as well as intensity
and duration of the exercise. Furthermore, it may be important to focus on the effect of
exercise on tissue levels of sex steroidogenesis rather than on the change of circulating sex
steroid hormone levels, because both acute and chronic exercise-induced alteration of serum
sex steroid hormone levels are systemic circulating sex steroid hormone levels and may not
accurately reflect intracellular sex steroid hormone levels [14245].
Effect of acute exercise on muscular sex steroidogenesis
Several previous studies have shown that the responses of muscle sex steroid hormone
levels and level of steroidogenic enzyme protein and mRNA expression were changes by a
single bout of exercise stimulation. Muscle free testosterone and DHT levels were acutely
elevated by exercise; treadmill running and intense swimming. Furthermore, it was
demonstrated that protein and mRNA expression of steroidogenic enzymes in skeletal
muscle were changed by a single bout of treadmill running in rats. However, the responses
to acute exercise of the muscle sex steroid hormones levels and steroidogenic enzyme
expression were different between males and females in normal healthy rats. Muscle
testosterone levels as well as 17beta-HSD, 3beta-HSD and 5alpha-reductase protein
expression were increased by acute aerobic exercise in both sexes. By comparison, muscle
estradiol levels increased in males following exercise, but remained unchanged in females.
Moreover, after acute exercise, the protein and mRNA expression of P450arom in the
skeletal muscle increased in males, but decreased in females. In addition, 5alpha-reductase
protein and mRNA expression significantly increased in both sexes even though the
expression of 5alpha-reductase at rest (pre-exercise) was lower in females. Therefore, acute
exercise may increase muscle estrogen synthesis in males, and may increase testosterone
synthesis in females. In fact, muscle estrogen levels were increased in males, while muscle
testosterone levels in females were increased by acute exercise. Thus, the responses of
muscle steroidogenesis were increased by acute exercise, and furthermore, sex differences
in the regulation of P450arom by exercise may contribute to compensate for insufficient local
levels of sex steroid hormones. Taking all data together, muscle steroidogenesis responses
to acute exercise in young subjects may differ between sexes as well as type of exercise
design. Further studies need to examine the effect of acute exercise response on muscle
928
steroidogenesis in older individuals [14245].
Effect of chronic exercise on muscular sex steroidogenesis
It was shown that chronic exercise training induces increase in mRNA levels encoding 3beta-
HSD, P450arom, and 5alpha-reductase in the skeletal muscle in normal young rats.
Moreover, exercise training increased protein expression of P450arom and 5alpha-reductase
in the skeletal muscle while muscle free testosterone and DHT concentrations were
increased by exercise training. In another study, chronic aerobic exercise training increased
the expression of mRNAs encoding these steroidogenic enzymes DHEA, free-testosterone
and DHT levels in muscle in high-sucrose induced obese rats. Moreover, 6-week aerobic
exercise training for Zucker diabetic fatty rats also elevated 17beta-HSD, 3beta-HSD and
5alpha-reductase protein expression levels in skeletal muscle as well as muscle DHEA, free-
testosterone and DHT levels, even though the protein expression and hormone levels were
significantly decreased in Zucker diabetic fatty rats. Interestingly, exercise training-induced
change in muscle DHT was significantly correlated with the change in the fasting blood
glucose level as well as the level of glucose transporter-4 (GLUT-4) translocation in muscle.
Accordingly, increased sex steroid hormone levels increased activation of the glucose
metabolism-signaling pathway in skeletal muscle. Moreover, the exercise-induced
improvement of muscle glucose metabolic signaling activity and fasting blood glucose levels
were significantly inhibited by exercise training. Moreover, 5alpha-reductase inhibitor
administration blocked the synthesis of DHT from testosterone. Thus, exercise training-
induced enhancement of muscle sex steroidogenesis may be associated with an
improvement of hyperglycemia and impaired muscle glucose metabolism in obesity and type
2 diabetic subjects. Since diabetes is one of the risk factors of coronary artery disease,
hypertension and hyperlipidemia, it is crucially important to prevent and treat patients with
hyperglycemia. To increase sex steroid hormone levels, routine exercise may be a
therapeutic strategy for improvement of hyperglycemia with insulin resistance [14245].
Effect of sex steroid hormone administration on muscle sex steroidogenesis
Sex steroid hormone administration is also one direct way to increase sex steroid hormone
levels. It was shown that the muscle DHEA and DHT levels restored by 6-week DHEA
administration improved hyperglycemia via enhancement of impaired glucose metabolic
signaling in the skeletal muscle. Thus, DHEA administration improved fasting blood glucose
levels via enhancement of impaired muscle glucose metabolic signaling in obese rats. DHEA
administration also increased muscle mass in obesity rats. Testosterone administration leads
to increases in satellite cell replication and inhibition of satellite cell apoptosis, and may
contribute to muscle hypertrophy. In human studies, it was shown that, for 6 months, 100
mg/day DHEA treatment in 50-60 year-old men induced an increase in muscle strength only
in men but not in women. According to another study, 12 months of 10 percent DHEA cream
application increased femoral muscle area assessed by computerized tomography (CT) in 15
women aged 60–70 years. On the contrary, it was reported that 50 mg daily treatment for
one year in a large study involving 140 women and 140 men had no effect on thigh muscle,
as assessed by magnetic resonance imaging (MRI), or on strength. Accordingly, DHEA or
testosterone administration improves hyperglycemia with enhancement of muscle glucose
metabolic signaling, and increases muscle mass and strength which that may contribute to
prevention and treatment of obesity, type 2 diabetes and sarcopenia. However, other studies
are required for a better understanding of the mechanisms involved [14245].
Effect of AAS on bodyweight
929
Although many strength athletes frequently report increments of about 10-15kg of
bodyweight due to AAS administration, such alterations have not been documented in well
designed prospective studies. Most studies show that bodyweight may increase by 2–5kg as
a result of short-term (<10 weeks) AAS use. The most pronounced average gain of
bodyweight was reported after 6 weeks of stanozolol administration. However, in a case
report, an increase of 12.7 kg over a 2-year AAS administration period was registered
[04002]
Effect of muscle oxygenation during resistance exercise
The mechanisms that underlie the affect of acute program variables on muscle growth and
strength development for strength/power athletes have been of great interest. One
investigation examined the affects of two different resistance exercise protocols on muscle
oxygenation, and the anabolic hormonal response to such exercise. Eleven experienced
resistance-trained male athletes performed four sets of the squat exercise using either a low-
intensity, high-volume (LI; 15 repetitions at 60 % one-repetition maximum, 1-RM) or high-
intensity, low-volume (HI; 4 repetitions at 90 % 1-RM) load. Venous blood samples were
obtained before (Pre), immediate (IP), 20- (20P), and 40-min (40P) postexercise.
Continuous-wave near-infrared spectroscopy was used to measure oxygen desaturation
during exercise. No differences in muscle deoxygenation were seen between LI and HI.
However, time-dependent postexercise reoxygenation was significantly different between the
two exercise sessions. Testosterone and growth hormone (GH) concentrations were
significantly elevated from Pre at IP, 20P, and 40P in both LI and HI. GH concentrations were
higher for LI than at HI at 20P and 40P. It was concluded that muscle oxygen recovery
kinetics appeared to be influenced by differences in the intensity and volume of exercise, and
delayed reoxygenation appears to affect the GH response to exercise [03042].
Effects of AAS on lean body mass
Apart from a single exception, no study was able to elucidate significant fat mass
decrements. Therefore, the alteration of bodyweight may be attributed mainly to an increase
of lean mass. In this light, Kouri and coworkers reported interesting preliminary data,
indicating that AAS users may be distinguished from non-users by calculation of the fat-free
mass index, a formula incorporating fat-free mass and height. The effects on lean body mass
have been shown to be dose dependent and regional differences in expression of the AAS-
induced gain in lean body mass have been demonstrated [04002].
Effects on fat mass
Since AAS were found to reduce fat mass in animal studies, athletes concluded that this
might also occur in humans. However, research could not support this claim, but revealed
the opposite. In three studies, a reduction in the percentage of fat could be observed;
however, this was not reflected in a decrease of fat mass. Therefore, the change in
percentage fat can be attributed to the increase of lean body mass. Conversely, personal
observations revealed that strength athletes combine low caloric intake with concurrent AAS
intake. The rationale for such regimens is that the athletes aim to reduce fat mass with
simultaneous maintenance of muscle mass [04002].
Effects on recovery after exercise of anabolic steroids
Many athletes report that AAS administration enhances recovery time from strenuous
training. Unfortunately, the research done on this issue only studied indirect parameters that
930
are associated with recovery time. Investigation have shown that exercise-induced
increments of heart rate and serum lactate levels were significantly delayed with use of
nandrolone decanoate. Additionally, after completion of the exercise session the return of the
heart rate and lactate level to baseline values was faster in the AAS users. On the other
hand, administration of injectable testosterone enantate had no effect on serum levels of
urea, ammonia, creatinine, creatine kinase (CK) and aspartate aminotransferase (ASAT),
indicating that this substance did not affect regeneration in well trained endurance athletes. It
has also been shown that higher androgen/cortisol ratios and lower plasma lactate levels in
AAS users compared with non-users after completing a strength-training work out. The
authors suggested that the lower subjective level of fatigue after training sessions could be
attributed to AAS. It was thus concluded that AAS administration resulted in a diminished CK
response and an altered stress response to a single bout of resistance exercise. Although in
animals an enhanced recovery has been demonstrated after AAS administration, research
on the recovery rate in humans is too limited to draw definite conclusions yet [04002].
Effect of AAS and physical activity on the hypothalamic-pituitary-gonadal axis
Analysing effects of pharmaceutical substances and training on feedback mechanisms of the

hypothalamic-pituitary-gonadal axis may be helpful to quantify the benefit of strategies
preventing loss of muscle mass, and in the fight against doping. In one study it was analysed
combined effects of anabolic steroids and training on the hypothalamic-pituitary-gonadal
axis. Therefore intact male Wistar rats were dose-dependently treated with metandienone,
estradienedione and the selective androgen receptor modulator (SARM) S-1. In serum
cortisol, testosterone, 17beta-estradiol (E2), prolactin, inhibin B, follicle-stimulating hormone
(FSH), luteinizing hormone (LH), Insulin-like growth factor 1 (IGF-1), and thyroxine (T4)
concentrations were determined. Six human volunteers were single treated with 1-
androstenedione. In addition abusing and clean body builders were analysed. Serum
concentrations of inhibin B, IGF-1, cortisol, prolactin, T4, thyroid-stimulating hormone (TSH),
testosterone and LH were determined. In rats, administration of metandienone,
estradienedione and S-1 resulted in an increase of muscle fiber diameter. Metandienone and
estradienedione but not S-1 administration significantly decreases LH and inhibin B serum
concentration. Administration of estradienedione resulted in an increase of E2 and S-1 in an
increase of cortisol. Single administration of 1-androstenedione in humans decreased cortisol
and inhibin B serum concentrations. LH was not affected. In abusing body builders a
significantly decrease of LH, TSH and inhibin B and an increase of prolactin, IGF-1 and T4
was detected. In clean body builders only T4 and TSH were affected [150149].
Effects on immunological function
The in vitro effect of testosterone on human neutrophil function was investigated. Blood
neutrophils from healthy male subjects were isolated and treated with 10 nM, 0.1 and 10
microM testosterone for 24 h. As compared with untreated cells, the testosterone treatment
produced a significant decrease of superoxide production as indicated by the measurement
of extra- and intracellular superoxide content. An increment in the production of nitric oxide
was observed at 0.1 and 10 microM testosterone concentrations, whereas no effect was
found for 10 nM. Intracellular calcium mobilization was significantly increased at 10 nM,
whereas it was reduced at 10 microM testosterone. There was an increase in phagocytic
capacity at 10 nM and a decrease of microbicidal activity in neutrophils treated with
testosterone at 10 microM. Glutathione reductase activity was increased by testosterone
treatment, whereas no effect was observed in other antioxidant enzyme activities. An
increase in the content of thiol groups was observed at all testosterone concentrations. Lipid
peroxidation in neutrophils evaluated by levels of TBARS was decreased at 10 nM and 0.1
931
microM testosterone. These results indicate the antioxidant properties of testosterone in
neutrophils as suggested by reduction of superoxide anion production, and lipid peroxidation,
and by the increase in nitric oxide production, glutathione reductase activity and the content
of thiol groups. Therefore, the plasma levels of testosterone are important regulators of
neutrophil function and so of the inflammatory response [10338].
Influence on endurance
Anabolic androgenic steroids (AAS) are doping agents that are mostly used for improvement
of strength and muscle hypertrophy. In some sports, athletes reported that the intake of AAS
is associated with a better recovery, a higher training load capacity and therefore an increase
in physical and mental performances. The purpose of one study was to evaluate, the effect of
multiple doses of AAS on different physiological parameters that could indirectly relate the
physical state of athletes during a hard endurance training program. In a double blind
settings, three groups (n = 9, 8 and 8) were orally administered placebo, testosterone
undecanoate or 19-norandrostenedione, 12 times during 1 month. Serum biomarkers
(creatine kinase, ASAT and urea), serum hormone profiles (testosterone, cortisol and LH)
and urinary catecholamines (noradrenalin, adrenalin and dopamine) were evaluated during
the treatment. Running performance was assessed before and after the intervention phase
by means of a standardized treadmill test. None of the measured biochemical variables
showed significant impact of AAS on physical stress level. Data from exercise testing on
submaximal and maximal level did not reveal any performance differences between the three
groups or their response to the treatment. In the present study, no effect of multiple oral
doses of AAS on endurance performance or bioserum recovery markers was found [06095].
The purpose of the study was to investigate the influence of a 14-week swimming training
program on psychological, hormonal, and performance parameters of elite women
swimmers. Ten Olympic and international-level elite women swimmers were evaluated 4
times along the experiment (i.e. in T1, T2, T3, and T4). On the first day at 8:00 am, before
the blood collecting at rest for the determination of hormonal parameters, the athletes had
their psychological parameters assessed by the profile of mood-state questionnaire. At 3:00
am, the swimmers had their anaerobic threshold assessed. On the second day at 3:00 am,
the athletes had their alactic anaerobic performance measured. Vigor score and testosterone
levels were significantly lower in T4 compared with T3. In addition, the rate between the peak
blood lactate concentration and the median velocity obtained in the alactic anaerobic
performance test significantly increased in T4 compared with T3. For practical applications,
the swimming coaches should not use a tapering with the present characteristics to avoid
unexpected results [10046].
The purpose of one study was to explore the mechanisms for increased exercise
performance in conditions of competition. Endurance trained subjects (n=14) performed
incremental treadmill running to exhaustion in control laboratory conditions (non-competition)
and in conditions of simulated competition to assess performance (running duration). Heart
rate and respiration gases were monitored continuously through each exercise condition.
Blood lactate, cortisol, growth hormone and testosterone concentrations were also
determined at pre- (rest) and postexercise in each condition. Results indicated competition
exercise performance was significantly increased as was peak VO2 response versus non-
competition. No significant differences were found in peak measurements of minute
ventilation, respiratory exchange ratio, ventilation threshold, post-exercise lactate, heart rate,
or the ventilation equivalent for O2 between the exercise conditions. In both conditions growth
hormone and testosterone concentrations increased significantly in response to exercise,
whereas cortisol responses post-exercise were significantly elevated in the competition but
not in the control condition. These findings support that in competitive situations the affective
932
state (motivation) experienced by athletes can enhance performance in exercise events, and
lead to an increased peak oxygen uptake. The magnitude of the improvement is of a
substantial nature and of a level seen with some training programs. Competitive conditions
also augment the cortisol response to exercise, suggesting that enhanced sympatho-adrenal
system activation occur in such situations which may be one of the key "driving forces" to
performance improvement [10047].
Strength is an important quality for many sports. Strength is relevant not only in specifically
strength sports (e.g. weightlifting), but also in many others such as rowing, sprinting and
cycling. Many researchers have investigated the effects of AAS on strength. Several of these
studies do not meet the quality standards for scientific research. Based on available well
designed studies it can be concluded that AAS enhance the effects of strength training. The
observed improvements were in the range of 5-20 percent of baseline strength, largely
depending on the drugs and dose used as well as the administration period. Although most
research has focused on absolute strength determined by one repetition maximum or
isokinetic strength, one study tested the effects on canoeing capacities. I has been reported
that in male canoeists, 6 weeks of Oral-Turinabol administration improved strength and
performance measured by canoe ergometry with 6 and 9 respectively, respectively [04002].
For many years it was assumed that AAS only exerted significant effects in experienced
strength athletes. However, it was later demonstrated that even in novice athletes a 10-week
strength training programme accompanied by testosterone administration may improve
strength more than strength training alone does. Previously, a literature review concluded
that strength improvements in experienced strength athletes may be only slightly larger than
in novice athletes. Moreover, injectable testosterone (600 mg/week intramuscularly) has
clearly been demonstrated to improve strength even without a concomitant exercise training
programme. From the available literature it seems impossible to predict which drugs and
dose will exert the best improvements in strength. For example, metandienone
(methandrostenolone) is the most frequently investigated drug and studies differed with
respect to dose and duration of drug administration. The majority of studies observed
significant strength gains, especially for bench press performance, during metandienone
administration, whereas in four studies no strength alterations were reported. The strength
gains did not seem to depend on duration of metandienone use since changes could already
be observed after 3 weeks’ administration of 10 mg/day. The impact of the administered
dose is difficult to assess because in most research a dose of 10 mg/day was investigated
with some reporting strength gains, while others could not find such improvements.
Nevertheless, it seems likely that dosages exceeding 10 mg/day will increase strength,
although the results from one study not reporting strength improvements are difficult to
explain [04002].
Few studies investigated the effects of injectable steroids. Testosterone enantate has been
demonstrated to consistently enhance strength after administration of daily dosages of 300
mg and over, or by using 3.5 mg/kg bodyweight. It was also demonstrated that androgenic
substances influence strength more than anabolic agents. With respect to strength
enhancement it remains debatable whether polydrug regimens are superior to single drug
administration, although the former increase muscle dimensions, which are closely related to
strength, more than the latter. However, we should emphasise again that laboratory studies
may not adequately mimic the actual AAS-induced improvements of strength since the drugs
and doses investigated in most studies are not in agreement with current steroid
administration regimens by AAS abusers [04002].
Effect of hydration
Exercise intensity powerfully influences testosterone, cortisol, and testosterone/cortisol ratio

933
(T/C) responses to endurance exercise. Hydration state may also modulate these hormones,
and therefore may alter the anabolic/catabolic balance in response to endurance exercise
and training. This study examined the effect of running intensity on testosterone, cortisol, and
T/C when exercise was initiated in a hypohydrated state. Nine male collegiate runners (age
20 years) completed four 10-min treadmill runs differing in pre-exercise hydration status
(euhydrated, or hypohydrated by 5 % of body mass) and exercise intensity (70 % or 85 %
VO2max). Body mass, urine osmolality, and urine-specific gravity documented fluid balance;
blood samples drawn pre-, immediately post-, and 20 min post-exercise were analyzed for
testosterone, cortisol, and T/C. Except for heart rate measured during the 70 percent VO 2max
trials, heart rate, VO2, and plasma lactate were similar between euhydrated and
hypohydrated conditions for a given intensity, suggesting hypohydration did not measurably
increase the physiological stress of the exercise bouts. Furthermore, hydration state had no
measurable effect on testosterone concentrations before, during, or after exercise at either
intensity. Regardless of exercise intensity, cortisol concentrations were greater during
hypohydration than euhydration pre-exercise and 20 min post-exercise. Additionally, T/C was
significantly lower 20 min post-exercise at 70 percent VO2max when subjects were initially
hypohydrated (T/C = 0.056) versus euhydrated (T/C = 0.072). These findings suggest that
depending on exercise intensity, T/C may be altered by hydration state, therefore influencing
the balance between anabolism and catabolism in response to running exercise performed at
typical training intensities [06096].
Influence on strength
Performing strength exercise, whether acutely or in a training programme, leads to

alterations at the hypothalamic-pituitary-testicular and hypothalamic-pituitary-adrenal axes.
One way to evaluate these changes is by analysis of the excretion of steroid hormones in the
urine. One study determined the variations in the urine profile of glucuroconjugated steroids
after a single session of strength exercise and after a 4-week programme of strength training.
The subjects were a group (n=20) of non-sportsman male university students who worked
out 3 days a week (Monday, Wednesday, and Friday), performing the exercises at 70-75
percent of one repetition maximum strength (1-RM). Four urine samples were collected per
subject: (A) before and (B) after a standard session prior to initiating the training programme,
and (C) before and (D) after the same standard session at the end of the study, and they
were assayed by gas chromatography coupled to mass spectrometry. The concentrations of
the different hormones were determined relatively to the urine creatinine level (ng steroid/mg
creatinine) to correct for diuresis. After the exercise sessions, both before and after the
training programme, there was a fall in the urine excretion of androgens and estrogens, but
no statistically significant changes in the excretion of tetrahydrocortisol (THF) and
tetrahydrocortisone (THE). The anabolic/catabolic hormones ratio also decreased after the
acute session, although only androstenodione + dehydroepiandrosterone (DHEA)/THE +
THF ratio had a significant decrease. After the training programme, there was a significant
improvement in the strength of the muscle groups studied, and an increased urinary
excretion of all the androgens with respect to the initial state of repose, with the difference
being significant in the case of epitestosterone (Epit). The androsterone (A) +
etiocholanolone (E)/THE + THF ratio increased significantly concerning the initial state. It
was therefore conclude that subjects suffer variations of the urine profile with regard to the
steroid hormones before and after the acute strength sessions and after the training period.
The alteration after the training programme seems to be due to the subjects' hypothalamic-
hypophysis-testicular and hypothalamic-pituitary-adrenal axes adaptations, which enable
them to increase physical strength [06094]
Previous studies with different results have suggested that total and bioavailable
testosterone levels are modified by physical exercise. Such changes may be related to
934
modifications in cortisol levels and could be reflected in some urine androgens. To determine
how weight lifting training may affect serum and urinary androgens, it was measured total
serum testosterone (T), cortisol, sex hormone binding globulin (SHBG) and urinary
testosterone, epitestosterone, androsterone, and etiocholanolone, in a group of 19 elite
weight lifters after 20 weeks of training. SHBG increased significantly (from 28 + 10 to 35 + 8
nM) whereas T/SHBG decreased significantly (from 1.1 + 0.4 to 0.9 + 0.3). Serum total
testosterone and cortisol did not change significantly. In urine, androsterone and
etiocholanolone decreased significantly, whereas testosterone and epitestosterone remained
unchanged. Changes in T/SHBG were related positively with changes in urinary androgens,
and changes in SHBG were negatively related with changes in urinary androgens. These
results suggest that intense physical activity may have an influence on the elimination of
androgenic hormones due mainly to changes in their transporting protein SHBG [10048].
One study assessed an enzyme-immunoassay (EIA) kit for measuring the salivary
testosterone (T) and cortisol (C) concentrations of weightlifters. Saliva samples (n=64) were
collected from male and female weightlifters during normal training procedures and analysed
for T and C using a commercial EIA kit and a criterion radioimmunoassay (RIA) method.
Significant correlations were demonstrated between the EIA and RIA measurements of
salivary T and C concentrations. Further examination by sample and gender revealed similar
relationships. The EIA concentrations of salivary T and C were found to be slightly greater
(10-13 %) than the RIA values. Similar discrepancies were noted when gender comparisons
were made, although the relative information on T (males > females) and C (males=females)
were consistent for both assay methods. In conclusion, a commercially available EIA kit
provided valid measures of the salivary T and C concentrations of male and female
weightlifters. Factors to consider when using an enzyme-immunoassay kit include the
hormone(s) of interest, the magnitude of the correlations, as well as the descriptive
information gained (e. g. absolute, relative) and its uses within sport [10049].
In weightlifters
One study examined the relationships between salivary testosterone (Sal-T) and cortisol
(Sal-C) concentrations and training performance in Olympic weightlifters. Four male and four
female Olympic weightlifters each provided saliva samples before and after four workouts
during a four-week training period. Training involved the same three exercises; snatch, clean
and jerk, and front squat with the one repetition maximum (1RM) calculated for each exercise
during each workout. Significant training improvements in 1RM performance (4.0-5.2 %)
were noted during the snatch and clean and jerk exercises, along with the Olympic total lift.
For male participants only, the pre-workout concentrations of Sal T were significantly
correlated with the snatch and clean and jerk 1RM, and the Olympic total lift. It was
concluded that short period of training improved the 1RM performance of Olympic
weightlifters in two exercises (snatch and clean and jerk) and the Olympic total. For male
participants, their Sal-T concentrations before each workout was also related to 1RM
performance during these exercises, thereby highlighting one possible short-term causative
mechanism [10332].
One study examined the acute hormonal responses to a single high power resistance
exercise training session. Four weight trained men participated as subjects in two randomly
ordered sessions. During the lifting session, serum samples were collected pre- and 5 min
post-exercise, and later analyzed for testosterone (Tes), cortisol (Cort), their ratio (Tes/Cort),
and lactate (HLa). The lifting protocol was 10 x 5 speed squats at 70 percent of system mass
(1 RM + BW) with 2 min inter-set rest intervals. Mean power and velocity were determined for
each repetition using an external dynamometer. On the control day, the procedures and
935
times (1600-1900 hrs) were identical except the subjects did not lift. Tes and Cort were
analyzed via EIA. Mean power and velocity was 1377 + 10 W and 0.79 + 0.01 m/s
respectively for all repetitions, and did not decrease over the 10 sets. Although not
significant, post-exercise testosterone exhibited a very large effect size. No changes were
observed for either cortisol or the Tes/Cort ratio. Lactate significantly increased post-
exercise. The exercise protocol resulted in no significant changes in Tes, Cort or the
Tes/Cort ratio. The acute increase for Tes is in agreement with previous reports that high
power activities can elicit a Tes response. High power resistance exercise protocols such as
the one used in the present study produce acute increases of Tes. These results indicate
that high power resistance exercise can contribute to an anabolic hormonal response with
this type of training, and may partially explain the muscle hypertrophy observed in athletes
who routinely employ high power resistance exercise [1033].
Influence of sprint
The purpose of this study was to investigate the effects of a 6-month sprint training program
on plasma androgens and catecholamine (CA) concentrations in response to a 6 s sprint in
adolescent boys [training group (TG), n=6; control group (CG), n=6]. A 6 s-sprint test was
performed on a cycle ergometer before and after training (Pre-T and Post-T, respectively).
Plasma total testosterone (TT), bioavailable testosterone (BT), and CA concentrations were
measured at rest, after a warm-up, immediately after a 6 s-sprint, and during the recovery (i.
e. 5 and 20 min). After training period, plasma TT concentrations increased significantly at
the end of the sprint and during the recovery in the TG. No effects for sampling times and
period were observed in BT levels. Plasma TT concentrations after 5 min of recovery were
positively correlated with the corresponding values of plasma lactate (La) concentrations and
with post-6 s-sprint plasma adrenaline (A) concentrations.. These results suggest that sprint
training increases plasma TT concentrations in response to sprint exercise in adolescent
boys. Plasma A and plasma La concentrations increases in response to sprint exercise could
be involved in this elevation of plasma TT concentrations [10050].
Influence of bodybuilders’ fasting periods
The purpose of one study was to investigate simultaneous effects of energy balance, caloric
intake, and the hormonal anabolic-catabolic balance in bodybuilders prior to competition.
Fourteen male bodybuilders took part in an 11-week energy-restricted period to reduce body
fat. The subjects were divided into the energy-restricted group (ERG) (n=7), who were
preparing for the competition, or the control group (CG) (n=7) who continued to train regularly
and did not change their dietary or training pattern. Participants were tested at 11 weeks
(T1), 5 weeks (T2), and 3 days (T3) before competition for diet, body composition, and
fasting hormonal assessment. Body mass and body fat percentage of ERG were significantly
(p < 0.05) decreased during the study period. In ERG, insulinlike growth factor-1 (IGF-1) and
insulin decreased significantly during the 11-week weight-reduction period. Testosterone was
decreased only from week 11 to week 5 (from 20 + 6 to 18 + 7 nmol/L). Changes in IGF-I
concentration were significantly related to changes in insulin, fat mass, lean body mass, and
body mass. Changes in insulin concentrations were significantly related to changes in fat
mass, and lean body mass. These data indicate that severe energy restriction to extremely
low body energy reserves decreases significantly the concentrations of 3 anabolic pathways
despite high protein intake. Monitoring of insulin and IGF-1 concentration is suggested to
prevent losses in muscle mass in energy-restricted conditions [10051].
Flywheel ergometer workouts
936
The purpose of one study was to compare blood lactate and hormonal responses with
flywheel ergometer (FERG) leg presses for preliminary assessment of workouts best suited
for future in-flight resistance exercise. Comprised of 10 repetition sets, the workouts entailed
3 sets of concentric and eccentric (CE3) actions, or concentric-only actions done for 3 (CO3)
or 6 (CO6) sets. Methods employed included assessment of blood lactate concentrations
(BLa) before and 5 minutes postexercise. Venous blood was also collected before and at 1
and 30 minutes postexercise to assess growth hormone, testosterone, cortisol
concentrations Results showed blood lactate concentrations had a time effect. Growth
hormone concentration showed gender x workout, gender x time, and workout x time
interactions, whereas testosterone had a 3-way interaction. Testosterone-cortisol ratio
yielded a gender x time interaction. It was concluded that, because CO6 and CE3 yielded
similar anabolic hormonal data but the latter had a lower cortisol 30 minutes postexercise,
CE3 served as the best workout. Although the FERG was originally designed for
microgravity, the effort put forth by current subjects was like that for workouts aimed at
greater athletic performance and conditioning. Practical applications suggest that eccentric
actions should be used for FERG workouts geared toward muscle mass and strength
improvement [10052].
Watching a previous victory
Previous research indicates that testosterone concentrations are highly responsive to human
competitive interactions and that winners have elevated testosterone concentrations relative
to losers. Also, there is some evidence that simply observing others compete can have a
similar effect on the endocrine system. Here, in two studies, it was examined the extent to
which elite male hockey players would demonstrate an increase in testosterone
concentrations after watching themselves engaged in a previous successful competitive
interaction. Results indicated that watching a previous victory produced a significant increase
in testosterone concentrations (42-44 % increase), whereas watching a previous defeat or a
neutral video did not produce a significant change in testosterone (17 % and 6 %,
respectively). Given that natural fluctuations in testosterone have been shown to influence
future competitive and aggressive behaviours, the current studies may have important
practical implications for individuals involved in competitive sports [10053].
Effects of magnesium supplementation
One study was performed to assess how 4 weeks of magnesium supplementation and
exercise affect the free and total plasma testosterone levels of sportsmen practicing tae
kwon do and sedentary controls at rest and after exhaustion. The testosterone levels were
determined at four different periods: resting before supplementation, exhaustion before
supplementation, resting after supplementation, and exhaustion after supplementation in
three study groups, which are as follows: Group 1-sedentary controls supplemented with 10
mg magnesium per kilogram body weight. Group 2-tae kwon do athletes practicing 90-120
min/day supplemented with 10 mg magnesium per kilogram body weight. Group 3-tae kwon
do athletes practicing 90-120 min/day receiving no magnesium supplements. The free
plasma testosterone levels increased at exhaustion before and after supplementation
compared to resting levels. Exercise also increased testosterone levels relative to sedentary
subjects. Similar increases were observed for total testosterone. The results show that
supplementation with magnesium increases free and total testosterone values in sedentary
and in athletes. The increases are higher in those who exercise than in sedentary individuals
[10056].
Effects of training on salivary levels

937
The aims of one study were to identify the time-course of change of salivary testosterone
(sT), cortisol (sC) and IgA (SIgA), mood state and performance capacity during a 2-week
taper in judo athletes, and to examine the diurnal variation in these salivary markers. Eleven
male judo athletes completed 5 weeks of training: 1 week of normal training (NORM), 2
weeks of intensified training (INT) and 2 weeks of exponential tapering (TAPER). Once per
week subjects completed vertical and horizontal countermovement jump tests, a grip
strength test, a Special Judo fitness test (SJFT), a multistage aerobic fitness test (MSFT), a
3x300-m run test and anthropometric measurement. Subjects also completed questionnaires
to assess mood state and muscle soreness. Two daily saliva samples (at 07:00 and 19:00)
were collected at the end of each week during NORM and INT and every day during TAPER.
Increased morning sT, decreased evening sC, lower muscle soreness and enhanced mood
state were evident by the early phases of TAPER. A significant 7 percent improvement in
3x300-m performance time, a 7 percent improvement in the vertical jump and increased
morning and evening SIgA secretion rate were observed during the middle-late phases of
TAPER. The higher values of salivary variables were observed in the morning. The study
indicates that salivary hormones display diurnal variation. Furthermore, changes in hormonal
responses, mood state and muscle soreness precede enhancements in performance and
mucosal immunity, suggesting that judo athletes taper for at least a week prior to competition
[12122].
Effects of androgens on IGF-1
The mechanism whereby anabolic androgens are associated with hypertrophy of skeletal
muscle is incompletely understood but may involve an interaction with locally generated
insulin-like growth factor (IGF) 1. The present investigation utilized a cell culture model of
human skeletal muscle-derived cell maturation to test the hypothesis that androgens
increase differentiation of human muscle precursor cells in vitro and to assess effects of
androgen with or without IGF-1 on IGF-1 messenger RNA (mRNA) expression in human
muscle precursor cells. Differentiation of muscle-derived cells was induced under standard
low-serum conditions. Cultures were then exposed to androgen (testosterone, T) at 50, 100,
and 500 nM or IGF-1 (10-50 ng/mL). Immunocytochemistry and real-time polymerase chain
reaction (RT-PCR) were used to assess effects of androgens and IGF-1 after 3- (early) or 7-
d (late) muscle differentiation, respectively; RT-PCR was used to quantify the effects on
androgen receptor expression. Under low-serum conditions, 3-d exposure to androgens or
IGF-1 or both resulted in no significant increase in cellular myogenic commitment. After 7-d
exposure, however, T and IGF-1 were both found to increase fusion index with no
observable synergistic effect. T also increased IGF-1 mRNA generation, whereas exogenous
IGF-1 reduced IGF-1 mRNA transcription relative to control. The T effect was reversible after
treatment with flutamide, an androgen receptor antagonist. Both T and IGF-1 increase
myogenic commitment after 7-d exposure to a differentiation medium. With T causing a
concomitant increase in IGF-1 mRNA underpinning IGF-1 as a central mediator in the
cellular pathways associated with muscle hypertrophy, including those affected by
androgens. The novel system described has the potential for elucidating the pattern of
growth factor effects associated with androgens in skeletal muscle [12123].
Influence on GABA of anabolic steroids
Anabolic androgenic steroids are synthetic derivatives of testosterone designed for

therapeutic uses, but now taken as drugs of abuse. Potential health risks associated with
anabolic androgenic steroid abuse are believed to be higher in adolescents than in adults,
but few studies have tested anabolic androgenic steroid effects in adolescent subjects or
938
determined if effects of these steroids differ between females and males. It was now studied
GABA(A) receptor expression and function in the medial preoptic nucleus of mice chronically
treated during adolescence with the anabolic androgenic steroid, 17alpha-methyl-
testosterone. Three-week treatment did not elicit significant differences the expression of
alpha1, alpha2 or alpha5 subunit mRNAs in animals of either sex, although there was a trend
toward decreases in all three subunit mRNAs in female mice, which was augmented and
attained significance for the alpha2 subunit mRNA in females treated for six weeks.
Immunocytochemical analysis revealed that treatment with 17alpha-methyltestosterone for 6
weeks also elicited a significant decrease in the number of alpha2-immunopositive neurons
in female subjects. To test if anabolic androgenic steroid treatment also promoted changes in
GABA(A) receptor function, spontaneous inhibitory synaptic currents were analyzed in
adolescent animals treated for 3-4 weeks. This treatment regimen promoted a significant
decrease in spontaneous inhibitory synaptic current frequency in female, but not male mice.
Finally, anabolic androgenic steroid treatment was found to have no effect on the numbers of
interneurons within the medial preoptic nucleus, as assessed by immunoreactivity for calcium
binding proteins, suggesting that the decrease in the frequency of spontaneous inhibitory
synaptic currents in female mice does not arise from an anabolic androgenic steroid-induced
loss of interneurons. Taken together, the results indicate that chronic exposure to 17alpha-
methyltestosterone elicits significant changes in GABAergic transmission in the medial
preoptic nucleus of female, but not male, mice effectively enhancing the sexually dimorphic
nature of GABAergic transmission in a forebrain region crucial for the expression of
aggression and sexual behaviors [05039].

therapeutic purposes, but now taken predominantly as drugs of abuse. The most common
behavioral effects associated with anabolic androgenic steroid use are changes in anxiety,
aggression and reproductive behaviors, including the onset of puberty and sexual receptivity.
GABAergic circuits in the forebrain underlie these behaviors and are regulated by gonadal
steroids. Work from one laboratory has shown that the expression and function of GABA(A)
receptors in the rat and mouse forebrain varies between the sexes and across the estrous
cycle. It has also been shown that there are significant changes in GABA(A) receptor
expression that occur with the progression through puberty to adulthood. Because
GABAergic systems are both steroid-sensitive and critical for the expression of behaviors
altered with anabolic androgenic steroid use, forebrain GABA(A) receptors are an attractive
candidate to assess how molecular actions of anabolic androgenic steroids may be
translated to known behavioral outcomes. The studies demonstrate that anabolic androgenic
steroids elicit both acute modulation of GABA(A) receptor-mediated currents, as well as
chronic regulation of GABA(A) receptor expression and forebrain GABAergic transmission.
Because anabolic androgenic steroid use has now become prevalent not only among
adolescent boys, but in an increasing number of adolescent girls, we have also been
particularly interested in determining age- and sex-specific effects of anabolic androgenic
steroids. The data show that the effects of chronic anabolic androgenic steroid exposure can
be greater for adolescent than adult animals and are more marked in females than in males.
These data have particularly important implications with respect to studies that have bben
done demonstrating that chronic anabolic androgenic steroid exposure alters the onset of
puberty, estrous cyclicity and sexual receptivity [05040].
Interactions with opioids
Over the past decades, research on doping agents, such as anabolic androgenic steroids
(AAS), has revealed that these compounds are often used in combination with other drugs of
abuse. It seems that misuse of AAS probably involves more than a desire to enhance
appearance or sports performance and studies have revealed that steroids are commonly
939
connected with alcohol, opioids, tobacco, and psychotropic drugs. It was observed that AAS
may interact with the endogenous opioids, excitatory amino acids, and dopaminergic
pathways involved in the brain reward system. Furthermore, studies provide evidence that
AAS may induce an imbalance in these signal systems leading to an increased sensitivity
toward opioid narcotics and central stimulants. In fact, studies performed in various clinics
have shown that individuals taking AAS are likely to get addicted to opioids like heroin
[12124].
Induction of nitric acid
Accumulating evidence indicates that abuse of anabolic androgenic steroids may cause
cardiovascular adverse side-effects, including endothelial dysfunction. The aim of the present
study was to investigate the effects of supra-physiological doses of testosterone on the
endothelial production of nitric oxide (NO) and oxidative stress in vitro and in vivo.
Testosterone enanthate was administrated as of a single 500 mg dose to healthy volunteers
(n=27). Gene expression was studied in human vascular endothelial cells exposed to
testosterone. The in vivo results show that the urinary NO level and the antioxidative capacity
were significantly decreased two days after testosterone administration. In agreement, our
in vitro studies show that testosterone inhibits the gene expression of endothelial NO
synthase (eNOS) after 48 hours. When the antioxidant seleno-L-methionine was added, the
down-regulation of mRNA specific eNOS was partly abrogated. The mRNA expression of
antioxidizing enzyme genes was significantly inhibited after eight hours and recovered 48
hours after testosterone treatment of endothelial cells. These results show that a
supraphysiological dose of testosterone decreases the expression of eNOS and
consequently the formation of NO, which could partly be explained by oxidative stress. These
results indicate that supraphysiological doses of testosterone may induce endothelial
dysfunction, which is of interest in relation to the cardiovascular adverse side-effects
observed in anabolic androgenic steroid abusers [13207].
Lack of influence of NSAID
When focusing on steroid glucuronides as diagnostic parameters in doping controls, the

influence of dietary components on relevant enzymes (i.e. UDP-glucuronosyltransferases)
involved in the conjugation reactions in vivo must be considered. A variety of reports
demonstrating glucuronide inhibiting properties of pharmaceuticals (e.g. non-steroidal anti-
inflammatory drugs, NSAIDs) and ingredients of green tea or red wine were published, the
majority of which however was done in vitro. It was therefore investigated the influence of
NSAIDs (ibuprofen and diclofenac) on the renal elimination of TG and epiTG in a controlled,
randomized cross-over study with 23 male volunteers with two (n=8), one (n=7), or no (n=8)
allele of the UGT2B17 gene, thus representing the above mentioned ins/ins, ins/del, and
del/del genotype, respectively. Both the baseline T/epiT ratios as well as the T/epiT values
following an intramuscular injection of 500 mg of testosterone enanthate were not
significantly influenced by repeated maximum daily doses of the NSAIDs, which suggests
that the commonly employed steroid profile approach is not compromised by NSAID
Testosterone is one of the most commonly abused anabolic androgenic steroids (AAS) within
doping in sports and for enhancement of physical performance. In 2011 anabolic agents
represented the most frequently reported adverse analytical findings and atypical findings (59
%) by accredited doping laboratories. Among these, elevated testosterone/epitestosterone
ratios represented 60 percent of the findings, although only 10 percent of these were adverse
analytical findings. The UDP Glucuronosyl Transferase (UGT) enzymes are important in the
940
pharmacokinetics, and conjugation, of a variety of drugs including non-steroidal anti-
inflammatory drugs (NSAIDs) as well as anabolic androgenic steroids (AAS). Testosterone
glucuronidation capacity is strongly associated with a deletion polymorphism in the UGT2B17
gene. As the use of high doses of NSAIDs has been observed in athletes there is a risk for a
drug-drug interaction that may influence the doping tests for AAS. In vitro studies show
inhibitory potential on UGT2B7, 2B15, and 2B17 enzymes by NSAIDs. To discriminate
exogenous testosterone from testosterone of endogenous origin the urinary ratio of
testosterone glucuronide (TG) to epitestosterone glucuronide (EG) (T/E ratio) is used. Based
on population studies a normal T/E ratio would be around 1.0 and a T/E ratio above six was
initially considered suspicious of doping. However, additional knowledge showed that Asian
individuals excreted low amounts of TG, and as a result low T/E ratios increasing the risk of
false negative doping test results. Due to these findings the T/E ratio was lowered to 4.0 in
2004. Testosterone is inactivated, and excreted in urine, mainly as glucuronide conjugates,
the formation of which is catalyzed by UDP-glucuronosyltransferases (UGTs). These
enzymes play a key role in the homeostasis of a number of endogenous molecules including
steroid hormones. The UGT super family is subdivided into UGT1A, UGT2A, and UGT2B
families based on sequence identity. The human UGT2B genes are clustered on
chromosome 4q13-21.1 and encode seven functional enzymes: UGT2B4, B7, B10, B11,
B15, B17, and B28. In vivo, UGT2B17 has been identified as the main enzyme in
testosterone glucuronidation where a gene deletion in UGT2B17 was associated with low, or
negligible, excretion of testosterone in urine. All subjects devoid of UGT2B17 had a T/E ratio
below 0.4. This polymorphism was considerably more common in a Korean Asian than in a
Swedish Caucasian population, with 67 and 9 percent deletion/deletion (del/del)
homozygotes, respectively. The UGT enzymes are important in the pharmacokinetics, and
conjugation, of a variety of drugs including non-steroidal anti-inflammatory drugs (NSAIDs).
NSAIDs are a class of therapeutic agents used in the treatment of pain and inflammation and
are commonly used by athletes. In fact, according to recent studies, inappropriate use of high
doses and concomitant use of several different NSAIDs has been observed in athletes. Since
steroids and NSAIDs are both inactivated by UGT enzymes there is a risk for a drug-drug
interaction. In vitro studies show inhibitory potential on UGT2B7, 2B15 and 2B17 enzymes by
NSAIDs. In the latter study both diclofenac and ibuprofen inhibited testosterone
glucuronidation in liver microsomes, as well as recombinant UGT2B15 and UGT2B17
enzymes. However, epitestosterone glucuronidation activity in human liver microsomes was
largely insensitive to ibuprofen and diclofenac. The aim of one study was to investigate if
concomitant use of NSAIDs and a single dose of testosterone enanthate would affect the
excretion rate of testosterone and epitestosterone glucuronide (TG and EG) as well as the
T/E ratio, thereby affecting the outcome of the testosterone doping test. The study was
designed as an open, randomized, cross-over study with subjects being their own control.
The 23 male healthy volunteers, with either two, one or no allele (ins/ins, ins/del, or del/del)
of the UGT2B17 gene, received the maximum recommended dose of NSAID (Ibuprofen or
Diclofenac) for 6 days. On day three, 500 mg of testosterone enanthate was administered.
Spot urine samples were collected for 17 days. After a wash-out period of 4 months the
volunteers received 500 mg testosterone enanthate only, with subsequent spot urine
collection for 14 days. The glucuronides of testosterone and epitestosterone were quantified.
NSAIDs did not affect the excretion of TG or EG before the administration of testosterone.
The concomitant use of NSAIDs and testosterone slightly increased the TG excretion while
the EG excretion was less suppressed compared to testosterone use only. The effects of the
NSAIDs on the TG and EG excretion did not differ between the UGT2B17 genotype groups.
In conclusion, the outcome of testosterone doping tests does not seem to be affected by the
use of NSAIDs [13208].
Body composition changes after withdrawal of anabolic steroids
941
After drug withdrawal the alterations of body composition fade away slowly, but may be
partially present for time periods up to 3 months. However, on the basis of scientific data, the
final net result of short-term AAS administration on body composition seems to be rather
small. This seems to be particularly true for recreational athletes as they are not capable of
maintaining the nutritional intake and training workload of the level required for significant
body composition changes.]On the other hand, short-term AAS self-administration in these
athletes results in fast gains and is, therefore, very attractive to them [04002].
Different effects on different parts of the body
In strength athletes, both short- and long-term AAS administration will increase lean body
mass significantly, which may contribute to an increase of muscle mass. In the upper body,
type II muscle fibres seem to increase more than type I fibres after short-term AAS use,
whereas in the thigh the opposite may occur. Conversely, type I fibres may grow after
persistent long-term AAS abuse. The hydration of the lean mass remains unaffected,
although small increments of blood volume cannot be ruled out. Also, fat mass does is not
altered by AAS use. The effects on lean body mass are dose dependent, although it is not
clear which drug administration regimen leads to the most pronounced results. The
administration of therapeutic doses of a single steroid for periods up to 10 weeks does not
seem to exert measurable effects on muscle mass, although body changes are observable.
The upper region of the body (thorax, neck, shoulders and upper arm) seems to be more
susceptible for AAS than other body regions because of predominance of androgen
receptors in the upper body [04002].
Effect of anabolic steroids on hematological variables
Soon after their introduction, AAS were registered for the treatment of several kinds of
anaemia. Long-term treatment with AAS has been demonstrated to increase serum
haemoglobin concentrations. Because of the relationship between hemoglobin and
endurance performance, athletes started to self-administer AAS. However, only two
investigations were able to register AAS-induced alterations of hematology in athletes. In one
study it was described increases in serum hemoglobin concentrations, aematocrit, mean
corpuscular hemoglobin concentration and mean corpuscular hemoglobin, the number of
white blood cells and platelets in athletes after 6 months’ self-administration of high doses of
AAS, whereas mean red cell volume remained unaltered. Later, in another study it was found
an increase of platelet count after 8 weeks of AAS use, whereas all other hematological
parameters remained unaffected [04002].
Another approach to detect testosterone use that is gaining widespread acceptance is

longitudinal studies of urinary steroid concentrations. The concept is based on the
observation that the T/E ratio for a single individual male typically varies by <30 percent,
whereas between-individual variability is considerably larger. Individual T/E values from at
least 3 test results are used to establish a baseline, and suspicious results that differ
significantly from baseline are proof of synthetic testosterone use. Several statistical
approaches have been used to detect outliers in longitudinal data. A Bayesian test using both
population data and individual athlete test results appears to be superior to other statistical
tests for detecting T/E ratio outliers. A Bayesian interpretation of T/E test results has been
shown to produce 0 false-positive results for 43 true positives using a p-value of 0.1 percent.
For the same data set, a population-based T/E cutoff of 4.0 resulted in 24 false positives and
942
34 true positives. Urine samples producing a test result significantly higher than baseline
could then be submitted for GC/C/IRMS confirmation testing. This approach can be used to
detect testosterone use in individuals with the UGT2B17 deletion polymorphism, possibly
negating the need for genetic testing. Athlete-specific baseline T/E ratios, along with
GC/C/IRMS testing on random and suspicious urine samples, were used at the Olympic
Games 2008 to catch athletes that use synthetic testosterone and have low a T/E ratio.
Longitudinal studies can also be applied to other urinary steroid metabolites to further
enhance detection of synthetic steroid use [08016].
Longitudinal profiling of urinary steroids was investigated by using a gas chromatography

/combustion/isotope ratio mass spectrometry (GC/C/IRMS) method. The carbon isotope ratio
of three urinary testosterone (T) metabolites: androsterone, etiocholanolone, 5beta-
androstane-3alpha,17beta-diol (5beta-androstanediol) together with 16(5alpha)-androsten-
3alpha-ol (androstenol) and 5beta-pregnane-3alpha,20alpha-diol (5beta-pregnanediol) were
measured in urine samples collected from three top-level athletes over 2 years. Throughout
the study, the subjects were living in Switzerland and were residing every year for a month or
two in an African country. 13C-enrichment larger than 2.5 per thousand was observed for one
subject after a 2-month stay in Africa. The findings reveal that 13C-enrichment caused by a
diet change might be reduced if the stay in Africa was shorter or if the urine sample was not
collected within the days after return to Switzerland. The steroids of interest in each sample
did not show significant isotopic fractionation that could lead to false positive results in anti-
doping testing. In contrast to the results obtained with the carbon isotopic ratio, profiling of
urinary testosterone/epitestosterone (T/E) ratios was found to be unaffected by a diet change
[05041].
Long-time effects of anabolic steroids
To study the long-term impact of anabolic androgenic steroid (AAS) abuse on the cholesterol
profile, and the potential to suppress endocrine activity in men working out at gym facilities a
studyof the relation between urinary biomarkers for testosterone and nandrolone abuse and
the UGT2B17 genotype and time profile wasz performed. Subjects (n=56) were recruited
through Anti-Doping Hot-Line. Serum levels of luteinizing hormone (LH), follicle-stimulating
hormone (FSH), plasma levels of low density lipoprotein (LDL), high density lipoprotein
(HDL) and urinary steroid profile were regularly measured for a period of up to one year after
cessation of intramuscular AAS abuse. A sustained suppression of LH, and FSH was
observed for several months. The nandrolone urinary biomarker 19-NA was detectable
several months after the last nandrolone intake and was correlated to the levels of LH and
FSH. Testosterone abuse on the other hand was detectable only for a few weeks, and some
of the testosterone abusers did not test positive due to a genetic deletion polymorphism of
the UGT2B17. Significantly increased levels of HDL and decreased levels of LDL were
observed for 6-months after cessation of AAS abuse. It was concluded that some individuals
had a sustained suppression of LH and FSH for a period of 1 year whereas the cholesterol
profile was normalized within 6 month. The long term consequences of these findings remain
to be established [11570].
Effects of previous strength training can be long-lived, even after prolonged subsequent
inactivity, and retraining is facilitated by a previous training episode. Traditionally, such
"muscle memory" has been attributed to neural factors in the absence of any identified local
memory mechanism in the muscle tissue. It was used in vivo imaging techniques to study
live myonuclei belonging to distinct muscle fibers and observe that new myonuclei are added
before any major increase in size during overload. The old and newly acquired nuclei are
943
retained during severe atrophy caused by subsequent denervation lasting for a considerable
period of the animal's lifespan. The myonuclei seem to be protected from the high apoptotic
activity found in inactive muscle tissue. A hypertrophy episode leading to a lasting elevated
number of myonuclei retarded disuse atrophy, and the nuclei could serve as a cell biological
substrate for such memory. Because the ability to create myonuclei is impaired in the elderly,
individuals may benefit from strength training at an early age, and because anabolic steroids
facilitate more myonuclei, nuclear permanency may also have implications for exclusion
periods after a doping offense [10306].
Anabolic androgenic steroids (AAS) use by adolescents is steadily increasing. Adolescence

involves remodeling of steroid-sensitive neural circuits that mediate social behaviors, and
previous studies using animal models document effects of AAS on male social behaviors.
The present experiments tested whether AAS have persistent and more pronounced
behavioral consequences when drug exposure occurs during adolescence as compared to
exposure in adulthood. Male Syrian hamsters were injected daily for 14 days with either
vehicle or an AAS cocktail containing testosterone cypionate (2 mg/kg), nandrolone
decanoate (2 mg/kg), and boldenone undecylenate (1 mg/kg), either during adolescence (27-
41 days of age) or adulthood (63-77 days of age). As adults, subjects were tested two or four
weeks after the last injection for either sexual behavior with a receptive female or male-male
agonistic behavior in a resident-intruder test. Compared with vehicle-treated males, AAS-
treated males, regardless of age of treatment, displayed fewer long intromissions and a
significant increase in latency to the first long intromission, indicative of reduced potential to
reach sexual satiety. Increased aggression was observed in males exposed to AAS
compared with males treated with vehicle, independently of age of AAS treatment. However,
unlike hamsters exposed to AAS in adulthood, hamsters exposed to AAS during
adolescence did not display any submissive or risk-assessment behaviors up to 4 weeks
after discontinuation of AAS treatment. Thus, AAS have long-lasting effects on male sexual
and agonistic behaviors, with AAS exposure during adolescence resulting in a more
pronounced reduction in submissive behavior compared to AAS exposure in adulthood
[09064].
Multi-parametric steroid profiling
Steroid profiling provides valuable information to detect doping with endogenous steroids.
Apart from the traditionally monitored steroids, minor metabolites can play an important role
to increase the specificity and efficiency of current detection methods. The applicability of
several minor steroid metabolites was tested on administration studies with low doses of oral
testosterone (T), T gel, dihydrotestosterone (DHT) gel and oral dehydroepiandrosterone
(DHEA). The collected data for all monitored parameters were evaluated with the respective
population based reference ranges. Besides the traditional markers T/E, T and DHT, minor
metabolites 4-OH-Adion and 6α-OH-Adion were found as most sensitive metabolites to
detect oral T administration. The most sensitive metabolites for the detection of DHEA were
identified as 16alpha-OH-DHEA and 7beta-OH-DHEA but longest detection up to three days
(after oral administration of 50 mg) was obtained with non-specific 5beta-steroids and its
ratios. Steroids applied as a gel had longer effects on the metabolism but were generally not
detectable with universal decision criteria. It can be concluded that population based
reference ranges show limited overall performance in detecting misuse of small doses of
natural androgens. Although some minor metabolites provide additional information for the
oral testosterone and DHEA formulations, the topical administered steroids could not be
detected for all volunteers using universal reference limits. Application of other population
based threshold limits did not lead to longer detection times [10307].
944
Steroid profiling is one of the most versatile and informative screening tools for the detection
of steroid abuse in sports drug testing. Concentrations and ratios of various endogenously
produced steroidal hormones, their precursors and metabolites including testosterone,
epitestosterone, dihydrotestosterone (DHT), androsterone, etiocholanolone, dehydro-
epiandrosterone (DHEA), 5alpha-androstane-3alpha,17beta-diol, and 5beta-androstane-
3alpha,17beta-diol as well as androstenedione, 6alpha-OH-androstenedione, 5beta-
androstane-3alpha,17alpha-diol, 5alpha-androstane-3alpha,17alpha-diol, 3alpha,5-cyclo-
5alpha-androstan-6beta-ol-17-one, 5alpha-androstanedione, and 5beta-androstanedione
add up to a steroid profile that is highly sensitive to applications of endogenous as well as
synthetic anabolic steroids, masking agents, and bacterial activity. Hence, the knowledge of
factors that do influence the steroid profile pattern is a central aspect, and pharmaceutical
(application of endogenous steroids and various pharmaceutical preparations), technical
(hydrolysis, derivatization, matrix), and biological (bacterial activities, enzyme side activities)
issues are reviewed [08184].
Aspects on anabolic steroids in different sports
Prominent doping cases in certain sports have recently raised public awareness of doping
and reinforced the perception that doping is widespread. Efforts to deal with doping in sport
have intensified in recent years, yet the general public believes that the 'cheaters' are ahead
of the testers. Therefore, there is an urgent need to change the antidoping strategy. For
example, the increase in the number of individual drug tests conducted between 2005 and
2012 was approximately 90 000 and equivalent to an increase of about 50 percent, yet the
number of adverse analytical findings remained broadly the same. There is also a strikingly
different prevalence of doping substances and methods in sports such as a 0.03 percent
prevalence of anabolic steroids in football compared to 0.4 percent in the overall WADA
statistics. Future efforts in the fight against doping should therefore be more heavily based
on preventative strategies such as education and on the analysis of data and forensic
intelligence and also on the experiences of relevant stakeholders such as the national
antidoping organisations, the laboratories, athletes or team physicians and related
biomedical support staff. This strategy is essential to instigate the change needed to more
effectively fight doping in sport [14246].
Use in Brazil
According to Silva and Moreau (2003), in the city of Sao Paulo, the prevalence of the use of
AAS prior to 2003 was 19 percent, of which 8 percent were using and 11 percent had already
used. In 2004, in the city of Porto Alegre, 11 percent of bodybuilders had used anabolic
steroids and, in a 2008 study, 123 bodybuilders in the state of Pernambuco, 33.3% reported
having used AAS [14247].
One cross-sectional, quantitative, exploratory study investigated the prevalence and profile of
anabolic-androgenic steroids (AAS) users amongst a convenience sample of 510
bodybuilders from 52 gyms, in João Pessoa, Brazil, with a structured questionnaire
containing selected questions about socioeconomic and training variables on the use of AAS.
Data were analyzed using frequency and chi-square tests. AAS prevalence use was 21
percent; mostly young men (98 %), of a low education level (47 %), who trained for more
than 4 years (50 %). The use of AAS was related to the use of dietary supplements. About
81 percent of consumed AAS consisted of Deca-Durabolin, Winstrol, and Sustanonn [14248].
945
Prevalence of missuse
The prevalence of anabolic-androgenic steroids use has risen dramatically over the last two
decades and filtered into all aspects of society. Support for AAS users has increased, but not
by the medical profession, who will not accept that AAS use dependency is a psychiatric
condition. Polypharmacy by self-prescription is prevalent in this sector. Most recently, AAS
use has filtered through to ”recreational street drug” users and is the largest growth of drugs
in this subdivision. There is a degree of contentiousness in the scenario of anabolic-
androgenic steroids drug use, both within and outside sport. AAS and associated doping
agents are not illegal per se. Possession is not an offence, despite contravening sporting
regulations and moral codes. Until AAS are classified in the same capacity as street drugs in
the UK, where possession becomes a criminal offence, they will continue to attract those
who want to win at any cost [08126].
Dependence
Anabolic-androgenic steroids (AAS) are widely used illicitly to gain muscle and lose body fat.
Here it was reviewed the accumulating human and animal evidence showing that AAS may
cause a distinct dependence syndrome, often associated with adverse psychiatric and
medical effects. It was presented an illustrative case of AAS dependence, followed by a
summary of the human and animal literature on this topic, based on publications known to us
or obtained by searching the PubMed database. About 30 percent of AAS users appear to
develop a dependence syndrome, characterized by chronic AAS use despite adverse effects
on physical, psychosocial or occupational functioning. AAS dependence shares many
features with classical drug dependence. For example, hamsters will self-administer AAS,
even to the point of death, and both humans and animals exhibit a well-documented AAS
withdrawal syndrome, mediated by neuroendocrine and cortical neurotransmitter systems.
AAS dependence may particularly involve opioidergic mechanisms. However, AAS differ
from classical drugs in that they produce little immediate reward of acute intoxication, but
instead a delayed effect of muscle gains. Thus standard diagnostic criteria for substance
dependence, usually crafted for acutely intoxicating drugs, must be adapted slightly for
cumulatively acting drugs such as AAS. It was concluded that AAS dependence is a valid
diagnostic entity, and probably a growing public health problem. AAS dependence may share
brain mechanisms with other forms of substance dependence, especially opioid dependence.
Future studies are needed to characterize AAS dependence more clearly, identify risk factors
for this syndrome and develop treatment strategies [09054].
It appears that about 30 percent of AAS users may develop AAS dependence, which in some
instances may be part of a larger pattern of dependence on PEDs, involving additional
agents such as hGH and CNS stimulants. Unlike most dependence-inducing drugs, which
typically deliver an immediate “reward” of intoxication, AAS produce few intoxicating effects,
and are instead taken primarily for the delayed reward of increased muscle mass and
decreased body fat. Despite these differences, AAS dependence may nevertheless become
a chronic and potentially dangerous disorder. One group has suggested that AAS
dependence may develop via any or all of three different pathways, namely a “body image”
pathway, a neuroendocrine pathway, and a hedonic pathway. The “body image” pathway
refers to the observation that many individuals initiate AAS use because they exhibit
symptoms of “muscle dysmorphia”, a form of body dysmorphic disorder where individuals
develop severe preoccupations that they are not adequately muscular. Muscle dysmorphia
appears closely associated with AAS use. Individuals with such concerns often become
extremely anxious if they stop AAS use and lose even a little muscular size. Thus, they often
quickly resume AAS, which contributes to the AAS dependence syndrome [14017].
946
Neuroendocrine factors also contribute to AAS dependence. Since exogenous AAS
suppresses hypothalamic-pituitary-testicular (HPT) function, users will gradually develop
suppressed testosterone levels, and may become hypogonadal upon discontinuation of AAS
use. Although illicit AAS users employ various techniques to minimize hypogonadism
associated with AAS withdrawal (e.g. self-administration of clomiphene and/or human
chorionic gonadotropin at the end of a “cycle” of AAS use, many will display profound
hypogonadism for weeks or months after discontinuing use. The associated symptoms of
fatigue, loss of libido, and depression may prompt some users to quickly resume using AAS
in order to “treat” these dysphoric symptoms [14017].
Finally, animal studies have provided strong support for a third, hedonic pathway to AAS
dependence, likely mediated by nongenomic pathways via membrane receptors rather than
by the classical genomic effects of AAS. Reports that AAS abusers often experience mental
effects within 15 to 20 minutes of AAS administration also favor the nongenomic effects
through membrane receptors rather than the classical AR-mediated genomic effects. In fact,
studies have reported steroid binding sites on both GABA and the N-methyl-D-aspartate
neurons. Studies have also reported interaction of AAS with sigma receptors. The function of
these receptors remains poorly understood, though there is some overlap with opioid system.
These sites are recognized by neurosteroids produced endogenously in the brain. AAS also
may interact with enzymes involved in neurosteroid metabolism, thereby modulating the
action of these neurosteroids, which are known to produce effects on various behaviors. Rats
and mice display conditioned place preference to testosterone, and male hamsters will self-
administer testosterone to the point of death. AAS enhance beta-endorphin in the ventral
tegmental area and may thereby activate the brain reward system. Interestingly, the opioid
antagonist naltrexone can block testosterone self-administration in hamsters. These
observations, combined with others, suggest that opioidergic mechanisms may be involved
in the hedonic pathway to AAS dependence [14017].
In a woman
Anabolic-androgenic steroids (AAS) are increasingly being used by athletes and youngsters
to become masculine and to loose body fat. Long-term consumption of AAS causes multiple
physical and psychological morbidities. Research has also concluded that AAS have
addictive potential and AAS abuse is commonly found with other substance abuse. Abuse of
AAS is rare in eastern countries. Abuse among women is even rarer. It was reported a case
of an Indian woman, who was prescribed nandrolone decanoate injections by an unqualified
medical practitioner to treat multiple non-specific somatic pains and reported weakness,
leading to dependence for nandrolonedecanoate. This case report supports the research
finding of abuse potential of AAS, raises concern about the need for spreading the
awareness about AAS abuse among medical professionals, regulating medical practice by
unqualified practitioners, and strict legal check against AAS availability in developing
countries [14057].
Influence on reaction on pain
The purpose of one study was to investigate the effects of acute and chronic administration
of anabolic-androgenic steroids (AAS) on nociception and morphine antinociception in acute
pain models, as well as on chronic inflammatory nociception. In Experiment 1, adult,
gonadally intact male rats were injected s.c. for 28days with either 5mg/kg testosterone (T),
dihydrotestosterone (DHT), stanozolol (STAN), or safflower oil vehicle (n=12-25/group). On
day 28, rats in each group were tested on acute thermal and mechanical nociceptive assays,
947
before and after morphine treatment. In Experiment 2, rats in each group (n=8-10/group)
were injected with mineral oil or complete Freund's adjuvant (CFA) into one hindpaw after 28
days of AAS treatment, and then tested for thermal hyperalgesia, mechanical allodynia,
inflammation and locomotor suppression intermittently for 28 days. Experiment 3 replicated
nociceptive measurements in Experiments 1 and 2, but with a single AAS or vehicle injection
occurring 3h prior to testing (n=10-12/group). While chronic AAS administration tended to
decrease body weight gain and alter reproductive organ weights in the expected manner, it
did not significantly alter acute nociception nor attenuate the development of various chronic
pain indices after CFA administration. Morphine antinociceptive potency was significantly
decreased by chronic DHT on the hotplate test only. Acute AAS administration also did not
significantly alter acute or chronic nociception, or morphine antinociceptive potency.
Comparisons between acute and chronic AAS administration suggest that steroid tolerance
did not occur in rats treated with AAS chronically. Taken together, these data do not support
the hypothesis that AAS exposure alters nociception or morphine antinociception in
gonadally intact males [11071].
Overview of detection of AAS abuse
AAS misuse can be conventionally detected by steroid profiling including precursors and
metabolites as well the urinary testosterone/epitestosterone (T/E) ratio, of which normal
levels are below or up to 4.0. Any sample displaying levels above this threshold should be
quantitatively analyzed for confirmation by tandem gas chromatography/mass spectroscopy
(GC/MS). Guidelines for confirmatory analysis by GC/MS and LC/MS/MS have been
released by WADA. High-performance liquid chromatography-tandem mass spectrometry
(LC/MS/MS), also employed in recent years in forensic toxicology, has been established as a
powerful and reliable tool for quantitative confirmatory analysis of drugs used for doping.
Epitestosterone administration is prohibited because it lowers the urinary testosterone/
epitestosterone ratio, a marker of testosterone administration. However, use of a gas
chromatography–combustion–isotope ratio mass spectrometry method resulted in
quantification of the delta C values for urinary epitestosterone as high in controls and lower in
the epitestosterone group. A two-step derivatization procedure has recently been introduced
to enhance performance of electrospray ionization liquid chromatography-mass spectroscopy
in detecting ASS, these being compounds that notably possess limited ionization efficiency.
Nevertheless, the newly initiated approach based on high resolution/high accuracy MS and
ion mobilityhas the capacity to analyze the gas phase dissociation behaviour of several new
drugs. The thus enabled cartography of fragmentation routes of new compounds may permit
a more rapid identification of metabolites and ‘‘tailor-made’’ analogues developed for doping
purposes. The detection of doping is moving away from checking for quantified exposure to
prohibited substance towards biologic assays detecting an effect of prohibited substances.
Cell-based biological assays comprise the future generation of assays which should be
implemented by anti-doping laboratories to detect presence of androgenic anabolic steroids
and other human AR ligands as well as assess the biological activity when the structure of
the compound is not known. Another method, the metabolomics-based approach that was
introduced as a high-tech strategy to determine the anabolic steroid urine profile in livestock
production, is an illicit use of natural steroids and is moreover hard to prove since the
metabolites are unknown. Despite the present lack of compelling data, metabolomics,
involving study of the fingerprints of ASS metabolites, is likely to be added to the arsenal of
anti-doping methods and control programs [12011].
Ratio between testosterone and epitestosterone

948
In addition to the development of secondary sex characteristics, testosterone (T) has
anabolic effects including increases in muscle size and strength and increases in lean body
mass. In the case of exogenous administration of testosterone, the ratio of testosterone to its
isomer, epitestosterone (E), is elevated. WADA has set a standard for T/E ratios of 4.0 as
indicative of possible exogenous testosterone administration. Typically, a sample that
screens for a T/E ratio above that threshold is then subjected to quantitative confirmation by
GC/MS. This methodology, however, can limited due to sensitivity issues as well as a limited
number of qualifying ions that can be used for unambiguous identification. It was therefore
developed a confirmation method which uses liquid/liquid extraction, followed by room
temperature Girard P derivatization, and analysis using LC/MS-ToF. Analysis time is
decreased. Sensitivity is increased, resulting in limits of detection of 2 and 0.5 ng/ml for
testosterone and epitestosterone, respectively. The number of diagnostic qualifier ions is
also increased allowing more confident identification of the analytes. Finally, while this
method has been developed on a QToF instrument, it should be easily transferable to any
tandem LC/MS/MS system [08181].
In an effort to control androgen use for enhanced sport performance, WADA screens
biological samples for the presence of androgens, metabolites, and/or masking agents.
Professional athletes are tested both during, and before, competition. There are a number of
routine screening tests that are used to detect exogenous androgen administration, including
measuring the testosterone to epitestosterone (T/E) ratio by GC-MS. Epitestosterone is a co-
secreted product of T and normally is present in urine at levels similar to testosterone. If
exogenous T is administered this will elevate the T level, but not the epitestosterone level.
This test is complicated by the fact that the values of testosterone and epitestosterone vary
greatly between individuals, and therefore, any sample that meets any one of the following
criteria will be sent off for further analysis using Isotope Ratio Mass Spectrometry (IRMS):
T/E value greater than or equal to 4, concentration of T or E greater than 200 ng/mL,
concentration of androsterone or etiocholanolone greater than 10,000 ng/mL, or
concentration of DHEA greater than 100 ng/mL [13084].
If a sample is submitted for further evaluation by IRMS, the 13C/12C ratio of the androgen will
be measured. This is because commercially produced androgens will have higher 13C levels
compared with endogenous androgens. Synthetic androgen use is screened by GC-MS. This
is possible because each synthetic androgen has a distinctive chemical structure on GC-MS
that is readily identifiable and can be matched to a catalogue kept by WADA. Even trace
amounts of synthetic androgen intake are detectable months after the last administration with
GC/MS able to detect concentrations in the pg/ml range. The GC-MS screening tests are
very sensitive and specific for known androgens on the WADA list. However, these screening
tests cannot provide a complete detection of all androgens because they are unable to detect
designer androgens [13084].
Detection of doping with endogenous steroids, such as testosterone, has been and continues
to be a challenge. To overcome the problem of separating testosterone doping from
endogenous testosterone the ratio between the glucuronides of testosterone and
epitestosterone (T/E) is used. This T/E ratio was introduced in doping tests with an
authorized upper limit of 4. Interestingly, the mean T/E ratio in Caucasian men is
approximately 1, whereas in Asians, the mean ratio is considerably lower. Glucuronidation of
androgens by UDP-glucuronosyltransferases (UGTs), i.e., phase II metabolism, is the major
route for androgen inactivation and excretion. UGT2B7 has been identified as the enzyme
responsible for epitestosterone conjugation whereas testosterone is a poor substrate for this
enzyme. Testosterone is mainly conjugated by UGT2B17 and to a minor extent by
UGT2B15. It was recently shown that testosterone glucuronidation activity in the liver is
949
significantly higher in men homozygous for the insertion (ins/ins) of UGT2B17 than in women
with the same genotype, It has also been shown that the ethnic disparity in the T/E ratio is
strongly associated with a deletion polymorphism in the UGT2B17 gene. Individuals
homozygous for this deletion (del/del) may not reach a T/E ratio of 4 when doped with
testosterone. The deletion polymorphism is much more common in East Asian populations
as compared to Caucasians and Africans. There are also individuals that have naturally high
T/E ratios due to decreased excretion of epitestosterone. In males, part of this low
epitestosterone excretion can be explained by a promoter polymorphism in the CYP17 gene,
resulting in 64 percent higher T/E ratios in men homozygous for the T allele [14432].
Evaluation of testosterone/epitestosterone ratio

The ratio of the concentration of testosterone glucuronide to the concentration of
epitestosterone glucuronide (T/E ratio) as determined in urine is the most frequently used
method to prove testosterone abuse by athletes. A T/E ratio higher than 6 has been
considered as proof of abuse in the past; however, cases of naturally occurring higher T/E
ratios have been described. Since the introduction of the T/E ratio in doping analysis, the
parameters that may or may not influence the T/E ratio, possibly leading to false-positive
results, have been debated. To achieve more insight on the influencing circumstances, an
overview was given to obtain an objective view on the merits of the urinary T/E ratio.
Relevant analytical aspects of the T/E ratio, potential parameters of endogenous and
exogenous origins, as well as some alternative methods to determine testosterone abuse,
such as the urinary testosterone/luteinizing hormone ratio, gas chromatography-combustion-
isotope-ratio mass spectrometry, hair analysis, and high-performance liquid chromatography-
mass spectrometry, are discussed [00003].
Comparative safety evaluation of SARM and anabolic androgenic steroids
Anabolic androgenic steroids (AASs) have been in use for decades for the treatment of short
stature, severe burns, HIV wasting syndrome, osteoporosis, and anemia. However, their lack
of selective effects on certain symptoms and unfavorable pharmacokinetic properties has
limited their long-term usage in clinics. Areas covered: Selective androgen receptor
modulators (SARMs) have some advantages over AASs; they are highly specific for
androgen receptors, are orally available, and, most importantly, act as strong receptor
agonists in skeletal muscle and bone, and as weak agonists or antagonists in androgen-
responsive tissues such as the prostate and sebaceous glands. The exact molecular
mechanism, however, has not been fully elucidated. One article included a toxicological
review of major AASs, and a comparative safety analysis of major AASs and SARMs in
clinical trials to evaluate the therapeutic potential of SARMs. The expert opinion based on the
robust tissue selectivity of SARMs over AASs, is that they are worth considering as a
promising therapeutic option for the treatment of various muscle-wasting diseases [150155].
Testosterone treatment of females to men
To assess the evolution of body composition and bone metabolism in trans men during the
first year of cross-sex hormonal therapy in a prospective controlled study, it was included 23
trans men (female-to-male trans persons) and 23 age-matched control women. In both
groups, we examined grip strength (hand dynamometer), biochemical markers of bone
turnover (C-terminal telopeptides of type 1 collagen (CTX) and procollagen 1 aminoterminal
propeptide (P1NP)), total body fat and lean mass, and areal bone mineral density (aBMD) by
dual-X-ray absorptiometry (DXA) and fat and muscle area at the forearm and calf, bone
950
geometry, and volumetric bone mineral density (vBMD) by peripheral quantitative computed
tomography (pQCT), before treatment and after 1 year of treatment with undecanoate
(1000 mg i.m./12 weeks). Before hormonal treatment, trans men had similar bone and body
composition compared with control women. Testosterone treatment induced in trans men a
gain in muscle mass (+10.4 %) and strength and loss of fat mass (-9.7 %) and increased the
levels of P1NP and CTX. Areal and volumetric bone parameters remained largely unchanged
apart from a small increase in trabecular vBMD at the distal radius and in BMD at the total
hip in trans men. None of these changes were observed in the control group. It was
concluded that short-term testosterone treatment in trans men increased muscle mass and
bone turnover. The latter may rather reflect an anabolic effect of testosterone treatment
rather than bone loss [150156].
Male hormonal contraception
New male contraceptive options are urgently needed. Safe, effective and fully reversible
methods of male contraception would be useful for monogamous couples who are trying to
regulate their family size. In addition, an effective male hormonal contraceptive that could be
implanted or injected as a long-acting formulation every 3-6 months would be useful in
countries where limiting population growth has become a public policy imperative. Male
hormonal contraception is based on the same principles as traditional oestrogen-progestin
female oral contraceptives. Both spermatogenesis and ovulation are dependent upon normal
secretion of the pituitary gonadotropins, follicle stimulating hormone (FSH) and luteinizing
hormone (LH). Exogenous gonadotropin-releasing hormone (GnRH) analogues and sex
steroid hormones such as testosterone (T) and progestins suppress gonadotropins and
spermatogenesis. Two large multicentre trials demonstrated that weekly administration of
high-dosage T was very effective in suppressing gonadotropins and spermatogenesis and
conferred an overall contraceptive efficacy comparable to female oral contraceptives. Studies
of combination regimens of lower-dosage T plus a progestin or a GnRH analogue have
demonstrated greater suppression of spermatogenesis than the World Health Organization
trials of high-dosage T. Most of these male hormonal contraceptives have been associated
with modest weight gain and suppression of serum high-density cholesterol (HDL) levels. In
one article, it was reviewed the new developments in male hormonal contraception [01037].
Altered gonadal steroidogenesis in critical illness
The physiology of the reproductive system changes dramatically with the onset of major
illness. The serum testosterone concentrations fall to pre-pubertal levels secondary to a
decreased secretion of gonadotropins and a decreased Leydig cell response to luteinizing
hormone. At the same time, the serum oestrogen concentration rises as the result of an
increased rate of peripheral aromatization. The clinical consequences of these marked
changes are not yet well understood. One line of evidence argues for the administration of
anabolic steroids (derivatives of testosterone) to critically ill patients to improve their catabolic
state. Another line of evidence in animal models suggests that testosterone may suppress
the immune system and myocardial function in critical illness. No clinical trials of oestrogen
administration to critically ill patients have been reported, although two animal studies
suggest that oestrogen may have a positive effect on survival. One chapter reviewed
changes in the physiology of the reproductive system in major illness as well as current
evidence regarding the clinical effects of androgens and oestrogens in critical illness and
their potential therapeutic roles [01038].
951
Anabolic steroids and opioids
Prolonged use of high-dose anabolic-androgenic steroids (AAS) may induce a dependence

syndrome, and emerging evidence suggests that AAS effects on endogenous opioid systems
may contribute to AAS abuse. The present study tested the hypothesis that high dose AAS
treatment enhances endogenous opioid activity in rhesus monkeys as revealed by 1)
tolerance to the antinociceptive effects of the mu opioid agonist morphine and 2) physical
dependence as indicated by evidence of opioid withdrawal following administration of the
opioid antagonist naloxone. Three rhesus monkeys were treated for 14 days with 3.2
mg/kg/day testosterone propionate, and the effects of morphine (0.32-10 mg/kg) and
naloxone (0.01-0.32 mg/kg) were examined both before and during treatment. Morphine
antinociception was evaluated using a warm-water tail-withdrawal procedure, and naloxone-
precipitated withdrawal was evaluated using checked behavioral signs and measures of
ventilatory rate. Chronic testosterone administration for 14 days produced a 100-fold
increase in mean plasma testosterone levels. However, testosterone treatment did not
significantly alter the antinociceptive effects of morphine, and naloxone did not precipitate
signs of opioid withdrawal either before or during testosterone treatment. These data do not
support the hypothesis that high-dose AAS treatment enhances endogenous opioid activity in
rhesus monkeys in a way that produces opioid tolerance or dependence [01039].
Changes in androgenic steroid profile due to urine contamination by

microorganisms
Urine contamination by microorganisms may affect the interpretation of urinalysis in different

areas of clinical diagnosis. This is particularly relevant in doping control. A prospective study
was designed to assess the effects of urine contamination by selected pathogens on the
endogenous androgenic steroid profile. Pooled urine from a healthy male volunteer with
standard steroid profile compared with reference values for the Caucasian population was
sterilized by filtration and stored in sterile glass tubes. Aliquots were inoculated with known
amounts of 15 different organisms (bacteria, fungi, and moulds) and incubated at 37 degrees
C for 2 weeks. Different markers of urine contamination, such as pH, deconjugation of
steroids, and metabolic by-products, were determined. Alkalization of urinary pH was not a
reliable indicator of urine contamination as several organisms grew in this medium and no
alteration of this parameter was found. In uncontaminated urine, less than 10 percent of
steroid glucuronide conjugates were spontaneously hydrolyzed. Higher rates of hydrolysis for
sulfate conjugates were found. An unconjugated fraction higher than 10 percent of the total
amount of testosterone was a reliable indicator of urine contamination. However, microbial
production of testosterone or epitestosterone was not detected. In contrast, a few organisms
were able to synthesize 5alpha-androstanedione, 5beta-androstanedione, and androstene-
dione using endogenous steroids as substrates [01040].
Anabolic steroids in pre-hibernating Arctic ground squirrels
Androgens have benefits, such as promoting muscle growth, but also significant costs,
including suppression of immune function. In many species, these trade-offs in androgen
action are reflected in regulated androgen production, which is typically highest only in
reproductive males. However, all non-reproductive Arctic ground squirrels, irrespective of
age and sex, have high levels of androgens prior to hibernating at sub-zero temperatures.
Androgens appear to be required to make muscle in summer, which, together with lipid, is
952
then catabolized during overwinter. By contrast, most hibernating mammals catabolize only
lipid. It was tested the hypothesis that androgen action is selectively enhanced in Arctic
ground squirrel muscle because of an upregulation of androgen receptors (ARs). Using
Western blot analysis, we found that Arctic ground squirrels have AR in skeletal muscle more
than four times that of Columbian ground squirrels, a related southern species that
overwinters at approximately 0°C and has low pre-hibernation androgen levels. By contrast,
AR in lymph nodes was equivalent in both species. Brain AR was also modestly but
significantly increased in Arctic ground squirrel relative to Columbian ground squirrel. These
results are consistent with the hypothesis that tissue-specific AR regulation prior to
hibernation provides a mechanism whereby Arctic ground squirrels obtain the life-history
benefits and mitigate the costs associated with high androgen production [14745].
Specific laboratory techniques for anabolic steroids
In the International Olympic Committee (IOC) accredited laboratories, specific methods have
been developed to detect anabolic steroids in athletes' urine. The technique of choice to
achieve this is gas-chromatography coupled with mass spectrometry (GC-MS). In order to
improve the efficiency of anti-doping programmes, the laboratories have defined new
analytical strategies. The final sensitivity of the analytical procedure can be improved by
choosing new technologies for use in detection, such as tandem mass spectrometry (MS-
MS) or high resolution mass spectrometry (HRMS). A better sample preparation using
immuno-affinity chromatography (IAC) is also a good tool for improving sensitivity. These
techniques are suitable for the detection of synthetic anabolic steroids whose structure is not
found naturally in the human body. The more and more evident use, on a large scale, of
substances chemically similar to the endogenous steroids obliges both the laboratory and the
sports authorities to use the steroid profile of the athlete in comparison with reference ranges
from a population or with intraindividual reference values [00038].
The list of prohibited substances of the World Anti-Doping Agency (WADA) classifies the
administration of several steroids in sports as doping. Their analysis is generally performed
using urine specimen as matrix. Lots of the steroids are extensively metabolised in the
human body. Thus, knowledge of urinary excretion is extremely important for the sensitive
detection of steroid misuse in doping control. The methods routinely used in steroid
screening mainly focus on substances, that are excreted unconjugated or as glucuronides.
Common procedures include deconjugation using a beta-glucuronidase enzyme. Following
extraction and concentration the analytes are submitted to LC-MS(/MS) analysis and/or GC-
MS(/MS) analyses. Besides the classical steroids, more and more products appear on the
market for "dietary supplements" containing steroids that have never been marketed as
approved drugs, mostly without proper labelling of the contents. To cover the whole range of
potential products comprehensive screening tools have to be utilised in addition to the
classical methods. Endogenous steroids, e.g. testosterone, represent a special group of
compounds. As classical chemical methodology is incapable of discriminating synthetic
hormones from the biosynthesised congeners, the method of steroid profiling is used for
screening purpose. Additionally, based on isotope signatures a discrimination of synthetic
and natural hormones can be achieved [10339].
A simple, rapid and sensitive method was developed for determining the presence of seven
anabolic steroids (boldenone, nandrolone, testosterone, methyltestosterone,
epiandrosterone, androsterone, and atnozolol) in human urine. Glucuronide-conjugates of
these compounds were hydrolyzed with beta-glucuronidase. The anabolic steroids were
analyzed by on-line in-tube solid-phase microextraction (SPME) coupled with liquid
953
chromatography-mass spectrometry (LC-MS). The steroids were separated within 14 min by
high performance liquid chromatography using a Chromolith RP-18e column and 5 mM
ammonium formate/methanol (35/65, v/v) as a mobile phase at a flow rate of 1.0 mL/min.
Electrospray ionization conditions in the positive ion mode were optimized for the MS
detection of these compounds. The optimum in-tube SPME conditions were 20 draw/eject
cycles with a sample size of 40 microL using a Supel-Q PLOT capillary column for the
extraction. The extracted compounds could be desorbed readily from the capillary column by
flow of the mobile phase, and no carryover was observed. Using the in-tube SPME LC-MS
with SIM mode detection, good linearity of the calibration curve (r>0.995) was obtained in the
concentration range of 0.5-20 ng/mL, except for stanozolol. The detection limits (S/N=3) of
anabolic steroids were in the range 9-182 pg/mL and the proposed method showed 20-33-
fold higher sensitivity than the direct injection method. The within-day and between-day
precisions were below 4.0 and 7.3 percent (n=5), respectively. This method was applied
successfully to the analysis of urine samples without the interference peaks. The recovery
rates of anabolic steroids spiked into urine samples were above 85 percent. This method is
useful to analyze the urinary levels of these compounds in anti-doping tests [10340].
Triple quadrupole (QqQ), time of flight (TOF) and quadrupole-time of flight (QTOF) analysers
have been compared for the detection of anabolic steroids in human urine. Ten anabolic
steroids were selected as model compounds based on their ionization and the presence of
endogenous interferences. Both qualitative and quantitative analyses were evaluated. QqQ
allowed for the detection of all analytes at the minimum required performance limit (MRPL)
established by the World Anti-Doping Agency (between 2 and 10 ng/mL in urine). TOF and
QTOF approaches were not sensitive enough to detect some of the analytes (3'-hydroxy-
stanozolol or the metabolites of boldenone and formebolone) at the established MRPL.
Although a suitable accuracy was obtained, the precision was unsatisfactory (RSD typically
higher than 20 %) for quantitative purposes irrespective of the analyser used. The methods
were applied to 30 real samples declared positives either for the misuse of boldenone,
stanozolol and/or methandienone. Most of the compounds were detected by every
technique, however QqQ was necessary for the detection of some metabolites in a few
samples. Finally, the possibility to detect non-target steroids has been explored by the use of
TOF and QTOF. The use of this approach revealed that the presence of boldenone and its
metabolite in one sample was due to the intake of androsta-1,4,6-triene-3,17-dione.
Additionally, the intake of methandienone was confirmed by the post-target detection of a
long-term metabolite [11073].
In routine screening, hormone residues of all known growth promoting agents are detected
by immuno assays or chromatographical methods in combination with mass spectrometry.
To overcome the detection by these routine screening methods new xenobiotic growth
promoters and new ways of application were developed, e.g. the combination of different
agents in hormone cocktails are employed. To enable an efficient tracing of misused
anabolic substances it is necessary to develop new screening technologies for a broad range
of illegal drugs including newly designed xenobiotic anabolic agents. The use of omic
technologies like, transcriptomics, proteomics or metabolomics is a promising approach to
discover the misuse of anabolic hormones by indirectly detecting their physiological action.
With the help of biostatistical tools it is possible to extract the quested information from the
data sets retrieved from the omic technologies [09065].
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A simple, low cost system for the backflushing of capillary gas chromatography (GC)
columns has been investigated and integrated into a method for the detection of anabolic
steroids in equine urine. The modification to the method was simple to make and quick to
setup and optimize. The use of backflushing technology was found to offer significant
benefits in terms of sample throughput and improved system robustness [10067].
A molecularly imprinted polymer (MIP), templated with methyltestosterone, had been

synthesized for the cleanup of hydrolyzed urine samples for subsequent testosterone (T)
quantification by LC-MS/MS. A concentration of 2 ng/mL testosterone could be quantified
after a single step extraction on the MIP. The limit of detection and quantification with the
criteria of a signal-to-noise ratio of 3 and 5 were 0.3 and 2 ng/mL, respectively. These values
meet the conditions set by the World Anti-Doping Agency for the minimum required
performance limits for doping controls, between 2 and 10 ng/mL. Epitestosterone (E) was
also separated on this polymer and could be detected at concentrations down to 0.3 ng/mL.
The quantification of T and E gives access to the determination of the T/E ratio, essential in
doping analysis. Hence, the polymers can offer a more specific extraction procedure,
resulting in increased sensitivity with limits of detection 10 times lower than the ones
achieved by the standard SPE C(18) sorbents employed in official testing laboratories
[10068].
Phase-II metabolism has a major contribution to androgen metabolism, converting the highly
non-polar compounds to a more easily excreted form prior to their excretion in urine. In the
human body the main phase-II metabolic reactions are glucuronidation and sulphonation.
These reactions are catalysed by enzymes, which are categorised into families and further
subfamilies based on their function and similarities of their amino-acid sequences. Due to
inter-individual variation of the metabolising enzymes and their activities, the metabolic
patterns of prohibited substances should be estimated for efficient doping control. In addition
to target analytes the phase-II reactions have an effect on the selection of sample
preparation procedure, chromatographic technique and ionisation method of the analysis
routine. For method development and identification purposes adequate reference material is
required, and to replace the laborious in vivo excretion studies, in vitro methodologies have
been implemented to produce intact phase-II metabolites of androgens [10069].
A simple, rapid and sensitive method was developed for determining the presence of seven
anabolic steroids (boldenone, nandrolone, testosterone, methyltestosterone, epiandro-
sterone, androsterone, and atnozolol) in human urine. Glucuronide-conjugates of these
compounds were hydrolyzed with beta-glucuronidase. The anabolic steroids were analyzed
by on-line in-tube solid-phase microextraction (SPME) coupled with liquid chromatography-
mass spectrometry (LC-MS). The steroids were separated within 14 min by high
performance liquid chromatography using a Chromolith RP-18e column and 5 mM
ammonium formate/methanol (35/65, v/v) as a mobile phase at a flow rate of 1.0 mL/min.
Electrospray ionization conditions in the positive ion mode were optimized for the MS
detection of these compounds. The optimum in-tube SPME conditions were 20 draw/eject
cycles with a sample size of 40 microL using a Supel-Q PLOT capillary column for the
extraction. The extracted compounds could be desorbed readily from the capillary column by
flow of the mobile phase, and no carryover was observed. Using the in-tube SPME LC-MS
with SIM mode detection, good linearity of the calibration curve (r>0.995) was obtained in the
concentration range of 0.5-20 ng/mL, except for stanozolol. The detection limits (S/N=3) of
anabolic steroids were in the range 9-182 pg/mL and the proposed method showed 20-33-
fold higher sensitivity than the direct injection method. This method was applied successfully
to the analysis of urine samples without the interference peaks. The recovery rates of
anabolic steroids spiked into urine samples were above 85 percent. This method is useful to
955
analyze the urinary levels of these compounds in anti-doping tests [10070].
One work presented the validation study of the comprehensive two-dimensional gas
chromatography (GC x GC)-time-of-flight mass spectrometry method performance in the
analysis of the key World Anti-Doping Agency (WADA) anabolic agents in doping control.
The relative abundance ratio, retention time, identification and other method performance
criteria have been tested in the GC x GC format to confirm that they comply with those set by
WADA. Furthermore, tens of other components were identified with an average similarity of
>920 (on the 0-999 scale), including 10 other endogenous sterols, and full mass spectra of
5,000+ compounds were retained. The testosterone/epitestosterone ratio was obtained from
the same run. A new dimension in doping analysis has been implemented by addressing
separation improvement. Instead of increasing the method sensitivity, which is accompanied
by making the detector increasingly "blind" to the matrix (as represented by selected ion
monitoring mode, high-resolution mass spectrometry (MS) and tandem MS), the method
capabilities have been improved by adding a new "separation" dimension while retaining full
mass spectral scan information. Apart from the requirement for the mass spectral domain
that a minimum of three diagnostic ions with relative abundance of 5% or higher in the MS
spectra, all other WADA criteria are satisfied by GC x GC operation. The minimum of three
diagnostic ions arises from the need to add some degree of specificity to the acquired mass
spectrometry data; however, under the proposed full MS scan method, the high MS similarity
to the reference compounds offers more than the required three diagnostic ions for an
unambiguous identification [10071].
Doping control screening based on the enhanced resolution of comprehensive two-

dimensional gas chromatography hyphenated to time of flight mass spectrometer was
investigated. The identification of anabolic agents (clenbuterol, norandrosterone,
epimetendiol, two methyltestosterone metabolites and 3'-hydroxystanozolol) contained in a
spiked urine sample (2 ng/ml) has been demonstrated. Special emphasis was given to 3'-
hydroxystanozolol, mainly considering the difficulty in its detection. In contrast to
conventional GC-MS approaches that must use single-ion monitoring, the GCxGC-TOFMS
method enabled the identification of that metabolite through the deconvolution of the full
mass spectrum and also resolved the co-eluted peaks of 3'-hydroxystanozolol and an
endogenous Components [08166].
Current threshold levels of steroids do not allow for the detection of all endogenous steroid
misuse due to great interindividual variations in urinary steroid concentrations. A method has
been developed and validated to screen for traditionally monitored endogenous steroids in
doping control as well as specific hydroxylated/oxygenated metabolites in order to enhance
the detection capabilities for the misuse of endogenous steroids [08167].
The urinary metabolism of the irreversible aromatase inhibitor androsta-1,4,6-triene-3,17-

dione was investigated. It is mainly excreted unchanged and as its 17beta-hydroxy analogue.
For confirmation, 17beta-hydroxyandrosta-1,4,6-trien-3-one was synthesized and
characterized by nuclear magnetic resonance (NMR) in addition to the parent compound. In
addition, several reduced metabolites were detected in the post-administration urines,
namely 17beta-hydroxyandrosta-1,4-dien-3-one (boldenone), 17beta-hydroxy-5beta-androst-
1-en-3-one (boldenone metabolite), 17beta-hydroxyandrosta-4,6-dien-3-one, and androsta-
4,6-diene-3,17-dione. The identification was performed by comparison of the metabolites
with reference material utilizing gas chromatography/mass spectrometry (GC/MS) of the
underivatized compounds and GC/MS and GC/tandem mass spectrometry (MS/MS) of their
trimethylsilyl (TMS) derivatives. Alterations in the steroid profile were also observed, most
obviously in the androsterone/testosterone ratio. Even if not explicitly listed, androsta-1,4,6-
triene-3,17-dione is classified as a prohibited substance in sports by the World Anti-Doping
956
Agency (WADA) due to its aromatase-inhibiting properties. In 2006 three samples from
human routine sports doping control tested positive for metabolites of androsta-1,4,6-triene-
3,17-dione. The samples were initially found suspicious for the boldenone metabolite 17beta-
hydroxy-5beta-androst-1-en-3-one. Since metabolites of androst-4-ene-3,6,17-trione were
also present in the urine samples, it is presumed that these findings were due to the
administration of a product like “Novedex Xtreme“, which could be easily obtained from the
sport supplement market [08168].
The detection of steroids originating from synthetic precursors in relation to their chemically
identical natural analogues has proven to be a significant challenge for doping control
laboratories accredited by WADA. Endogenous steroid abuse may be confirmed by utilising
the atomic specificity of gas chromatography-combustion-isotope ratio mass spectrometry
(GC-C-IRMS) that enables the precise measurement of differences in stable isotope ratios
that arise as a result of fractionation patterns inherent in the source of steroids. A
comprehensive carbon isotope ratio (delta13C) profiling study (n=1262) of urinary
ketosteroids is reported that demonstrates the inter-individual variation that can be expected
from factors such as diet, ethnicity, gender and age within and between different populations
(13 countries). This delta13C distribution is shown by principal component analysis (PCA) to
provide a statistical comparison to delta13C values observed following administration of
testosterone enanthate [08169].
The product Orastan-A from Gaspari Nutrition was analyzed for its steroid content. According
to the labeling, it is supposed to contain "5a-androstano[2,3-c]furazan-17b-tetrahydropyranol
ether", also called furazadrol-THP ether. The GC-MS analyses of the liberated steroids (after
extraction from the capsule matrix and cleavage of the THP ether, TMS-derivative and
underivatized) revealed mass spectra of two components, both inconsistent with the labeling.
Thus, the steroids were characterized by different analytical techniques such as mass
spectrometry, nuclear magnetic resonance spectroscopy and X-ray crystal structure analysis.
They were identified as 17beta-hydroxyandrostano[3,2-c]isoxazole and -[2,3-d]isoxazole
[08170].
The use of natural and synthetic anabolic steroids in animal fattening has been prohibited in
many countries because of their potential toxic effect on human health. one paper describes
a newly developed gas chromatography-ion trap-mass spectrometry (GC-IT-MS) method for
the quantitative determination of various residual anabolic steroids in meat. Anabolic steroid
was derivatized with N-methyl-N-trimethylsilytrifluoroacetamide prior to GC-IT-MS analysis.
MS2 was employed for quantitative measurement. In addition, 2d-estradiol was used as an
internal standard. Quantitative determination was based on the ratio of peak area of steroid
derivative to peak area of internal standard derivative. Good linearity of each compound,
0.03-1.0mug/ml, was determined. Solvent extraction was used to extract residual anabolic
compounds in meat samples and a solid phase extraction procedure was utilized for sample
cleanup and pre-concentration. The limits of detection of anabolic compounds approximately
ranged from 0.1 to 0.4 mug/kg. The detection limit was comparable with or better than
reported methods and was below the minimum required performance limits established by
the European Community (EC). The application of this newly developed method was
demonstrated by analyzing various beef, pork, chicken and several animal internal organ
samples from local markets [08171].
Carbon isotope ratio (CIR) analysis of urinary steroids using gas chromatography-
combustion isotope ratio mass spectrometry (GCC-IRMS) is a recognized test to detect illicit
doping with synthetic testosterone. There are currently no universally used steroid isotopic
standards (SIS). It was adapted a protocol to prepare isotopically uniform steroids for use as
a calibrant in GCC-IRMS that can be analyzed under the same conditions as used for
957
steroids extracted from urine. Two separate SIS containing a mixture of steroids were
created and coded CU/USADA 33-1 and CU/USADA 34-1, containing acetates and native
steroids, respectively. CU/USADA 33-1 contains 5alpha-androstan-3beta-ol acetate (5alpha-
A-AC), 5alpha-androstan-3alpha-ol-17-one acetate (androsterone acetate, A-AC), 5beta-
androstan-3alpha-ol-11, 17-dione acetate (11-ketoetiocholanolone acetate, 11k-AC) and
5alpha-cholestane (Cne). CU/USADA 34-1 contains 5beta-androstan-3alpha-ol-17-one
(etiocholanolone), 5alpha-androstan-3alpha-ol-17-one (androsterone), and 5beta-pregnane-
3alpha, 20alpha-diol (5betaP). Each mixture was prepared and dispensed into a set of about
100 ampoules using a protocol carefully designed to minimize isotopic fractionation and
contamination. A natural gas reference material, NIST RM 8559, traceable to the
international standard Vienna PeeDee Belemnite (VPDB) was used to calibrate the SIS.
Absolute delta13C(VPDB) and Deltadelta13C(VPDB) values from randomly selected ampoules
from both SIS indicate uniformity of steroid isotopic composition within measurement
reproducibility. This procedure for creation of isotopic steroid mixtures results in consistent
standards with isotope ratios traceable to the relevant international reference material
[08172].
The applicability of comprehensive two-dimensional gas chromatography (GCxGC) for sterol

analysis was investigated by separation and identification of endogenous sterols in
standards, and spiked in human urine. The modulation temperature was optimized to
achieve the best separation and signal enhancement. The separation pattern of trimethylsilyl
derivatives of sterols was compared on two complementary column sets. Whilst the
BPX5/BPX50 column set offers better overall separation, BPX50/BPX5 provides better peak
shape and sensitivity. The average match quality for 19 analysed sterols on the
BPX50/BPX5 column set was 950/1000 when searched against the in-house library; only
four were identified against the NIST05 library, at a match threshold of 800. The study shows
that GCxGC-TOFMS yields high specificity for steroids derived from urine, with detection
limits appropriate for use in doping control [08173].
Exogenous testosterone is known to decrease the urinary excretion rate of epitestosterone

glucuronide due to suppression of the secretion of luteinizing hormone [08271-08273]. In one
study the epitestosterone glucuronide levels decreased after a testosterone injection. There
were large interindividual differences, but six days after the testosterone administration 92
percent of all subjects had epitestosterone glucuronide levels below 30 percent of baseline
leading to even higher increases of T/E ratios [08011].
By means of gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS)

urinary steroids obtained from a reference population of 56 subjects were analyzed for their
C13/C12-ratios. Androsterone and etiocholanolone represent androgen metabolites,
whereas 11beta-hydroxyetiocholanolone, 11beta-hydroxyandrosterone, and 5beta-pregnane-
3alpha,20alpha-diol have sources independent from androgen metabolism. The deltaC13-
values of the latter compounds may be compared to those of androgen metabolites in order
to detect doping with synthetic androgens and thus may serve as endogenous reference
compounds (ERC). In order to allow for classification of conspicuous samples, reference
ranges and limits were calculated for deltaC13-values of selected steroids and differences
hereof (DeltaC13-values). When androsterone is compared to ERCs, deltaC13-values larger
than 3 per thousand are very unlikely. A set of additional parameters was surveyed by a
questionnaire. Several factors turned out to exert significant influence on the deltaC13-values
of urin ary steroids. These encompass the identity of the steroid itself, sex, oral
contraception, travels, and physical activity [08174].
The detection of new anabolic steroid metabolites and new designer steroids in urine is a
challenge in doping analysis. An approach based on precursor ion scanning for the detection
958
of unknown anabolic steroids and metabolites is proposed. The study of the MS/MS spectra
of selected anabolic steroids revealed different fragmentation pathways at low and medium
collision energy depending on the steroid structure. However, after analysis at high collision
energy three common ions at m/z 105, m/z 91, and m/z 77 were found for all studied
anabolic steroids. These ions can be explained by the fragmentation of the steroid structure
and corresponded to the methyl tropylium, tropylium, and phenyl ions, respectively. Because
of the theoretical low specificity of these ions, the simultaneous presence of all of them was
used as a starting point to consider a substance as a possible anabolic steroid. Hence, the
developed approach is based on the simultaneous acquisition of the precursor ion scan of
m/z 105, 91, and 77. The specificity of this approach has been checked by the injection of
several doping agents including beta-agonists, corticosteroids, beta-blockers, and diuretics.
In general, only compounds with a steroidal structure showed a signal at all three selected
m/z values although some exceptions have been found. The applicability of the method was
tested for three different scenarios: the detection of steroid metabolites, the detection of
unknown steroids, and the analysis of prohormones. In metabolic studies, several recently
reported fluoxymesterone metabolites were also found using this method. For detection of
unknown steroids, some negative urine samples were spiked with the designer steroid THG
and 33 other anabolic steroids and treated as blind samples. Finally, the applicability of the
developed approach for the analysis of dietary supplements was checked by the analysis of
a prohormone where several impurities and/or degradation products were found [08175].
In recent years products containing 6alpha-methylandrost-4-ene-3,17-dione have appeared

on the sport supplement market. Scientific studies have proven aromatase inhibition and
anabolic and mild androgenic properties; however, no preparation has been approved for
medical use up to now. In sports 6alpha-methylandrost-4-ene-3,17-dione has to be classified
as a prohibited substance according to the regulations of WADA. For the detection of its
misuse the metabolism was studied following the administration of two preparations obtained
from the Internet (Formadrol and Methyl-1-Pro). Several metabolites as well as the parent
compounds were synthesized and the structures of 3alpha-hydroxy-6alpha-methyl-5beta-
androstan-17-one, 6alpha-methylandrost-4-ene-3,17-dione, and 5beta-dihydromedroxy-
progesterone were confirmed by nuclear magnetic resonance (NMR) spectroscopy. The
main metabolite, 3alpha-hydroxy-6alpha-methyl-5beta-androstan-17-one, was found to be
excreted as glucuronide and was still detectable in microg/mL amounts until urine collection
was terminated (after 25 h). Additionally, samples from routine human sports doping control
had already tested positive for the presence of metabolites of 6alpha-methylandrost-4-ene-
3,17-dione. Screening analysis can be easily performed by the existing screening procedure
for anabolic steroids using 3alpha-hydroxy-6alpha-methyl-5beta-androstan-17-one as target
substance (limit of detection <10 ng/mL). Its discrimination from the closely eluting
drostanolone metabolite, 3alpha-hydroxy-2alpha-methyl-5alpha-androstan-17-one, is
possible as the mono-TMS derivative [08176].
Nine anabolic steroids (androsterone, nandrolone, estradiol, testosterone propionate,

nandrolone-17 propionate, dydrogesterone, testosterone, epitestosterone, boldenone) and
alpha-cholestane as internal standard were studied by gas chromatography coupled with
mass spectrometry (GC/MS). Anabolic steroids can be derivatised into one or two forms,
mainly for androsterone into A-monoTMS and A-diTMS. The aim of one study was to
research the optimization conditions of the derivatisation process (maximum yield of silylation
reaction) of each anabolic steroid into only one form. The interaction "temperature-reaction
time" is significant and has a positive effect on the improvement of the effectiveness of the
derivatisation. Considering the large amount of information, often not convergent, a global
desirability function was applied for multi-responses optimization. Thus, the optimized
temperature and the reaction time of silylation were 85 degrees C and 24 min, respectively.
Several GC/MS analytical parameters were also studied: linearity (regression coefficient
959
upper than 0.99 for each compound, sensibility (range of concentration 0.05-0.30mug/ml).
Confirmatory experiments were applied to check the predicted values and to validate the
model. The confirmatory assay responses are relatively close to the responses predicted. It
was observed satisfactory resolutions by GC/MS and a run lower than 12 min [08177].
An isocratic HPLC method for the determination with screening purposes of anabolic
androgenic steroids (fluoxymesterone, boldenone, nortestosterone, metandrostenolone,
norethindrone, methyltestosterone and bolasterone), used as growth promoting agents, in
finishing pig feed samples has been developed and validated. The separation was achieved
by using a reversed-phase Chromolith RP-18e column at controlled temperature, UV-
detection at 245nm and epitestosterone as internal standard. The method development
involved optimization of different aqueous-organic mobile phases using methanol or
acetonitrile as organic modifiers, flow-rate and temperature. The optimized method was
applied to the analysis of anabolic steroids in finishing pig feed samples. The extraction
efficiencies, decision limits (CCalpha) and detection capabilities (CCbeta) for these
compounds were in the range 83-96 percent, 27-37 and 32-47 per microgkg range,
respectively. The within-laboratory reproducibility at 1, 1.5 and 2 CCbeta concentration levels
were smaller than 13, 10 and 8 percent, respectively. Finally, the proposed method was
successfully applied to nine different kinds of animal feed [08178].
An approach based on precursor ion scanning for the detection of unknown anabolic steroids
and metabolites was proposed. The study of the MS/MS spectra of selected anabolic
steroids revealed different fragmentation pathways at low and medium collision energy
depending on the steroid structure. However, after analysis at high collision energy three
common ions at m/z 105, m/z 91, and m/z 77 were found for all studied anabolic steroids.
These ions can be explained by the fragmentation of the steroid structure and corresponded
to the methyl tropylium, tropylium, and phenyl ions, respectively. Because of the theoretical
low specificity of these ions, the simultaneous presence of all of them was used as a starting
point to consider a substance as a possible anabolic steroid. Hence, the developed approach
is based on the simultaneous acquisition of the precursor ion scan of m/z 105, 91, and 77.
The specificity of this approach has been checked by the injection of several doping agents
including beta-agonists, corticosteroids, beta-blockers, and diuretics. In general, only
compounds with a steroidal structure showed a signal at all three selected m/z values
although some exceptions have been found. The applicability of the method was tested for
three different scenarios: the detection of steroid metabolites, the detection of unknown
steroids, and the analysis of prohormones. In metabolic studies, several recently reported
fluoxymesterone metabolites were also found using this method. For detection of unknown
steroids, some negative urine samples were spiked with the designer steroid THG and 33
other anabolic steroids and treated as blind samples. Finally, the applicability of the
developed approach for the analysis of dietary supplements was checked by the analysis of
a prohormone where several impurities and/or degradation products were found [08179].
A GC-MS method for the determination of anabolic steroids used as growth promoting
agents using SIM in piglet feed samples has been developed and validated, using
testosterone as internal standard. The formation of volatile steroid derivatives was carried out
by derivatization with N-methyl-N-(trimethylsilyl)trifluoroacetamide. The optimum separation
was achieved using a Zebron ZB-5 column under a gradient temperature elution, allowing
the separation of steroids in 18 min. The extraction efficiencies, CCalpha and CCbeta for
these compounds were in the ranges 78-98 percent, 10-21 and 18-35 mug/kg, respectively.
The repeatability and the within-laboratory reproducibility at 1, 1.5, and 2 CCbeta
concentration levels were smaller than 8.2, 7.5, and 5.8 percent and 12.2, 9.5, and 7.5
percent, respectively. Accuracy was in the 99-103 percent range. The robustness was
evaluated using the Youden robustness test. The proposed method was applied to the
960
analysis of steroids spiked in different kinds of animal feed samples with satisfactory results
[08180].
One paper presents the development, optimization and validation of a methodology to

determine nine key steroid hormones (viz. pregnenolone, progesterone, dehydroepi-
androsterone, androstenedione, testosterone, dihydrotestosterone, estrone, 17alpha-
estradiol and 17beta-estradiol) expressed in the steroidogenesis in biological fluids. The
analytical method allows for the determination of steroid hormones in blood plasma and
serum down to 0.08-0.16 ng/mL for estrogens, 0.20-0.36 ng/mL for androgens and 0.36-0.43
ng/mL for progestagens. These limits of detection were obtainable using a two-step solid-
phase clean-up for fractionation and elimination of interfering lipids (fatty acids,
phospholipids, glycerides and sterols) from the steroid hormones. The accuracy of the
method was 50-112 percent in the range 0.10 to 2.00 ng/mL [11075].
Trimethylsilylation of anabolic agents and their metabolites is frequently achieved by using

the derivatization mixture N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA)/NH4I/2-
mercaptoethanol. Nevertheless, artifacts were formed when this mixture was employed in the
monitoring of exemestane and its main metabolite 17β-hydroxyexemestane prior to GC-MS
analysis. These artifacts were identified as the N-methyltrifluoroacetamide (MTFA) and
trimethylsiloxyethylmercapto products of the respective trimethylsilyl (TMS) derivatives.
Furthermore, artifact formation was evaluated taking the structure (1,4-diene-3-keto-6-
exomethylene) of the compounds into account. Although these artifacts are relevant for
investigations regarding the derivatization process and may be of interest in many fields, they
are detrimental to cope with the requirements of the World Anti-Doping Agency (WADA) in
terms of the limits of detection (LODs) required. To overcome this issue, a method using an
alternative derivatization was proposed: formation of methyloxime-TMS derivatives through
double derivatization using O-methylhydroxylamine/pyridine and MSTFA/TMS imidazole after
enzymatic hydrolysis and liquid-liquid extraction. Samples from an excretion study after
administration of exemestane to healthy volunteers were analyzed by the proposed method
and detection of both exemestane and its main metabolite was possible. This method
showed excellent results for both analytes meeting the LODs required for antiestrogenic
agents (50 ng/mL) established by WADA. The method was validated for the main metabolite,
it was robust and cost-effective for qualitative and quantitative purposes, with LOD and LOQ
of 10 ng/mL and 25 ng/mL, respectively [10449].
The application of a comprehensive gas chromatography/combustion/isotope ratio mass

spectrometry-based method for the measurement of stable carbon isotopes of endogenous
urinary steroids excreted as sulphates is presented. The key element in sample preparation
is the consecutive cleanup with high-performance liquid chromatography of underivatized
and acetylated steroids, which allows the isolation of seven analytes (pregn-5-ene-
3β,17α,20α-triol, etiocholanolone, androsterone, epiandrosterone, dehydroepiandrosterone
(DHEA), androst-5-ene-3β,17β-diol and androst-5-ene-3β,17α-diol) from a single urine
specimen. These steroids are of particular importance to doping controls as they should
enable the sensitive and retrospective detection of DHEA abuse by athletes. Depending on
the biological background, the determination limit for all steroids ranges from 5 to 10 ng/mL
for a 10 mL specimen. The method is validated by means of linear mixing models for each
steroid, which covers the items, repeatability and reproducibility. The specificity was further
demonstrated by gas chromatography/mass spectrometry for each analyte, and no influence
of the sample preparation or the quantity of analyte on carbon isotope ratios was observed.
In order to determine naturally occurring 13C/12C ratios and urinary concentrations of all
implemented steroids, a reference population of 67 subjects was measured to enable the
calculation of reference limits for all relevant steroidal Δ values. The applicability of the
developed method was tested by means of a DHEA excretion study. Despite the fact that
961
orally ingested DHEA is preferentially converted into sulphated metabolites and that the renal
clearance of sulphated steroids is slow, only the 13C/12C ratio of EpiA demonstrated the
potential to prolong the detection time for DHEA misuse [10450].
The use of two separate derivatization procedures with the formation of oxime (hydroxyl
ammonium pretreatment) and picolinoyl (mixed anhydride method) derivates of anabolic
steroids following HPLC-MS/MS analysis was proposed. The main product ions of obtained
derivatives for 21 anabolic steroids were evaluated and fragmentation pathways were
compared. The analysis of MS/MS spectra for underivatized steroids versus oxime or
picolinoyl derivatives showed that in case of analytes containing conjugated double bonds in
sterane core all of the observed MS/MS spectra contained abundant product ions of
diagnostic value. The implementation of derivatization procedures to such compounds is
useful for upgrading sensitivity or selectivity of the evaluated method. On the other hand,
MS/MS spectra of underivatized and oxime analytes without conjugated double bonds in
sterane core produce spectra with large amounts of low abundant product ions. Picolinoyl
derivatives formation leads to highly specific spectra with product ions of diagnostic value
coupled with sensitive and selective analysis at the same time. The intra- and inter-group
comparison analysis revealed that fragmentation pathways for underivatized steroids and
correspondent oxime derivatives are similar. The obtained oxime and picolinoyl derivatives
provided 10-15 times higher ESI response in the HPLC-ESI-MS-selected reaction monitoring
(SRM) when compared to those of underivatized molecules in positive HPLC-ESI-MS mode.
Due to the laborious sample preparation it was suggested to use the performed strategy for
confirmation analysis purposes, metabolic studies or while the identification of new steroids
or steroid-like substances [10452].
A relatively selective, chemically and physically robust SPME fiber was developed in a
simple way with testosterone-imprinted polymer, and then directly coupled with gas
chromatography-mass spectrometry (GC-MS) for selective extraction and analysis of
anabolic steroids. The factors influencing polymerization (i.e. cross-linker, polymerization
solvent, polymerization time) were optimized in detail and the polymer was characterized by
scanning electron microscope, infrared spectrometer and thermogravimetric analyzer.
Furthermore, the extraction performance of the MIP-coated SPME fibers such as extraction
ability and selectivity was evaluated. Moreover, the interaction mode between target analytes
and fiber coating was deducted. Finally, the method for extraction and determination of
androsterone, stanolone, androstenedione and methyltestosterone by the homemade MIP-
coated SPME fibers with GC-MS was obtained. It was applied to the simultaneous analysis
of four anabolic steroids in the spiked human urine with the satisfactory recoveries [10451].
Testing for the illicit use of AAS consists of measurement of synthetic testosterone and
determining the ratio of testosterone to epitestosterone in the urine. A normal ratio in a man
rarely exceeds 1.3 and only 1 in 1000 men will have a ratio of 4.12 [07002].
Steroids with large conjugated or cross-conjugated electron systems such as trenbolone and
gestrinone or boldenone, respectively, have demonstrated marginal gas chromatographic
properties under commonly employed derivatization and analytical conditions. These
problems have resulted in relatively high detection limits using GC-MS approaches.
However, their particular structures provide them with considerable proton affinities and so
they are well suited to LC-MS/MS approaches. Consequently, anabolic steroids as well as
glucocorticosteroids that are difficult to assay using GC have been analyzed using LC–
MS/MS methods that yield detection limits matching the minimum required performance
limits (MRPL) as defined by WADA. Also, the designer steroid tetrahydrogestrinone (THG),
which bears the same steroidal nucleus as gestrinone, has been determined using LC-
962
MS/MS ever since it was discovered by Catlin et al. The fact that THG was not detected for
several years illustrates a drawback of the doping control screening protocols usually used,
which are based on target analysis. Known drugs and/or metabolic products are determined
via precursor/product ion pair measurements, which provide the utmost sensitivity but reduce
the analytical result to a limited number of compounds. Drugs that have unknown molecular
weights and dissociation pathways under conventional collision-induced dissociation (CID)
conditions are provided with a cloak of invisibility and remain undetected. Hence,
complementary analyses have been suggested that are based on precursor ion scanning of
the product ions that characterize particular steroid structures or that utilize androgen
bioassays in concert with high resolution MS (HRMS), allowing broader views of urinary
steroids. These proposals provide a deeper insight into potentially misused anabolic
androgenic steroids, but they can still not ensure the determination of surreptitiously altered
steroids prepared solely for doping purposes, as primarily metabolic reactions may reduce or
even impede the effectiveness of these assays [07050].
A qualitative liquid chromatography-tandem mass spectrometry method for the analysis of 22

sporting federation-banned anabolic agents (or their metabolite markers) and anti-estrogens
in urine that are refractory to analysis by gas chromatography-mass spectrometry is
presented. In addition, a quantitative method built around World Anti-Doping Agency (WADA)
guidelines for the confirmatory analysis of 19-norandrosterone, the primary metabolite of
nandrolone with a WADA-specified minimum required performance limit of 1 ng/mL, is
included. Hydrolysis of glucuronide conjugates, liquid-liquid extraction, no clean-up
derivatization with Girard's Reagent P, and analysis by quadrupole-time-of-flight mass
spectrometry provide sensitivity and selectivity well beyond that required by the WADA
[07079].
Characteristic of the preceding WADA prohibited lists, anabolic agents (in particular anabolic-
androgenic steroids, AAS) are most frequently reported concerning adverse analytical
findings in doping control samples. Despite the well-documented health risks attributed to the
abuse of AAS and the reoccurring case reports of AAS-associated fatalities, the attraction of
anabolic agents seems to be unconfined among cheating athletes. Consequently numerous
studies have been conducted to improve anti-doping efforts concerning this prime category
of substances monitored in sports drug testing programs. Enhanced/expanded screening
methods, improved steroid profiling approaches, new/complementary confirmation assays
based on either conventional mass spectrometric methodologies or isotope-ratio mass
spectrometry (IRMS) [13012].
Androgenic anabolic steroids (AAS) are prohibited in sports due to their anabolic effects.
Doping control laboratories usually face the screening of AAS misuse by target methods
based on MS detection. Although these methods allow for the sensitive and specific
detection of targeted compounds and metabolites, the rest remain undetectable. This fact
opens a door for cheaters, since different AAS can be synthesized in order to evade doping
control tests. This situation was evidenced in 2003 with the discovery of the designer steroid
tetrahydrogestrinone. One decade after this discovery, the detection of unknown AAS still
remains one of the main analytical challenges in the doping control field. Although important
steps have been made in order to minimize this analytical problem and different analytical
strategies have been proposed, there are still some drawbacks related to each approach
[13163].
Besides chromatographic-mass spectrometric approaches, alternative methodologies have

been assessed regarding their capability of detecting anabolic agents in urine. Such
approaches included for instance capillary electrophoresis (CE), the aforementioned MALDI-
MS(/MS), androgen bioassays, or a combination of bioassay and mass spectrometry. Wang
963
et al. described the analysis of five AAS (testosterone, epitestosterone, androstenedione,
boldenone, and clostebol) from human urine by means of CE utilizing a so-called sweeping
stacking method. Several analytical parameters such as buffer concentration, pH, and
percentage of organic solvent were varied to allow baseline separation of the model
substances. For several reasons the study can however not be considered relevant for
doping controls including the facts that (1) the method's sensitivity was limited to 30 ng/mL,
(2) analytical run times were > 30 min, and (3) the sample preparation (LLE without
hydrolysis) as well as the chosen compounds (active/intact AAS) do not represent the main
target analytes of sports drug testing approaches. Both phase-I and phase-II metabolic
reactions were not considered and thus the presented results can only serve as proof-of-
principle for the capability of CE to separate steroidal agents. Moreover, the applicability of
CE separation was reported to strongly depend on the analyte's physicochemical properties
and due to limitations such as intolerance of ESI interfaces to selected CE-additives, a wider
applicability of CE in routine applications seems restricted [14009].
The detection of anabolic androgenic steroids (AAS) is one of the most important topics in
doping control analysis. Gas chromatography coupled to (tandem) mass spectrometry (GC-
MS(/MS)) with electron ionization and liquid chromatography coupled to tandem mass
spectrometry have been traditionally applied for this purpose. However, both approaches still
have important limitations, and, therefore, detection of all AAS is currently afforded by the
combination of these strategies. Alternative ionization techniques can minimize these
drawbacks and help in the implementation of a single method for the detection of AAS. In the
present work, a new atmospheric pressure chemical ionization (APCI) source
commercialized for gas chromatography coupled to a quadrupole time-of-flight analyzer has
been tested to evaluate the ionization of 60 model AAS. Underivatized and trimethylsylil
(TMS)-derivatized compounds have been investigated. The use of GC-APCI-MS allowed for
the ionization of all AAS assayed irrespective of their structure. The presence of water in the
source as modifier promoted the formation of protonated molecules ([M+H](+)), becoming the
base peak of the spectrum for the majority of studied compounds. Under these conditions,
[M+H](+), [M+H-H2O](+) and [M+H-2·H2O](+) for underivatized AAS and [M+H](+), [M+H-
TMSOH](+) and [M+H-2·TMSOH](+) for TMS-derivatized AAS were observed as main ions in
the spectra. The formed ions preserve the intact steroid skeleton, and, therefore, they might
be used as specific precursors in MS/MS-based methods. Additionally, a relationship
between the relative abundance of these ions and the AAS structure has been established.
This relationship might be useful in the structural elucidation of unknown metabolites [14257].
Sweeping-MEKC sample concentration technique

A reliable, convenient, and sensitive on-line sweeping-MEKC sample concentration
technique has been applied for the simultaneous separation of six steroids including two
pairs of epimer with 10mM phosphate buffer (pH 7.0) that contains 80 mM sodium dodecyl
sulfate (SDS), 14 mM beta-cyclodextrin (beta-CD), and 4 percent (v/v) methanol. The column
length was 105 cm (effective length, 90 cm). Samples were hydrostatically injected for 600 s.
The separation was performed at ambient temperature under an applied voltage of 25kV.
The external standard calibration curves of the six steroidal hormones proved good linearity
within the concentration range 0.025-1.0 μg/mL. The limit of detections of the on-line
sweeping-MEKC with the ultraviolet detector at 220nm for estrone, alpha-estradiol, beta-
estradiol, androstenedione, epitestosterone, and testosterone were 10, 24, 28, 53, 73, and
11 ng/mL and were 240, 125, 93, 47, 32, and 200 times more sensitive than MEKC,
respectively. The Saccharomyces cerevisiae mediated simultaneous stereoselective
reduction of estrone and androstenedione exhibited a 100 percent stereoselectivity toward
beta-estradiol and testosterone. The accuracy and precision achieved for the spiking
experiments of the sweeping-MEKC were 95-98 beta and less than 3.8 percent (RSD),
respectively [11573].
964
A simple means of detecting the abuse of steroids that also occur naturally is a problem
facing doping control laboratories. Specific markers are required to allow the detection of the
administration of these steroids. These markers are commonly measured using a set of data
obtained from the screening of samples by gas chromatography-mass spectrometry (GC-
MS). Doping control laboratories further need to confirm identified abuse using techniques
such as gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). An
interesting urinary species was found while following the pharmacokinetics and changes to
the steroid profile from single and multiple oral doses of the International Olympic
Committee/World Anti Doping Agency (IOC/WADA) prohibited substance, dehydroepiandro-
sterone (DHEA). The urine samples collected from the administration studies were subject to
GC-MS and GC-C-IRMS steroid analysis following cleanup by solid phase extraction
techniques. A useful urinary product of DHEA administration was detected in the urine
samples from each of the administration studies and was identified by GC-MS experiments
to be 3alpha,5-cyclo-5alpha-androstan-6beta-ol-17-one (3alpha,5-cyclo). This compound
occurs naturally but the concentrations of 3alpha,5-cyclo were elevated following both the
single DHEA administration (up to 385 ng/mL) and multiple DHEA administrations (up to
1240 ng/mL), in relation to those observed prior to these administrations (70 and 80 ng/mL,
respectively). A reference distribution of urine samples collected from elite athletes (n=632)
enabled the natural concentration range of 3alpha,5-cyclo to be established (0-280 ng/mL),
with a mean concentration of 22 ng/mL. Based on this an upper 3alpha,5-cyclo concentration
limit of 140 ng/mL is proposed as a GC-MS screening marker of DHEA abuse in athletes.
GC-C-IRMS analysis revealed significant 13C depletion of 3alpha,5-cyclo following DHEA
administration. In the single administration study, the delta13C value of 3alpha,5-cyclo
changed from -24.3 per thousand to a minimum value of -31.1 per thousand at 9 h post-
administration, before returning to its original value after 48 h. The multiple administration
study had a minimum delta13C 3alpha,5-cyclo of -33.9 per thousand during the
administration phase in contrast to the initial value of -24.2 per thousand. Preliminary studies
have shown 3alpha,5-cyclo to most likely be produced from DHEA sulfate found at high
levels in urine. The complementary use of GC-MS and GC-C-IRMS to identify new markers
of steroid abuse and the application of screening criteria incorporating such markers could
also be adapted by doping control laboratories to detect metabolites of androstenedione,
testosterone and dihydrotestosterone abuse [04051].
Internal standard
A facile six-step synthesis of 2,2,3,4,4-d5-androsterone-beta-D-glucuronide (1) starting from

epiandrosterone (2) in 63 percent yield is described and compared with several alternative
synthetic pathways. Compound 1 can be used as an internal standard in screening
procedures for anabolic steroids to monitor the hydrolysis step of the steroid glucuronides
prior to gas chromatography-mass spectrometry (GC-MS) analysis. Thus, a time consuming
solid-phase extraction step to remove possible hydrolysis inhibitors can be omitted [03035].
Initial testing procedures

Gas chromatography-(tandem) mass spectrometry, GC-MS(/MS) has been the primary tool
for analytical approaches aiming at steroidal agents (with few exemptions) for decades.
Nevertheless, small but relevant modifications to established assays have been applied to
tweak methods and gain a competitive edge, for example in terms of sensitivity, robustness,
or specificity. Employing conventional sample preparation and chromatography strategies as
well as established target analytes, the use of the triple-quadrupole mass analyzer enabled
LODs for clenbuterol at 0.01 ng/ml and for the steroidal agents between 0.2 and 1 ng/ml on a
routine basis. In order to strengthen and expand the detection capabilities of initial testing
965
procedures particularly regarding the extension of detection windows, in-depth investigations
revealing potential long-term metabolites of anabolic agents are of great importance [13012].
Interlaboratory comparisons
It was evaluated and compared four in vitro assays to detect androgen agonists and
antagonists in an international interlaboratory study. Laboratory 1 used a cell proliferation
assay (assay 1) with human mammary carcinoma cells stably transfected with human
androgen receptor. The other laboratories used reporter gene assays, two based on stably
transfected human prostate carcinoma cells (assay 2) or human mammary carcinoma cells
(assay 4), and the third based on transient transfection of Chinese hamster ovary cells
(assay 3). Four laboratories received four coded compounds and two controls: two steroidal
androgens, two antiandrogens, an androgenic control, 5alpha-dihydrotestosterone (DHT),
and an antiandrogenic control, bicalutamide (ICI 176,334). All laboratories correctly detected
the androgenic activity of 4-androsten-3,17-dione and 17alpha-methyltestosterone. For both
compounds, the calculated androgenic potencies relative to the positive control (RAPs)
remained within one order of magnitude. However, laboratory 3 calculated a 50-fold higher
RAP for 4-androsten-3,17-dione. All assays detected and quantified the antiandrogenic effect
of vinclozolin [median inhibitory concentration (IC50) values ranging from 1.1 times symbol
10-7 M to 4.7 times symbol 10-7 M]. In assays 2 and 3, vinclozolin showed partial androgenic
activity at the highest concentrations tested. For vinclozolin, calculated antiandrogenic
potencies relative to bicalutamide (RAAPs) differed no more than a factor of 10, and IC50
values matched those of bicalutamide. Similarly, we found antiandrogenic activity for tris-(4-
chlorophenyl)methanol. RAAP values were between 0.086 and 0.37. Three assays showed
cytotoxicity for this compound at or above 1 times symbol 10-5 M. In summary, all assays
proved sensitive screening tools to detect and quantify androgen receptor-mediated
androgenic and antiandrogenic effects of these chemicals accurately, with coefficients of
variation between 8 and 90 percent [04065].
Prolongation of the detection window for exogenous AAS
The detection of exogenous AAS is commonly based on the detection of their urinary phase-I
and phase-II metabolites. Owing to the long lasting effects of AAS on athletic performance,
the recent strategy of antidoping research is mainly focused on the search for long-term
metabolites (LTMs) of exogenous AAS. The implementation of LTMs into the analytical
screening procedures allows the prolongation of the detection window and an increased
retrospectivity for AAS. In 1996 the first publications focused on LTMs of exogenous AAS
were published. The prolongation of the detection windows is not only due to the
implementation of their LTMs into sports drug testing but also to the use of highly sensitive
detection methods employing chromatographic/mass spectrometric techniques (e.g. LC-
MS/MS, GC-MS/MS, HRMS) in screening procedures.. These strategies of exploiting new
analytical technologies and alternative target compounds have led to an enormous increase
of adverse analytical findings (AAFs) in the past. For instance, the implementation of the
LTM for metandienone (18-nor-17beta-hydroxymethyl, 17α-methyl-androst-1,4,13-trien-3-
one) in the screening procedure of the WADA accredited laboratory Cologne in 2006 resulted
in an increase of AAFs with metandienone of more than 400 percent from approximately 12–
15 AAFs in the years 2003–2005 to 68 AAFs in 2006, although the number and origin of the
analysed samples was nearly constant. As Cologne was the first WADA accredited
laboratory in the world to screen for this new metabolite, the number of AAFs for
metandienone in Cologne in 2006 was higher than the sum of AAFs for metandienone of all
other WADA accredited laboratories. This effect of the implementation of new LTMs was
even more pronounced with regard to the detection of AAFs for dehydrochloromethyl-
966
testosterone. In January 2013, three new LTMs for dehydrochloromethyltestosterone were
implemented in the screening procedures of the Cologne laboratory. This led to an increase
of AAFs from an annual average number of 1 to 82 cases in the first 11 months of 2013. Only
one or two of these 82 cases would have been detected with the methods used till the end of
2012 in the Cologne laboratory [14029].
Doping control and analytical factors
Factors that are not dependent on technical aspects and linked to the urinary steroid profile
data acquisition have been well discussed above. Nevertheless, analytical techniques that
are used in WADA-accredited laboratories should also be considered when a longitudinal
steroid profiles follow-up is evaluated and interpreted by anti-doping stakeholders [14450].
Traditionally, anabolic androgenic steroids and their representative metabolites have been
analysed by GC-MS-based methods, and the analysis of exogenous steroids has been
qualitative of origin. The analysis of “total” (i.e. free and glucuronide-conjugated) fraction of
steroids is indirect, since glucuronide-conjugated analytes are enzymatically hydrolysed
before the next step of the procedure, which is typically liquid–liquid extraction (LLE) in
alkaline environment, for example, tert-butyl methyl ether. For GC separation, the analytes
are converted to trimethylsilyl (TMS) derivatives and in order to accomplish this effectively
also to ketosteroids, the reaction mixture includes components which allow for in situ
formation of trimethyl iodosilane. Along the developments in instrument technologies, the
routine GC methods apply also tandem mass spectrometric (MS/MS) approaches to steroid
analysis, which provide improved selectivity and often also higher sensitivity for the detection
of analytes [14450].
In the new situation where quantitative data are based on initial testing procedure (ITP) and
are originating from different anti-doping laboratories, harmonised methodologies and
uniform reporting are prerequisites for the steroid profiling. For that purpose, WADA has
recently compiled a new technical document, TD2014EAAS, which is a mandatory
operational procedure for laboratories to support the steroid profile quantification of the
steroidal module of the ABP. The document gives an introduction to the steroid profiling and
detailed description on the requirements for ITP and confirmation analysis. All critical steps of
the analytical procedure and monitoring of their success are covered by the document.
Issues connected to sample matrix include the adjustment of the sample aliquot volume in
case of diluted samples or based on the gender of the athlete with the driving force to be
able to provide a reliable steroid profile for each urine sample. At later stages of the process,
the laboratory should also monitor the sample integrity, for example, for the presence of
microbial degradation. Most often the analytical procedure for steroid profile parameters
involves hydrolysis of glucuronide-conjugated steroids, extraction of free steroids and
deliberated steroid aglycons, derivatisation and GC-MS or GC-MS/MS analysis. In the
method characteristics, the type of glucuronidase enzyme is specified as purified preparation
for Escherichia coli to avoid by-products during the hydrolysis, the efficiency of which should
also be controlled. Amineptine is an example of a drug which may inhibit beta-glucuronidase
activity, and, furthermore, one of its metabolites yield in MS fragmentation which may
interfere the screening of androsterone and etiocholanolone. Other specific substances and
factors that have been reported to affect (mainly to interfere with) the hydrolysis of steroid
glucuronides, and to offer some references to the corresponding literature, include ascorbic
acid, aspartic acid, malic acid and high concentrations of salicylic acid, chlorinated
hydroquinones and benzoquinones, as well as glucosaccharic acid derivatives (e.g.
saccharic acid 1,4-lactone), which have been reported to inhibit beta-glucuronidase activity
under in vitro conditions [14450].
967
According to the technical document and GC separation, formation of TMS derivatives is
required and the completeness of derivatisation step should be verified by monitoring mono-
O-TMS and di-O-TMS derivative of androsterone. The document sets quality requirements
with respect to instrument operation and data collection by instructing the verification of the
stability of calibration standards, incorporation of quality control sample with each analytical
sequence and calculation of the T/E ratio, as well as by setting the requirements for the
sensitivity (limits of quantitation) and quantitative performance (relative standard combined
uncertainty, uc(%)) of the method. In confirmation analysis, the analytical approach
incorporates also information from GC-C-IRMS analysis (see below) and the results,
quantitation and identification of the relevant steroid profile marker(s) and/or T/E ratio. For
the result interpretation, the laboratories should also monitor the sample for the presence of
5alpha-reductase inhibitors (e.g. finasteride), which are not prohibited substances but may
alter the steroid profile due to their mechanism of action. In confirmation analysis, the
additional tests are applied to determine the presence of ethanol metabolites, ketoconazole
or signs of microbial degradation, to reveal the potential external interfering factors before
issuing the results into ADAMS and adaptive model purposes [14450].
Bayesian based screening
In elite sports, indirect testing of testosterone abuse is mainly based on the testosterone over
epitestosterone (T/E) ratio. Since this marker is characterized by a small ratio of intra- to
inter-individual variation, it is surprising that current anti-doping strategy uses a screening
test based on a population-based limit. From a database of more than 15,000 steroid profiles
obtained from routine controls, the collection of steroids profiles of 11 elite athletes followed
during 2 years, and a longitudinal study involving 17 amateur athletes, 8 of which were orally
administrated testosterone undecanoate pills, we selected 12 case studies to represent the
possible scenarios to which the anti-doping laboratories are confronted. Various detection
strategies at the disposal of the laboratories are employed and discussed, including isotope
ratio mass spectrometry (IRMS) analysis and a Bayesian interpretation of the T/E-time
profile. The weak sensitivity versus specificity relation of a population-based limit for the T/E
ratio is outlined. As a result, it was propose a Bayesian screening test whose T/E threshold
progressively evolves from a population basis to a subject basis as the number of individual
test results increases. It was found that this screening test heightens drastically the capacity
to detect testosterone abuse, at no additional financial and administrative expenses for anti-
doping authorities [07080].
It was developed a test that compares sequential measurements of a biomarker against

previous readings performed on the same individual. A probability mass function expresses
prior information on interindividual variations of intraindividual parameters. Then, the model
progressively integrates new readings to more accurately quantify the characteristics of the
individual. This Bayesian framework generalizes the two main approaches currently used in
forensic toxicology for the detection of abnormal values of a biomarker. The specificity is
independent of the number n of previous test results, with a model that gradually evolves
from population-derived limits when n = 0 to individual-based cutoff thresholds when n is
large. We applied this model to detect abnormal values in an athlete's steroid profile
characterized by the testosterone over epitestosterone (T/E) marker. A cross-validation
procedure was used for the estimation of prior densities as well as model validation. The
heightened sensitivity/specificity relation obtained on a large data set shows that longitudinal
monitoring of an athlete's steroid profile may be used efficiently to detect the abuse of
testosterone and its precursors in sports. Mild assumptions make the model interesting for
other areas of forensic toxicology [07081].
968
Purity certified reference materials
The need for certified reference materials (CRM) of anabolic-androgenic steroids reference
materials was emphasized by the Beijing 2008 Olympic game as a tool to improve
comparability, ensuring accuracy and traceability of analytical results for competing athletes.
The China National Institute of Metrology responded to the state request by providing seven
anabolic-androgenic steroids (AAS) reference materials for Beijing Olympic anti-doping. It
was described the production of the series of AAS CRMs, according to ISO Guides 34 and
35, which comprises the material processing, homogeneity and stability assessment, CRMs'
characterization including moisture content, trace metal content. The AASs' purity values
were assigned with collaborative study involved eight laboratories applying high resolution
liquid chromatography-diode array detector (HPLC-DAD). Homogeneity of the AAS CRMs
were determined by an in-house validated liquid chromatographic methodology. Potential
degradation during storage was also investigated and a shelf-life based on this value was
established. The certified values of CRMs were 99.8, 99.8, 99.6, 99.7, 98.8, 96.3, and 99.7
percent for methyltestosterone, testosterone propionate, nandrolone, nandrolone 17-
propionate, boldenone, trenbolone acetate and testosterone respectively. It was concluded
that the certified values for all the studied AAS reference materials are traceable to the
international system of units (SI). The CRMs developed were applied by 32 laboratory
including sports organizations and analytical laboratories during the 2008 Olympic game for
anti-doping control [12150].
Alkaline hydrolysis of steroid metabolites

The alkaline hydrolysis (as opposed to commonly employed enzymatic deconjugation) of
steroid metabolites has recently revealed additional analytes serving as potential markers for
the abuse of natural steroids. The utility of these markers concerning the detection of orally
administered testosterone undecanoate (120 mg) or dehydroepiandrosterone (DHEA) as well
as transdermally applied dihydrotestosterone (DHT) or testosterone (T) was presented.
Prolonged detection windows for testosterone undecanoate administration were recognized
particularly when employing androsta-1,4-dien-3,17-dione (ADION) as one variable of the
monitored steroid metabolite ratios. In cases of transdermal DHT and oral DHEA application,
no advantage over established steroid profile ratios was observed; however, the detection of
transdermally administered T was substantially improved when the ratio of ADION and
androst-15-en-3,17-dione (15-AD) was monitored [13012].
Stacking method of repetitive large volume sample injection
In one research, a novel stacking capillary electrophoresis method, repetitive large volume
sample injection and sweeping MEKC (rLVSI-sweeping MEKC) were developed to analyze
the presence of three androgenic steroids considered as sport doping drugs, testosterone
(T), epitestosterone (E) and epitestosterone glucuronide (EG) in urine. This method provides
better sensitivity enhancement than the traditional large volume sample stacking-sweeping
strategies due to sensitivity enhancement by repetitive injections. This multiple sampling
method enhances sensitivity of monitoring of urine samples by UV detection (254 nm).
Firstly, the phosphate buffer was filled into an uncoated fused silica capillary and the
samples were injected into the capillary at 10 psi for 20s, and then stacked at -10 kV for 1
min using phosphate buffer containing SDS. The above injecting and stacking steps were
repeated five times. Finally, separation was performed at -20 kV, using phosphate buffer
containing methanol, SDS and (2-hydroxypropyl)-beta-cyclodextrin. Method validation
showed that calibration plots were linear over a range of 5-200 ng/mL for T, 20-200 ng/mL for
E and 0.5-500 ng/mL for EG. The limits of detection were 1.0 ng/mL for T, 5.0 ng/mL for E
and 200.0 pg/mL for EG. When evaluating precision and accuracy, values of RSD and RE in
969
intra-day (n=3) and inter-day (n=5) analysis were found to be less than 10.0 percent.
Compared with the simple LVSS-sweeping, which is also a stacking strategy, this method
further improves sensitivity up to 25 folds (about 2500 folds with MEKC without
preconcentration). This method was applied to monitor 10 athletes' urine, and did not detect
any analyte. The novel stacking method was feasible for monitoring of doping by sportsmen
[12156].
The presence of microorganisms in urine samples, under favourable conditions of storage

and transportation, may alter the concentration of steroid hormones, thus altering the correct
evaluation of the urinary steroid profile in doping control analysis. According to the rules of
the World Anti-Doping Agency (WADA technical document TD2004 EAAS), a testosterone
deconjugation higher than 5 percent and the presence of 5alpha-androstane-3,17-dione and
5beta-androstane-3,17-dione in the deconjugated fraction, are reliable indicators of urine
degradation. The determination of these markers would require an additional quantitative
analysis since the steroids screening analysis, in anti-doping laboratories, is performed in the
total (free+conjugated) fraction. The aim of this work is therefore to establish reliable
threshold values for some representative compounds (namely 5alpha-androstane-3,17-dione
and 5beta-androstane-3,17-dione) in the total fraction in order to predict directly at the
screening stage the potential microbial degradation of the urine samples. Preliminary
evidence on the most suitable degradation indexes has been obtained by measuring the
urinary concentration of testosterone, epitestosterone, 5alpha-androstane-3,17-dione and
5beta-androstane-3,17-dione by gas chromatography-mass spectrometric every day for 15
days in the deconjugated, glucuronide and total fraction of 10 pools of urines from 60 healthy
subjects, stored under different pH and temperature conditions, and isolating the samples
with one or more markers of degradation according to the WADA technical document
TD2004EAAS. The threshold values for 5α-androstane-3,17-dione and 5beta-androstane-
3,17-dione were therefore obtained correlating the testosterone deconjugation rate with the
urinary concentrations of 5alpha-androstane-3,17-dione and 5beta-androstane-3,17-dione in
the total fraction. The threshold values suggested as indexes of urine degradation in the total
fraction were: 10 ng/mL for 5α-androstane-3,17-dione and 20 ng/mL for 5beta-androstane-
3,17-dione. The validity of this approach was confirmed by the analysis of routine samples
for more than five months (i.e. on a total of more than 4000 urine samples): samples with a
concentration of total 5α-androstane-3,17-dione and 5β-androstane-3,17-dione higher than
the threshold values showed a percentage of free testosterone higher than 5 of its total
amount; whereas free testosterone in a percentage higher than 5 of its total amount was not
detected in urines with a concentration of total 5alpha-androstane-3,17-dione and 5beta-
androstane-3,17-dione lower than the threshold values [11072].
Minimum package of anabolic steroids in urine

One method comprises the screening of two groups of anabolic compounds, the stilbenes
and several steroids. All compounds, inclusive their metabolites when possible, for which gas
chromatography-mass spectrometry (GC-MS) currently is the preferred analytical technique,
are included. Two different derivatives are prepared. One group, including the stilbenes, is
detected as HFB derivative (Method 1), the second group is detected as TMS derivative
(Method 2). The method is used to perform a qualitative and semi-quantitative analysis of a
minimum package of anabolic steroids to be included in National Residue Control Plans
based on Council Directive 96/23 and complies with the current Minimum Required
Performance Limits. The method has been validated according to Commission Decision
2002/657/EC. The CCalpha and CCbeta values are based on the detection of the most
abundant ion. Results of validation experiments are presented. The method is flexible and
due to the non-specific sample clean-up more and new anabolic compounds can be easily
added in order to new monitoring requirements [06098].
970
Endogenous steroids
Anabolic steroids are mainly excreted through the urinary route, requiring modifications of
their hydrophobic chemical structures. Phase I and phase II metabolic reactions are
responsible for, respectively, functionalisation and addition of conjugates (i.e. glucuronides or
sulfates) to steroids, thereby increasing their hydrophilicity and allowing their dissolution and
elimination in urine mixture. Since steroid conjugates analysis is not compatible with GC-MS,
the only analytical technique recognised by WADA for endogenous steroids quantification in
urine, deconjugation of the conjugated moiety by enzymatic hydrolysis (beta-glucuronidase)
is a crucial step during sample preparation and prior to GC-MS measurement. The in-
competition and out-of-competition testing programmes are the best strategies to screen and
confirm adverse analytical findings of exogenous and endogenous steroids. From the basis
of these routine analyses, the WADA-accredited laboratories provide harmonised and robust
analytical data for steroid profile. Recently, a new technical document TD2014EAAS12 has
been edited to ensure this harmonisation and is in force from January 2014. A detailed
description of selected aspects of TD2014EAAS is given later in this review. The application
of these rules should enable a suitable application of steroid module of the athlete biological
passport (ABP) and the assessment of steroid profile using the adaptive model [14450].
Distinguishing between endogenous and exogenous steroids

The contamination of commonly used supplements by unknown steroids as well as their
metabolites (parent compounds) become a challenge for the analytical laboratories. Although
the determination of steroids profile is not trivial because of the complex matrix and low
concentration of single compound, one of the most difficult current problem is to distinguish,
during analytical procedure, endogenous androgens such as testosterone, dehydro-
testosterone or dehydroepiandrosterone from their synthetic equivalents. The aim of one
work was to develop and validate an analytical procedure for determination of the steroid
profile in human urine by gas chromatography-combustion-isotope ratio mass spectrometry
(GC/C/IRMS) toward distinguishing between endogenous and exogenous steroids. Beside
the optimization of the experimental parameters for gas chromatography separation and
mass spectrometry, attention was focused on urine sample preparation. Using an optimized
sample preparation protocol it was possible to achieve better chromatographic resolutions
and better sensitivity enabling the determination of 5 steroids, androsterone, etiocholanolone,
testosterone, 5-androstandiol, 11-hydroxyandrdostane, pregnandiol, with the expanded
uncertainty below 0.1 percent. This enable to evaluate the significant shift of the delta 13C/12C
values for each of examined steroids (excluding ERC). The analytical protocol described in
this work was successfully used for the confirmation of positive founding urine by evaluation
T/E ratio after GC/C/IRMS analysis [150157].
ELISA
A multianalyte enzyme-linked immunosorbent assay (ELISA) has been developed for the
simultaneous detection of anabolic androgenic steroids (AAS) in human serum. The
multiplexed method was developed according to a planar strategy in which the analytes are
identified by their location in the microtiter plate. In the immunochemical procedure
established here, human serum samples are mixed with a cocktail of antibodies and added
to the distinct sections of a microplate biofunctionalized with different haptenized
biomolecules. The cocktail of antibodies consists of a mixture of polyclonal antibodies raised
against stanozolol (ST), boldenone (B), and tetrahydrogestrinone (THG). The whole
immunochemical analytical procedure takes around 2 h including sample preparation, and
many samples can be processed simultaneously to screen for the presence of the three AAS
in a single run. Using this ELISA, ST, B, and THG can be detected and quantified
971
individually. When used as a screening method, due to the cross-reactivity profiles of the
immunoreagents used, the presence of up to 11 AAS can be detected simultaneously. The
detectabilities achieved by this method in human serum are below the MRPLs (minimum
required performance limits) proposed by WADA (World Anti-Doping Agency) and reference
laboratories of the European Community [12151].
Liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS)

method for simultaneous and direct detection of 12 glucuronide-conjugated anabolic
androgenic steroid (AAS) metabolites in human urine was described. The compounds
selected were the main metabolites detected in human urine after dosing of the most widely
abused AAS in sports, e.g. methandienone, methenolone, methyltestosterone, nandrolone
and testosterone, and certain deuterium-labeled analogs of these metabolites. Sample
preparation and the LC-ESI-MS/MS method were optimized, validated, and the overall
process was implemented and the results between seven laboratories were compared. All
the metabolites were extracted simultaneously by solid-phase extraction (SPE) and analyzed
by LC-ESI-MS/MS with positive ionization mode and multiple reaction monitoring (MRM).
Recovery of the SPE for the anabolic androgenic steroid glucuronides was 89-100 percent
and ten out of twelve compounds had detection limits in the range of 1-10 ng/ml in urine. The
results for inter/intraday repeatability were satisfactory and the interlaboratory comparison
with authentic urine samples demonstrated the ease of method transfer from one instrument
setup to another. When equivalent triple quadrupole analyzers were employed the overall
performance was independent from instrument manufacturer, electrospray ionisation (ESI) or
atmospheric pressure chemical ionization (APCI) and liquid chromatohraphic (LC) column,
whereas major differences were encountered when changing from one analyzer type to
another, especially in the analysis of those AAS glucuronides ionized mainly as adducts
[08182].
Liquid chromatography-tandem mass spectrometry (LC-MS/MS)

In order to improve the detection capabilities of anabolic androgenic steroids (AAS) in sports,
a liquid chromatography-tandem mass spectrometry (LC-MS/MS) screening method for the
simultaneous detection of AAS phase I and phase II intact urinary metabolites (glucuronides
and sulfates) was developed. A total of 36 metabolites (7 unconjugated; 19 glucuronides and
10 sulfates) corresponding to 15 of the most reported AAS were included. Analytes were
extracted from urine using C18 cartridges. LC and MS conditions were studied in-depth to
determine the most sensitive and selective conditions for each analyte. A selected reaction
monitoring method was set up. The optimization of the experimental parameters for 13
metabolites not available as standards was performed using excretion study urines.
Extraction recoveries were above 77 percent for all 23 validated analytes. Intra-day precision
was lower than 21 percent, and LODs were in the range 0.25-4 ng/mL for 18 of the 23
analytes. Matrix effect was evaluated using post column infusion and ranged from 92 to 147
percent. The method was successfully applied to excretion study urines of different
exogenous AAS. The suitability of the strategy was demonstrated with methyltestosterone
and stanozolol excretion study urines by achieving detection times of 22 and 21 days,
respectively. The method is compliant with the World Antidoping Agency requirements for
most of the studied compounds. It represents a cost-effective approach that improves the
detection capabilities of AAS by increasing the sensitivity for some metabolites and by
including recently described phase II long-term metabolites not detectable using the current
screening strategy [150160].
972
Liquid chromatography-electrospray ionization tandem mass spectrometry
A qualitative liquid chromatography-electrospray ionization tandem mass spectrometry
method was developed for screening of the abuse of 4-chlorodehydromethyltestosterone,
danazol, fluoxymesterone, formebolone, metandienone, oxandrolone, and stanozolol. The
introduced method measures simultaneously nine different 17-alkyl-substituted anabolic
androgenic steroids or their unconjugated metabolites in human urine, using
methyltestosterone as an internal standard. Sample preparation involved one-step liquid
extraction. Liquid chromatographic separation was achieved on a reversed-phase column
with methanol-water gradient containing 5 mmol/l ammonium acetate and 0.01 percent (v/v)
acetic acid. Compounds were ionized in the positive mode and detected by multiple reaction
monitoring. All steroids within the study could be selectively detected in urine with detection
limits of 0.1-2.0 ng/mL. The method showed good linearity up to 250 ng/ml with correlation
coefficients higher than 0.9947. With simple and fast sample preparation, low limits of
detection, and high selectivity and precision, the developed method provides advantages
over the present testing methods and has the potential for routine qualitative screening
method of unconjugated 17-alkyl-substituted anabolic steroids in human urine [04066].
The relationships between the ionization profile, sensitivity, and structures of 64 exogenous
anabolic steroids (groups I-IV) was investigated under electrospray ionization (ESI)
conditions. The target analytes were ionized as [M + H]+ or [M + H-nH2 O]+ in the positive
mode, and these ions were used as precursor ions for selected reaction monitoring analysis.
The collision energy and Q3 ions were optimized based on the sensitivity and selectivity. The
limits of detection (LODs) were 0.05-20 ng/mL for the 64 steroids. The LODs for 38
compounds, 14 compounds and 12 compounds were in the range of 0.05-1, 2-5 and 10-20
ng/mL, respectively. Steroids including the conjugated keto-functional group at C3 showed
good proton affinity and stability, and generated the [M + H]+ ion as the most abundant
precursor ion. In addition, the LODs of steroids using the [M + H] + ion as the precursor ion
were mostly distributed at low concentrations. In contrast, steroids containing
conjugated/unconjugated hydroxyl functional groups at C3 generated [M + H - H2 O]+ or [M +
H - 2H2 O]+ ions, and these steroids showed relatively high LODs owing to poor stability and
multiple ion formation. An LC-MS/MS method based on the present ionization profile was
developed and validated for the determination of 78 steroids (groups I-V) in human urine
[150159].

A screening method for 18 frequently measured exogenous anabolic steroids and the
testosterone/epitestosterone (T/E) ratio in forensic cases has been developed and validated.
The method involves a fully automated sample preparation including enzyme treatment,
addition of internal standards and solid phase extraction followed by analysis by liquid
chromatography-tandem mass spectrometry (LC-MS-MS) using electrospray ionization with
adduct formation for two compounds. Urine samples from 580 forensic cases were analyzed
to determine the T/E ratio and occurrence of exogenous anabolic steroids. Extraction
recoveries ranged from 77 to 95 percent, matrix effects from 48 to 78 percent, overall
process efficiencies from 40 to 54 percent and the lower limit of identification ranged from 2
to 40 ng/mL. In the 580 urine samples analyzed from routine forensic cases, 17 (2.9 %) were
found positive for one or more anabolic steroids. Only seven different steroids including
testosterone were found in the material, suggesting that only a small number of common
steroids are likely to occur in a forensic context. The steroids were often in high
concentrations (>100 ng/mL), and a combination of steroids and/or other drugs of abuse
were seen in the majority of cases. The method presented serves as a fast and automated
screening procedure, proving the suitability of LC-MS-MS for analyzing anabolic steroids
[150158].
973
High-temperature liquid chromatography-isotope ratio mass spectrometry
Generally, compound-specific isotope analysis of steroids is carried out by gas
chromatography combined with isotope ratio mass spectrometry. Thus, a derivatization of the
steroids prior to the measurement is compulsory, and a correction of the isotopic data is
often necessary. To overcome this limitation, it was presented a new approach of high-
temperature liquid chromatography coupled with photodiode array detection and isotope ratio
mass spectrometry (HT-LC/PDA/IRMS) for the carbon isotope ratio analysis of unconjugated
steroids. A steroid mixture containing 19-norandrosterone, testosterone, epitestosterone,
androsterone, and 5beta-pregnane-3alpha,17alpha,20alpha-triol was fully separated on a C4
column under high-temperature elution with water as the sole eluent. The accuracy for
isotope analysis (± 0.5 ‰) was around 20 microg g(-1) for testosterone, epitestosterone (79
ng steroid absolute on column), and 30 μg7g for 19-norandrosterone, androsterone, and 5β-
pregnane-3alpha,17alpha,20alpha-triol (119 ng steroid absolute on column). The applicability
of the method was tested by measuring a pharmaceutical gel containing testosterone. With
this work, the scope of LC/IRMS applications has been extended to nonpolar compounds
[14059].
Mass spectrometry of steroid glucuronide conjugates

Owing to the developments of analytical instruments and interfaces (e.g. coupling high-
performance liquid chromatography to mass spectrometry), there has been increased
interest in new reference materials, for example in doping analysis with steroid glucuronide
conjugates. The synthesized reference material has to pass several characterization steps
including the use of gas chromatography/mass spectrometry (GC/MS) for its structure
confirmation. In the present study, the fragmentation and mass spectrometric behaviour of
several steroid glucuronide conjugates of endogenous and anabolic steroids after
derivatization to pertrimethylsilylated products and to methyl ester pertrimethylsilylated
products were investigated using GC/MS ion trap and GC/MS quadrupole instruments. The
mass spectra of the derivatives of androsterone glucuronide, d5-androsterone glucuronide,
epiandrosterone glucuronide, etiocholanolone glucuronide, 11beta-hydroxy etiocholanolone
glucuronide, 19-norandrosterone glucuronide, d4-19-norandrosterone glucuronide and
1alpha-methyl-5alpha-androstan-3alpha-ol-17-one glucuronide are presented and the origin
of typical fragment ions of the glycosidic and steroidal moieties is proposed, based on
different derivatization techniques including derivatization with d18-bistrimethylsilyl-
acetamide, methyl ester and trimethylsilyl ester derivatization and selected reaction
monitoring. Typical fragmentation patterns which are related to the steroid structure are
discussed [01041].
Gas chromatography/mass spectrometry
The control on use of anabolic agents in meat producing animals is generally based on urine,
faeces or hair analysis. This exercise, which is usually performed in slaughterhouses or on
farms, is not relevant to imported carcasses or retail meat. A single sensitive method for a
wide range of anabolic steroids was developed. After extraction of the lyophilised meat,
enzymatic hydrolysis was used for deconjugation. Solid-phase extraction on a polymeric
stationary phase was performed prior to hydrolysis of ester residues under alkaline
conditions. Liquid-liquid partitioning was used to separate the analytes into two main
categories: phenol containing molecules, such as phenolic steroids, resorcylic acid lactones
and stilbenes, and delta4-3-one containing molecules, such as most androgens and
progestagens. Solid-phase extraction on silica columns was performed before applying a
specific derivatisation for each compound sub-group. The combination of high-resolution
chromatography with a quadrupole mass spectrometer permitted detection of 23 steroids in
974
the 5-100 ng/kg range. Ion chromatograms for residue positive samples are shown and
discussed [00039].
An efficient procedure is described for the simultaneous determination of 9 androgen

glucuronides including androsterone, etiocholanolone, 11-ketoandrosterone, 11-
ketoetiocholanolone, 11beta-hydroxyandrosterone, 11beta-hydroxyetiocholanolone, and
dehydroepiandrosterone (DHEA) in 3-glucuronide form and dihydrotestosterone (DHT) and
testosterone in 17-glucuronide form from urine specimens. The method involves solid-phase
extraction of the urinary steroids using Serdolit PAD-1 resin, with subsequent conversion to
methyl ester-trimethylsilyl (Me-TMS) ether derivatives for the direct analysis by gas
chromatography-mass spectrometry (GC-MS) using high temperature MXT-1 (Silcosteel-
treated stainless steel) capillary column. Upon split injection of Me-TMS steroids at 330
degrees C into the MXT-1 capillary column initially maintained at 300 degrees C then
programmed to 322 degrees C at 2 degrees C/min, each androgen glucuronide was well
separated in excellent peak shape. The characteristic ions at m/z 217 constituting the base
peaks in the electron-impact (20 eV) mass spectra for most steroids permitted their sensitive
detection by GC-MS with selected-ion monitoring (SIM), whereas base peak ion at m/z 271
was used for the SIM of dehydroepiandrosterone-3-glucuronide. The detection limits for SIM
of most of the steroids were 15 pg except for the 3-glucuronides of 11-ketoandrosterone and
11-ketoetiocholanolone, which could be detected down to 20 pg. The SIM responses were
linear with correlation coefficients varying from 0.981 to 0.993 in the concentration range of
20 to 3000 ng/ml for the androgens studied. When applied to urine samples, the present
method allowed rapid screening for the 7 androgens in their glucuro-conjugated forms
simultaneously with good overall precision and accuracy within the normal concentration
ranges of 15.1 to 3124.6 ng/mL [00040].
Gas chromatography/mass spectrometry (GC/MS)

Helium is considered to be the ideal carrier gas for gas chromatography/mass spectrometry
(GC/MS) in general, and for use with an ion trap in particular. Helium is an inert gas, can be
used without special precautions for security and, moreover, it is needed as a damping gas
in the trap. A disadvantage of helium is the high viscosity resulting in long GC run times. In
this work hydrogen was tested as an alternative carrier gas for GC in performing GC/MS
analyses. A hydrogen generator was used as a safe source of hydrogen gas. It is
demonstrated that hydrogen can be used as a carrier gas for the gas chromatograph in
combination with helium as make-up gas for the trap. The analysis time was thus shortened
and the chromatographic performance was optimized. Although hydrogen has proven useful
as a carrier gas in gas chromatography coupled to standard detectors such as ECD or FID,
its use is not mentioned extensively in the literature concerning gas chromatography-ion trap
mass spectrometry. However, it is worth considering as a possibility because of its
chromatographic advantages and its advantageous price when using a hydrogen generator
[01042].
One study was carried out qualitatively and quantitatively to investigate the presence and the
concentrations of anabolic steroids in urine collected from orally administered humans.
Microanalysis of conjugated steroids by gas chromatography and mass spectrometry
(GC/MS) has been carried out. Following oral administration three major metabolites of
anabolic steroid drugs have been detected and partially characterized. The six steroids can
be analysed at the same time in 17 min. The lower detection limit was 10 ng/mL in 5 mL of
urine. The conjugated steroids from urine were centrifuged to 2,430 g for 10 min, the
supernatant solution passed through Amberlite XAD-2 column and the steroids eluted
fraction esterified by using MSTFA and TMSI. The rate of metabolism and urinary excretion
seem to be reasonably fast [043].
975
Gas chromatographic-mass spectrometric analysis of urinary anabolic steroids
One paper presents an automated method for extracting anabolic agents from urine samples
for their GC-MS analysis by selected-ion monitoring. The sample preparation was carried out
in a Hewlett-Packard 7686 SPE PrepStation system. Each 0.6-ml aliquot was hydrolyzed,
extracted, dried and trimethylsilyl (TMS) derivatized in a 2-ml vial without any hands-on labor.
When sample preparation was finished 2 microl of the extract was injected into the gas
chromatograph by split (1:10) mode. Due to the small amount of free space in the 2-ml vials
for handling the sample, parameters like time of hydrolysis, type of shaking, number of
extractions and some TMS derivatization parameters had to be adjusted to achieve the best
recovery for all of the compounds in the screening. Manual and automated sample
preparation schemes were compared in terms of linearity, precision, accuracy, limit of
detection and recovery data. When large concentrations were analyzed using the automated
method no carry-over effect was observed [01044].
Gas chromatography/tandem mass spectrometry
A fast and sensitive method for the comprehensive screening of anabolic agents and other
banned doping substances using gas chromatography/tandem mass spectrometry
(GC/MS/MS) with an external ionization ion trap mass spectrometer is presented. The
method takes advantage of the resolving power of MS/MS to eliminate background
interferences, thus speeding up the chromatographic analysis. For each compound, different
fragmentation reactions were studied and their collision energies optimized to obtain the best
sensitivity in terms of their signal-to-noise ratio (S/N). A dramatic reduction in overall analysis
time was achieved compared with other common approaches. More than 50 substances
could finally be monitored in less than 7.4 min with detection limits (S/N >3) lower than 0.5
ng/mL for most of the compounds with special sensitivity requirements according to the
International Olympic Committee (IOC). A validation procedure for qualitative analysis was
performed. The selectivity of the method showed that no interfering peaks were observed at
the retention time of the analytes. Good intermediate precision, below 25 percent for most of
the compounds, and robustness were observed. The optimized method was successfully
applied to analyse more than 100 real human urine samples with optimum sensitivity and
specificity rates [02050].
Solid-phase extraction purification of steroid sulfates
Steroid sulfates are a major class of steroid metabolite that are of growing importance in
fields such as anti-doping analysis, the detection of residues in agricultural produce or
medicine. Despite this, many steroid sulfate reference materials may have limited or no
availability hampering the development of analytical methods. It was reported simple
protocols for the rapid synthesis and purification of steroid sulfates that are suitable for
adoption by analytical laboratories. Central to this approach is the use of solid-phase
extraction (SPE) for purification, a technique routinely used for sample preparation in
analytical laboratories around the world. The sulfate conjugates of sixteen steroid
compounds encompassing a wide range of steroid substitution patterns and configurations
are prepared, including the previously unreported sulfate conjugates of the designer steroids
furazadrol (17beta-hydroxyandrostan[2,3-d]isoxazole), isofurazadrol (17beta-hydroxy-
androstan[3,2-c]isoxazole) and trenazone (17beta-hydroxyestra-4,9-dien-3-one). Structural
characterization data, together with NMR and mass spectra are reported for all steroid
sulfates, often for the first time. The scope of this approach for small scale synthesis is
highlighted by the sulfation of 1 microg of testosterone (17beta-hydroxyandrost-4-en-3-one)
as monitored by liquid chromatography-mass spectrometry (LCMS) [14759].
976
recoveries ranged from 77 to 95 percent, matrix effects from 48 to 78 percent, overall
process efficiencies from 40 to 54 percent and the lower limit of identification ranged from 2
to 40 ng/mL. In the 580 urine samples analyzed from routine forensic cases, 17 (2.9 %) were
[14760].
A rapid, sensitive and robust gas chromatography-triple quadrupole mass spectrometry

method was developed for the determination of seven anabolic agents in human urine. The
selection of analytes includes the main metabolites of all anabolics with higher sensitivity
requirements. After optimizing the fragmentation conditions for each compound, a validation
procedure for qualitative analysis was performed. The selectivity of the method showed that
no interfering peaks were observed at the retention time of the compound. Adequate
intermediate precision, below 14 percent, was observed for all of the compounds at the lower
concentration tested. The concentrations assayed were in accordance with the performance
limits required by the World Anti-Doping Agency (WADA). Unlike a previously published
GC/QqQ method, detection of 17alpha-methyl-5beta-androstane-3alpha,17beta-diol (the
main metabolites of methyltestosterone) at 2 ng/mL was accomplished under routine
conditions. The qualitative method was applied to the analysis of 1367 samples in the span
of 2 weeks, as part of the doping control of the XVI Pan American Games which took place in
Mexico (14th-30th October, 2011). The high sensitivity was maintained during the analysis of
all analytical batches, proving for the first time the excellent ruggedness of GC/QqQ methods
[12152].
Gas chromatography/combustion/isotopic ratio mass spectrometry (GC/C/IRMS)

It was presented a method for the analysis of urinary 16(5alpha)-androsten-3alpha-ol
together with 5beta-pregnane-3alpha,20alpha-diol and four testosterone metabolites:
androsterone (Andro), etiocholanolone (Etio), 5alpha-androstane-3alpha,17beta-diol
(5alphaA), 5beta-androstane-3alpha,17beta-diol (5betaA) by means of gas
chromatography/combustion/isotopic ratio mass spectrometry (GC/C/IRMS). The within-
assay and between-assay precision S.D.s of the investigated steroids were lower than 0.3
and 0.6 per thousand, respectively. A comparative study on a population composed of 20
subjects has shown that the differences of the intra-individual delta(13)C-values for
16(5alpha)-androsten-3alpha-ol and 5beta-pregnane-3alpha,20alpha-diol are less than 0.9
per thousand. Thereafter, the method has been applied in the frame of an excretion study
following oral ingestion of 50 mg DHEA initially and oral ingestion of 50mg pregnenolone 48
h later. Our findings show that administration of DHEA does not affect the isotopic ratio
977
values of 16(5alpha)-androsten-3alpha-ol and 5beta-pregnane-3alpha,20alpha-diol, whereas
the isotopic ratio values of 5beta-pregnane-3alpha,20alpha-diol vary by more 5 per thousand
upon ingestion of pregnenolone. We have observed delta(13)C-value changes lower than 1
per thousand for 16(5alpha)-androsten-3alpha-ol, though pregnenolone is a precursor of the
16-ene steroids. In contrast to 5beta-pregnane-3alpha,20alpha-diol, the 16-ene steroid may
be used as an endogenous reference compound when pregnenolone is administered
[04067].
Sensitivity of GC-EI/MS, GC-EI/MS/MS, LC-ESI/MS/MS, and GC-ESI/MS/MS for AAS

One study compared the sensitivity of various separation and ionization methods, including
gas chromatography with an electron ionization source (GC-EI), liquid chromatography with
an electrospray ionization source (LC-ESI), and liquid chromatography with a silver ion
coordination ion spray source (LC-Ag(+) CIS), coupled to a mass spectrometer (MS) for
steroid analysis. Chromatographic conditions, mass spectrometric transitions, and ion source
parameters were optimized. The majority of steroids in GC-EI/MS/MS and LC-Ag(+)
CIS/MS/MS analysis showed higher sensitivities than those obtained with other analytical
methods. The limits of detection (LODs) of 65 steroids by GC-EI/MS/MS, 68 steroids by LC-
Ag(+) CIS/MS/MS, 56 steroids by GC-EI/MS, 54 steroids by LC-ESI/MS/MS, and 27 steroids
by GC-ESI/MS/MS were below cut-off value of 2.0 ng/mL. LODs of steroids that formed
protonated ions in LC-ESI/MS/MS analysis were all lower than the cut-off value. Several
steroids such as unconjugated C3-hydroxyl with C17-hydroxyl structure showed higher
sensitivities in GC-EI/MS/MS analysis relative to those obtained using the LC-based
methods. The steroids containing 4, 9, 11-triene structures showed relatively poor
sensitivities in GC-EI/MS and GC-ESI/MS/MS analysis. The results of this study provide
information that may be useful for selecting suitable analytical methods for confirmatory
analysis of steroids [150162].
Liquid chromatography-tandem mass spectrometry method (LC-MS/MS)
The applicability of liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the

detection of the free anabolic steroid fraction in human urine was examined. Electrospray
ionization (ESI), atmospheric pressure chemical ionization and atmospheric pressure
photoionization methods were optimized regarding eluent composition, ion source
parameters and fragmentation. The methods were compared with respect to specificity and
detection limit. Although all methods proved suitable, LC/ESI-MS/MS with a methanol-water
gradient including 5 mM ammonium acetate and 0.01 percent acetic acid was found best for
the purpose. Multiple reaction monitoring allowed the determination of steroids in urine at low
nanogram per milliliter levels. LC/MS/MS exhibited high sensitivity and specificity for the
detection of free steroids and may be a suitable technique for screening for the abuse of
anabolic steroids in sports [02051].
The liquid chromatography-tandem mass spectrometry method (LC-MS/MS) was developed

and validated to detect androgenic steroids: trenbolone, nortestosterone, boldenone,
methylboldenone, testosterone, methyltestosterone, 17beta-1-testosterone and their
metabolites in bovine urine. Sample preparation before LC-MS/MS analysis involved an
enzymatic hydrolysis with glucuronidase AS-HP, isolation of free hormones from urine on
C(18) SPE column and purification of the extract using liquid-liquid extraction with n-pentane
and SPE NH(2) column. For the chromatographic separation of steroids, the Poroshell 120-
EC C18 column (150 × 2.1 mm, 2.7 microm) was used. Mass spectrometric measurement
was achieved using the API 4000 triple quadrupole instrument with a TurboIon-Spray source
operating in positive electrospray ionization mode. The procedure was validated according to
the Decision 2002/657/EC. Recovery ranged from 77 to 119 percent for all examined
978
compounds. The repeatability was below 20 percent and reproducibility did not exceed the
25 percent. The linearity was good for all analytes in the whole range of tested
concentrations, as proved by the correlation coefficients greater than 0.99. The application of
an innovative Poroshell column allowed for very good chromatographic separation of steroids
with a much shorter time of analysis [12153].
A fast and sensitive LC-MS/MS method for the quantitative analysis of seven steroid
hormones in 150 microL of human serum was developed and validated. The following
compounds were included: 17alpha-hydroxypregnenolone, 17alpha-hydroxyprogesterone,
androstenedione, dehydroepiandrosterone, testosterone, pregnenolone, and progesterone.
Individual stable isotope-labeled analogues were used as internal standards. Sample
preparation was performed by liquid-liquid extraction, followed by oxime derivatization to
improve the ionization efficiency of the analytes. In contrast to the common derivatization-
based methods, the reaction was incorporated into the sample preparation process and the
only additional step due to the derivatization was a short heating of the autosampler vials
before the sample injection. Chromatographic separation was achieved on a reversed-phase
column using a methanol-water gradient. For the analyte detection, a triple quadrupole
instrument with electrospray ionization was used. Total run time was 7.0 min and the lower
limits of quantification were in the range of 0.03-0.34 nM (0.01-0.10 ng/mL), depending on
the analyte. The method was validated using human serum samples from both sexes and
applied for the serum steroid profiling of endometriosis patients [11572].
A specific and sensitive multi-method based on liquid chromatography-tandem mass

spectrometry using atmospheric pressure chemical ionization (LC-APCI-MS/MS) has been
developed for the determination of 20 anabolic steroids in muscle tissue (diethylstilbestrol,
beta-estradiol, ethynylestradiol, alpha/beta-boldenone, alpha/beta-nortestosterone,
methyltestosterone, beta-trenbolone, triamcinolone acetonide, dexamethasone,
flumethasone, alpha/beta-zearalenol, alpha/beta-zearalanol, zearalenone, melengestrol
acetate, megestrol acetate and medroxyprogesterone acetate). The procedure involved
hydrolysis, extraction with tert-butyl methyl ether, defattening and final clean-up with solid
phase extraction (SPE) on Oasis HLB and Amino cartridges. The analytes were analyzed by
reversed-phase LC-MS/MS, in positive and negative multiple reaction monitoring (MRM)
mode, acquiring two diagnostic product ions from each of the chosen precursor ions for the
unambiguous confirmation of the hormones. The method was validated at the validation level
of 0.5ng/g. The accuracy and precision of the method were satisfactory. The decision limits
CC-alpha ranged from 0.03 to 0.14ng/g while the detection capabilities CC-beta ranged from
0.05 to 0.24ng/g. The developed method is sensitive and useful for detection, quantification
and confirmation of these anabolic steroids in muscle tissue and can be used for residue
control programs [09067].

Anabolic androgenic steroids (AAS) are an important class of doping agents. The
metabolism of these substances is generally very extensive and includes phase-I and phase-
II pathways. In this work, a comprehensive detection of these metabolites is described using
a 2-fold dilution of urine and subsequent analysis by liquid chromatography-high resolution
mass spectrometry (LC-HRMS). The method was applied to study 32 different metabolites,
excreted free or conjugated (glucuronide or sulfate), which permit the detection of misuse of
at least 21 anabolic steroids. The method has been fully validated for 21 target compounds
(8 glucuronide, 1 sulfate and 12 free steroids) and 18 out of 21 compounds had detection
limits in the range of 1-10 ng/mL in urine. For the conjugated compounds, for which no
reference standards are available, metabolites were synthesized in vitro or excretion studies
were investigated. The detection limits for these compounds ranged between 0.5 and 18
ng/mL in urine. The simple and straightforward methodology complements the traditional
979
methods based on hydrolysis, liquid-liquid extraction, derivatization and analysis by gas
chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry
(LC-MS) [14259].
Liquid chromatography/electrospray ion trap mass spectrometry

A fast and selective LC/MS/MS method for the screening of four anabolic steroids in human
urine has been developed and validated. Liquid-liquid extraction with diethyl ether was
applied after enzymatic hydrolysis. Analyses were performed on an ion trap mass
spectrometer equipped with electrospray ionisation. MS/MS was applied for all compounds.
The analytical run time was 11 min. The LOD for all compounds varied between 1 and 10
ng/mL. Left-over A samples, which were declared positive by GC/MS for the presence of 3'-
hydroxystanozolol, were assessed using the described method [06099].
Ultra high performance liquid chromatography tandem mass spectrometry

The use of doping agents, once restricted to professional athletes, has nowadays become a
problem of public health, since it also concerns young people and non-competing amateurs
in different sports. The use is also diffused in social life for improving physical appearance
and enhancing performance and even dietary supplements assumed to improve
performance often contain anabolic steroids. While decades ago the so-called "classical
doping agents" (like stimulants and narcotics) were used, to-day anabolic steroids are more
widely diffused. Anabolic steroids are synthetic substances prepared by introducing
modifications in the molecular structure of testosterone, the main natural androgenic anabolic
steroid that forms in testes interstitial cells. Over the years, always new doping substances
are synthesized and, as a consequence, the list of prohibited compounds is continuously
updated and new suitable analytical methods for their detection and determination in
biological matrices are continuously required. In doping control analysis the knowledge of
steroid metabolism pathway in human body is of primary importance and the analytical
methods must permit the simultaneous detection and determination not only of the forbidden
precursor agents but also of their metabolites. In addition, the potential presence and amount
in the biological samples of species that can interfere in the analysis should be evaluated.
Also the several anabolic steroids, specifically designed to circumvent doping control, put on
the market have been incorporated in the list of the prohibited substances of the World Anti-
Doping Agency (WADA). In WADA list steroids figure in three main classes, namely anabolic
steroids, corticosteroids and substances with anti-estrogenic properties. It must be strongly
reminded that assumption of doping agents not only leads to athletes the possible failing of
doping tests but causes important health risk and WADA prohibited list establishes criteria to
highlight the alteration of the natural steroid profile caused by exogenous administration.
Doping control analyses are generally performed in urine, a matrix that provides a prolonged
detection time window, and less often in blood, serum, plasma, hair, saliva, and nails. To
identify the chemical structures of anabolic steroids the use of mass spectrometry detection
is very advantageous. Gas chromatography-mass spectrometry (GC-MS) techniques allowed
for the development of comprehensive screening methods. GC-MS methods are sensitive
and robust but present the disadvantages of time-consuming sample pretreatment, that is
often based on hydrolysis and derivatisation reactions. Liquid chromatography-mass
spectrometry (LC-MS) methods have been successfully used to identify and determinate
steroids in different matrices, as well as to study their metabolisms. Nowadays, automatic
rapid ultra high performance liquid chromatography (UHPLC) tandem mass spectrometry has
become the technique of choice for steroid analysis. Due to its generally higher speed,
sensitivity, reproducibility and specificity with respect to HPLC, it can be used to
simultaneously separate and determinate multi component steroid mixtures. The technique is
of huge interest to separate conjugates anabolic androgenic steroids, as it allows efficiency
enhancement due to the small particle (sub-2μm) column packing, which provides high peak
capacity within analysis times even 5-10 fold shorter than conventional HPLC methods.
980
Modern multiplex instruments can analyze thousands of samples per month so that,
notwithstanding the generally high instrumental costs, the cost of the individual assay is
affordable. In addition, the improved specificity and resolution offered by time-of-flight or
quadrupole time-of-flight mass spectrometry allow their application in doping control analysis
or in steroid profiling for accurate and sensitive full mass range acquisition. Aim of one
review was to consider, compare and discuss the applications of the UHPLC/MS methods
present in literature for the identification and determination of forbidden steroids and their
metabolites in human biological matrices [13004].
Automated solid phase extraction and LC-MS-MS

recoveries ranged from 77 to 95 percent, matrix effects from 48 to 78percent, overall process
efficiencies from 40 to 54 percent and the lower limit of identification ranged from 2 to 40
ng/mL. In the 580 urine samples analyzed from routine forensic cases, 17 (2.9 %) were
[14643].
Turbulent flow solid-phase extraction coupled with LC-MS-MS

A novel method for automated and sensitive analysis of testosterone, androstenedione,
methyltestosterone and methenolone in urine samples by online turbulent flow solid-phase
extraction coupled with high performance liquid chromatography-tandem mass spectrometry
was developed. The optimization and validation of the method were discussed in detail. The
Turboflow C18-P SPE column showed the best extraction efficiency for all the analytes.
Nanogram per liter (ng/L) level of AAS could be determined directly and the limits of
quantification (LOQs) were 0.01 ng/mL, which were much lower than normally concerned
concentrations for these typical anabolic androgenic steroids (AAS) (0.1 ng/mL). The linearity
range was from the LOQ to 100 ng/mL for each compound, with the coefficients of
determination ranging from 0.9990 to 0.9999. The intraday and interday relative standard
deviations (RSDs) ranged from 1.1 to 14.5 percent (n=5). The proposed method was
successfully applied to the analysis of urine samples collected from 24 male athletes and 15
patients of prostate cancer. The proposed method provides an alternative practical way to
rapidly determine AAS in urine samples, especially for clinical monitoring and doping control
[14644].
Ultra-liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS)
Anabolic-androgenic steroids (AASs) have been illegally used in counterfeit drugs to improve
the performance of athletes. In addition, AASs have been used for cosmetic purpose by non-
athletes. To determine the presence of 26 AASs, an analysis method using ultra-liquid
chromatography-tandem mass spectrometry (UHPLC-MS/MS) was developed and validated.
The validated method was applied to 19 counterfeit drugs collected from the Internet and off-
981
line markets during 2014. Nearly 50 percent (9/19) of the samples contained one of these 26
AASs. In addition, the concentration ranges of the AASs ranged from 0.09 to 119,228.57
mg/kg in the suspected samples. The determined AASs primarily consisted of testosterone
and testosterone 17-propionate (26 %) followed by boldenone (21 %). These results indicate
the adulteration of over-the-counter counterfeit drugs, and the continuous monitoring of
counterfeit drugs or dubious dietary supplements containing anabolic steroids is warranted
[150165].
Ultra-high-performance supercritical-fluid chromatography hyphenated tandem MS
Profiling conjugated urinary steroids to detect anabolic-steroid misuse is recognized as an

efficient analytical strategy in both chemical-food-safety and anti-doping fields. The relevance
and robustness of such profiling rely on the analysis of glucuronide and sulfate steroids,
which is expected to have properties including accuracy, specificity, sensitivity, and, if
possible, rapidity. In this context, the ability of ultra-high-performance supercritical-fluid
chromatography (UHPSFC) hyphenated tandem mass spectrometry (MS-MS) to provide
reliable and accurate phase II analysis of steroids was assessed. Four stationary phases
with sub-2 microm particles (BEH, BEH 2-ethyl-pyridine, HSS C18 SB, and CSH
fluorophenyl) were screened for their capacity to separate several conjugated steroid
isomers. Analytical conditions including stationary phase, modifier composition and
percentage, back pressure, column temperature, and composition and flow rate of make-up
solvent were investigated to improve the separation and/or the sensitivity. Thus, an analytical
procedure enabling the analysis of eight glucuronide and 12 sulfate steroids by two different
methods in 12 and 15 min, respectively, was optimized. The two procedures were evaluated,
and UHPSFC-MS-MS analysis revealed its ability to provide sensitive (limits of quantification:
0.1 ng/mL and 0.5 ng/mL for sulfate and glucuronide steroids, respectively) and reliable
quantitative performance through the use of suitable labeled internal standards. Comparison
with UHPLC-MS-MS was performed, and UHPSFC-MS-MS obtained better performance in
terms of sensitivity. Finally, as a proof of concept, this so-called green technology was used
in a chemical-food-safety context to profile steroid conjugates in urine samples from bovines
treated with estradiol. Graphical Abstract Glucuronide and sulfate steroids analysis in urine
by ultra-high performance supercritical fluid chromatography hyphenated tandem mass
spectrometry [150167].
Direct measurement of urinary testosterone and epitestosterone conjugates
Measurement of the ratio of testosterone (T) and epitestosterone (E) in urine has been used
as an indication of “natural” steroid supplementation for a decade. The direct measurement
of the glucuronide and sulfate conjugates of testosterone and epitestosterone by high-
performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) should
resolve a number of issues regarding unusual metabolism due to either genetic disposition or
attempts to avoid detection of abuse. Determination of nanomoles per liter (0.1 ppb)
concentrations of analytes in a complex biological matrix by HPLC/MS/MS is complicated by
sample matrix-specific ion suppression during ESI. Deuterated internal standards of all
compounds were used to overcome the effects of suppression. Comparison of the
HPLC/MS/MS method with a two-part gas chromatographic/mass spectrometric method
showed statistical equivalence in urine samples. Analysis of urine samples with elevated T-
glucuronide to E-glucuronide ratios did not show that a significant number could be explained
by an elevated excretion of epitestosterone sulfate. The HPLC/MS/MS method was also
used further to characterize genetic and metabolic factors that give rise to unusual T/E ratios
[00037].
982
Ligand and structure-based virtual screening
Parallel ligand- and structure-based virtual screenings of 269 steroids with anabolic activity
evaluated in vivo were performed. The quantitative structure-activity relationship (QSAR)
model expressed by selected descriptors as the octanol-water partition coefficient, the molar
volume and the quantum mechanical calculated charge values on atoms C1, C2, C5, C9,
C10, C14 and C17 of the steroid skeleton, expresses structural features of anabolic steroids
(AS) contributing to the transport and steroid-receptor interaction. On the other hand,
computational simulations of a candidate ligand binding to a receptor study (a "docking"
procedure) predict the association of these AS with the human androgen receptor (AR).
Fourteen compounds were identified as lead; the most potent was the 7alpha-methylestr-4-
en-3, 17-dione. It was concluded that a good anabolic activity requires hydrogen bonding
interactions between both Arg752 and Gln711 residues in the cycles A with O3 atom of the
steroid and either Asn705 and Thr877 residues in the cycles D of steroid with O17 atom
[13171].
Dried blood spot
In recent years, there has been an increase of doping with “endogenous-like substances”,
such as testosterone and some of its metabolic precursors. Discriminating the exogenous
intake of these substances from their endogenous origin constitutes a challenge in today’s
sports drug testing. Pharmaceutical testosterone preparations are commercially available for
oral, percutaneous, and intramuscular administration. The International Olympic Committee
criterion for suspecting exogenous testosterone intake, a urinary concentration ratio of
testosterone glucuronide to epitestosterone glucuronide (TG/EG) >6, needs elaborate follow-
up for definitive decisions. There are a few subjects who have physiologically increased
urinary TG/EG ratios, whereas other individuals do not show a TG/EG ratio >6 even after
exogenous intake of testosterone. In addition, oral administration of testosterone produces a
high but brief increase in the urinary TG/EG ratio, which probably would be difficult to detect
(as judged by the criterion TG/EG >6) as an indication of exogenous testosterone intake
when urine is collected after a sporting event (which usually includes a 1-h delay plus an
additional waiting period for the final collection of urine). Apart from measurement of the 13C
content of urinary testosterone metabolites by isotope-ratio mass spectrometry, a
theoretically better approach might be to analyze one drop of blood collected immediately
after the sporting event. The sample may be obtained by fingerprick or earprick, which
currently are used for lactic acid and other measurements [00041].
Blood sampling is not a common practice for sports drug testing. Our aim was to investigate
whether dried blood spots on filter paper could be an alternative to plasma samples for
monitoring steroid profiles in dope testing. It was collected dried blood spots and plasma
from six healthy Caucasian subjects after an oral 120-mg dose of testosterone undecanoate
(TU). Nonconjugated testosterone, testosterone glucuronide (TG), androsterone glucuronide
(AG), and etiocholanolone glucuronide (EtG) were measured by gas chromatography-mass
spectrometry in both matrices. 17alpha-Hydroxyprogesterone (17alphaOHP) and luteinizing
hormone (LH) also were measured in the plasma samples. For comparison, similar
measurements were done on samples obtained from the same subjects given 25 mg of
testosterone propionate (TP) plus 110 mg of testosterone enanthate (TE) intramuscularly
after a wash-out period. After oral TU intake, TG, AG, and EtG increased sharply, whereas
nonconjugated testosterone did not change significantly. Results on dried blood spots
correlated well with those on plasma. The TG/testosterone ratio in blood or plasma was
verified to be a sensitive and specific marker (significantly increased for up to 8 h after
intake) for oral TU intake but not for intramuscular administration of TP plus TE. Little
983
suppression of plasma LH and 17alphaOHP was observed after a single oral dose of TU.
One subject did not show a significant increase of blood TG after oral TU intake. It was
concluded that the measurement of glucuronide conjugates in blood and plasma samples is
relevant for sports drug testing when analyzing the steroid profile. Dried blood spots collected
on filter paper are a suitable alternative to plasma for detecting testosterone abuse [00041].
3-Oxo-steroidal agents
Focusing on 3-oxo-steroidal agents (methyltestosterone, nandrolone, boldenone, trenbolone,

fluoxymesterone, mesterolone, and bolasterone), a MALDI-MS-based protocol was
presented, reporting on the detection of the intact steroids as extracted from spiked urine at 2
ng/mL. Using a proprietary derivatization reagent with hydrazine-based chemistry,
substantial sensitivity was accomplished enabling the above mentioned detection limits of the
assay; however, as in the aforementioned study on CE and AAS, the target analytes (i.e. the
intact drugs) were not an appropriate choice of compounds to provide proof-of-concept for a
potential doping control method. Besides the fact that steroid analysis in sports drug testing
necessitates an extraordinary comprehensive picture of xenobiotic and natural/endogenous
steroids and their metabolites, which is not yet achievable by the MALDI-MS approach, the
method would benefit from demonstrating the capability of the developed reagent to convert
17-oxo groups. Aiming at the facilitated data interpretation, the same group presented a
software supporting the library-assisted identification of steroidal agents measured by means
of MALDI-MS(/MS) [13009].
Focusing on 3-oxo-steroidal agents (methyltestosterone, nandrolone, boldenone, trenbolone,

fluoxymesterone, mesterolone, and bolasterone), a MALDI-MS-based protocol was
presented, reporting on the detection of the intact steroids as extracted from spiked urine at 2
ng/mL. Using a proprietary derivatization reagent with hydrazine-based chemistry,
substantial sensitivity was accomplished enabling the above mentioned detection limits of the
assay; however, as in the aforementioned study on CE and AAS, the target analytes (i.e. the
intact drugs) were not an appropriate choice of compounds to provide proof-of-concept for a
potential doping control method. Besides the fact that steroid analysis in sports drug testing
necessitates an extraordinary comprehensive picture of xenobiotic and natural/endogenous
steroids and their metabolites, which is not yet achievable by the MALDI-MS approach, the
984
method would benefit from demonstrating the capability of the developed reagent to convert
17-oxo groups. Aiming at the facilitated data interpretation, the same group presented a
software supporting the library-assisted identification of steroidal agents measured by means
of MALDI-MS(/MS) [14009].
Stable isotope dilution liquid chromatography electrospray ionization
Prostate cancer is the most frequently diagnosed form of cancer in males in the United
States. The disease is androgen driven and the use of orchiectomy or chemical castration,
known as androgen deprivation therapy (ADT) has been employed for the treatment of
advanced prostate cancer for over 70 years. Agents such as GnRH agonists and non-
steroidal androgen receptor antagonists are routinely used in the clinic, but eventually
relapse occurs due to the emergence of castration-resistant prostate cancer. With the
appreciation that androgen signaling still persists in these patients and the development of
new therapies such as abiraterone and enzalutamide that further suppresses androgen
synthesis or signaling, there is a renewed need for sensitive and specific methods to quantify
androgen precursor and metabolite levels to assess drug efficacy. It was described the
development, validation and application of a stable isotope dilution liquid chromatography
electrospray ionization selected reaction monitoring mass spectrometry (SID-
LC/ESI/SRM/MS) method for quantification of serum keto-androgens and their sulfate and
glucuronide conjugates using Girard-T oxime derivatives. The method is robust down to 0.2-
4 pg on column, depending on the androgen metabolite quantified, and can also quantify
dehydroepiandrosterone sulfate (DHEA-S) in only 1 microL of serum. The clinical utility of
this method was demonstrated by analyzing serum androgens from patients enrolled in a
clinical trial assessing combinations of pharmacological agents to maximally suppress
gonadal and adrenal androgens (Targeted Androgen Pathway Suppression, TAPS clinical
trial). The method was validated by correlating the results obtained with a hydroxylamine
derivatization procedure coupled with tandem mass spectrometry using selected reaction
monitoring that was conducted in an independent laboratory [13173].
Microflow tile technology and LC-MS/MS
A novel microfluidic chromatography device coupled with tandem mass spectrometry (LC-
MS/MS) was utilized for the multiplex analysis of 5 steroids (testosterone,
dihydrotestosterone, progesterone, cortisol, cortisone) in human serum. The use of
microfluidics allowed for reduction of the chromatographic flow rate to 3 microl/min with
overall method run times comparable to standard flow LC-MS/MS methods reported in the
literature, corresponding to a 150 fold decrease in solvent consumption. Furthermore, a
simple sample preparation protocol was employed requiring injection of only 0.5 microl of
sample, corresponding to a 100-400 fold increase in on-column sensitivity as compared to
published standard flow assays. The measured LOQ for both testosterone and progesterone
was 0.4 ng/mL, representing an improvement over reported literature values obtained by
standard flow methods employing comparable sample preparation and large injection
volumes. The LOQs for cortisol (1.9 ng/mL), cortisone (0.3n g/mL), and dihydrotestosterone
(1.4 ng/mL) were all within a biologically relevant range. A comparison of clinical serum
samples was performed for the analysis of testosterone using this microfluidic LC-MS/MS
assay and the Beckman Access II automated antibody-based measurement system. The
immunoassay results were systematically higher due to matrix interference which was easily
resolved with the increased chromatographic resolution obtained in the microflow LC-MS/MS
assay [13174].
Effects of sample storage condition steroid hormones in saliva

985
Measurement of steroid hormones in saliva is increasingly common in elite sport settings.
However, this environment may enforce handling and storage practices that introduce error
in measurement of hormone concentrations. It was assessed the influence of storage
temperature and duration on reproducibility of salivary steroid levels. Nine healthy adults
provided morning and afternoon saliva samples on two separate occasions. Each sample
was divided into identical saliva aliquots which were stored long-term (i.e. 28 and 84 days) at
- 80°C or - 20°C (testing day 1), and short-term (i.e. 1, 3, 7 and 14 days) at 4°C or 20°C
(testing day 2). Samples were analyzed for cortisol, testosterone and estradiol using ELISA.
In non-freezer conditions, there was a decrease from baseline to 7 days in testosterone (- 26
± 15 %) and estradiol (- 58 ± 17 %) but not cortisol concentrations. This decrease was larger
in samples stored at room temperature than in the refrigerator. There were small but
significant changes in measured concentrations of all hormones after 28 and/or 84 days of
storage in freezer conditions, but these were generally within 12 percent of baseline
concentrations, and may be partly explained by inter-assay variability. Whole saliva samples
to be analyzed for cortisol, testosterone and estradiol should be frozen at -20°C or below
within 24 h of collection, and analyzed within 28 days. Storage of samples for measurement
of testosterone and estradiol at temperatures above -20°C can introduce large error variance
to measured concentrations [13175].
IRMS
Following suspicious initial test results obtained by steroid profile analyses, confirmatory
testing procedures preferably employing gas chromatography-combustion-isotope ratio mass
spectrometry (GC-C-IRMS) are indicated. However, since IRMS analyses are comparably
time consuming and costly, optimized screening methods providing appropriate sensitivity
and specificity for natural/endogenous steroid abuse are desirable [14715].
Nowadays, the importance of IRMS in sports drug testing is substantial and, as recently
concluded, its relevance is continuously growing and new instrumental options allow for
enhanced detection assays. In doping controls, IRMS is currently applied mainly to the
analysis of natural/endogenous anabolic-androgenic steroids and its metabolites; however,
additional candidates such as cortisone or 5-aminoimidazole-4-carboxamide-ribonucleoside
(AICAR) have been subject of recent research projects and are conceivable future target
analytes. IRMS represents a comparably complex and challenging analytical methodology
that necessitates thorough consideration of information indicating the need for an IRMS-
based analysis of a doping control sample and factors influencing the analytical result as well
as its interpretation [13009].
Cross-reactivity of steroid hormone immunoassays

Immunoassays are widely used in clinical laboratories for measurement of plasma/serum
concentrations of steroid hormones such as cortisol and testosterone. Immunoassays can be
performed on a variety of standard clinical chemistry analyzers, thus allowing even small
clinical laboratories to do analysis on-site. One limitation of steroid hormone immunoassays
is interference caused by compounds with structural similarity to the target steroid of the
assay. Interfering molecules include structurally related endogenous compounds and their
metabolites as well as drugs such as anabolic steroids and synthetic glucocorticoids. Cross-
reactivity of a structurally diverse set of compounds were determined for the Roche
Diagnostics Elecsys assays for cortisol, dehydroepiandrosterone (DHEA) sulfate, estradiol,
progesterone, and testosterone. These data were compared and contrasted to package
insert data and published cross-reactivity studies for other marketed steroid hormone
immunoassays. Cross-reactivity was computationally predicted using the technique of two-
986
dimensional molecular similarity. The Roche Elecsys Cortisol and Testosterone II assays
showed a wider range of cross-reactivity than the DHEA sulfate, Estradiol II, and
Progesterone II assays. 6-Methylprednisolone and prednisolone showed high cross-reactivity
for the cortisol assay, with high likelihood of clinically significant effect for patients
administered these drugs. In addition, 21-deoxycortisol likely produces clinically relevant
cross-reactivity for cortisol in patients with 21-hydroxylase deficiency, while 11-deoxycortisol
may produce clinically relevant cross-reactivity in 11beta-hydroxylase deficiency or following
metyrapone challenge. Several anabolic steroids may produce clinically significant false
positives on the testosterone assay, although interpretation is limited by sparse
pharmacokinetic data for some of these drugs. Norethindrone therapy may impact
immunoassay measurement of testosterone in women. Using two-dimensional similarity
calculations, all compounds with high cross-reactivity also showed a high degree of similarity
to the target molecule of the immunoassay. It was thus concluded that compounds producing
cross-reactivity in steroid hormone immunoassays generally have a high degree of structural
similarity to the target hormone. Clinically significant interactions can occur with structurally
similar drugs (e.g. prednisolone and cortisol immunoassays; methyltestosterone and
testosterone immunoassays) or with endogenous compounds such as 21-deoxycortisol that
can accumulate to very high concentrations in certain disease conditions. Simple similarity
calculations can help triage compounds for future testing of assay cross-reactivity [14640].
Gas chromatography coupled to IRMS
Nowadays, the importance of IRMS in sports drug testing is substantial and, as recently
concluded, its relevance is continuously growing and new instrumental options allow for
enhanced detection assays. In doping controls, IRMS is currently applied mainly to the
analysis of natural/endogenous anabolic-androgenic steroids and its metabolites; however,
additional candidates such as cortisone or 5-aminoimidazole-4-carboxamide-ribonucleoside
(AICAR) have been subject of recent research projects and are conceivable future target
analytes. IRMS represents a comparably complex and challenging analytical methodology
that necessitates thorough consideration of information indicating the need for an IRMS-
based analysis of a doping control sample and factors influencing the analytical result as well
as its interpretation. Consequently, quality assurance is of utmost importance. In this context,
the characterization of reference material, i.e. carbon isotope ratios of steroids in freeze-dried
human urine, was reported. In addition, studies concerning the impact of carbon isotope
ratios of derivatization reagents (such as acetic anhydride) on the analytical result (i.e. the
isotopic signature of a target analyte) were conducted, demonstrating that delta13C values of
steroids as calculated from the mass balance equation are independent from the delta13C
value of the derivatization reagent. Moreover, applying conventional derivatization conditions
of acetylation, the net kinetic isotope effect was not found to significantly affect the
determined steroid delta13C value [14009].
The administration of anabolic steroids is one of the most important issues in doping control
and is detectable through a change in the carbon isotopic composition of testosterone and/or
its metabolites. Gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-
IRMS), however, remains a very laborious and expensive technique and substantial amounts
of urine are needed to meet the sensitivity requirements of the IRMS. This can be
problematic because only a limited amount of urine is available for anti-doping analysis on a
broad spectrum of substances. In this work we introduce a new type of injection that
increases the sensitivity of GC-C-IRMS by a factor of 13 and reduces the limit of detection,
simply by using solvent vent injections instead of splitless injection. This drastically reduces
the amount of urine required. On top of that, by only changing the injection technique, the
detection parameters of the IRMS are not affected and there is no loss in linearity [12154].
987
The application of a comprehensive gas chromatography/combustion/isotope ratio mass
spectrometry (GC/C/IRMS)-based method for stable carbon isotopes of endogenous urinary
steroids was presented. The key element in sample preparation is the consecutive cleanup
with high-performance liquid chromatography (HPLC) of underivatized and acetylated
steroids, which allows the isolation of ten analytes (11beta-hydroxyandrosterone, 5alpha-
androst-16-en-3beta-ol, pregnanediol, androsterone, etiocholanolone, testosterone,
epitestosterone, 5alpha-androstane-3alpha,17beta-diol, 5beta-androstane-3alpha,17beta-
diol and dehydroepi-androsterone) from a single urine specimen. These steroids are of
particular importance to doping controls as they enable the sensitive and retrospective
detection of steroid abuse by athletes. Depending on the biological background, the
determination limit for all steroids ranges from 5 to 10 ng/mL for a 10 mL specimen. The
method is validated by means of linear mixing models for each steroid, which covers
repeatability and reproducibility. Specificity was further demonstrated by gas
chromatography/mass spectrometry (GC/MS) for each analyte, and no influence of the
sample preparation or the quantity of analyte on carbon isotope ratios was observed. In order
to determine naturally occurring 13C/12C ratios of all implemented steroids, a reference
population (n=61) subjects was measured to enable the calculation of reference limits for all
relevant steroidal delta values [08183].
An alternative calibration procedure for the gas chromatography-combustion-isotope ratio

mass spectrometry (GC-C-IRMS) measurements of the World Antidoping Agency (WADA)
accredited laboratories is presented. To alleviate the need for externally calibrated CO 2 gas
for GC-C-IRMS analysis of urinary steroid metabolites, calibration using an external standard
mixture solution of steroids with certified isotopic composition was investigated. The
reference steroids of the calibration mixture and routine samples underwent identical
instrumental processes. The calibration standards bracketed the entire range of the relevant
delta13C values for the endogenous and exogenous steroids as well as their chromatographic
retention times. The certified delta13C values of the reference calibrators were plotted in
relation to measured m/z 13CO2/12CO2 (i.e. R(45/44)) mass spectrometric signals of each
calibrator. delta13C values of the sample steroids were calculated from the least squares fit
through the calibration curve. The effect of the external calibration on delta13C values, using
the same calibration standards and set of urine samples but different brands of GC-C-IRMS
instruments, was assessed by an interlaboratory study in the WADA Accredited Laboratories
of Sydney, Australia and Athens, Greece. Relative correspondence between the laboratories
for determination of androsterone, etiocholanolone, 5beta-androstane-3alpha,17beta-
diacetate, and pregnanediacetate means were SD(delta13C) 0.012, 0.058, -0.034, and -0.040
percent, respectively. These data demonstrate that accurate intralaboratory external
calibration with certified steroids provided by United States Antidoping Agency (USADA) and
without external CO2 calibration is feasible and directly applicable to the WADA Accredited
Laboratories for the harmonization of the GC-C-IRMS measurements [11076].
An alternative calibration procedure for the gas chromatography-combustion-isotope ratio

mass spectrometry (GC-C-IRMS) measurements of the World Antidoping Agency (WADA)
Accredited Laboratories is presented. To alleviate the need for externally calibrated CO₂ gas
for GC-C-IRMS analysis of urinary steroid metabolites, calibration using an external standard
mixture solution of steroids with certified isotopic composition was investigated. The
reference steroids of the calibration mixture and routine samples underwent identical
instrumental processes. The calibration standards bracketed the entire range of the relevant
δ¹³C values for the endogenous and exogenous steroids as well as their chromatographic
retention times. The certified delta¹³C values of the reference calibrators were plotted in
relation to measured m/z ¹³CO₂/¹²CO₂ mass spectrometric signals of each calibrator. delta¹³C
values of the sample steroids were calculated from the least squares fit through the
calibration curve. The effect of the external calibration on delta¹³C values, using the same
988
calibration standards and set of urine samples but different brands of GC-C-IRMS
instruments, was assessed by an interlaboratory study in the WADA Accredited Laboratories
of Sydney, Australia and Athens, Greece. Relative correspondence between the laboratories
for determination of androsterone, etiocholanolone, 5beta-androstane-3alpha,17beta-
diacetate, and pregnanediacetate means were SD(delta¹³C) 0.12‰, 0.58‰, -0.34‰, and -
0.40‰, respectively. These data demonstrate that accurate intralaboratory external
calibration with certified steroids provided by United States Antidoping Agency (USADA) and
without external CO₂ calibration is feasible and directly applicable to the WADA Accredited
Laboratories for the harmonization of the GC-C-IRMS measurements [11432].
IRMS is a powerful device that allows the source determination of the investigated
compounds based on variations of stable isotopes. IRMS has many applications such as
pharmacology, food research, archaeology, environment sciences and forensic science.
Doping is also a domain in which IRMS can provide informative data as one of the main
challenges for T doping detection is to establish the origin of this hormone as it could be
found either produced endogenously by the body or by misuse through an exogenous
administration. The first application in doping was published in 1994 of GC coupled to IRMS
for the determination of carbon isotope ratio of T extracted from human urine. This significant
work was then followed by numerous studies that explored doping detection based on the
carbon isotopic ratio of endogenous hormones linked to T metabolism. Recently, some T
preparations were reported as having a similar carbon isotopic ratio compared with T
produced endogenously, pushing scientists to find alternative methods based on hydrogen
and deuterium ratio to discern naturally produced T from synthetic formulations. Since its
introduction in the anti-doping laboratories, GC-C-IRMS has provided robust and reliable
data to convict many athletes for T misuse in sports. Until now, GC-C-IRMS analysis was
performed whenever a urine sample showed a T/E ratio above the threshold and was
considered as the ultimate proof of doping if the carbon isotopic ratio of T or its metabolites
was significantly different from one of the defined endogenous reference compounds. New
technical document TD2014EAAS has been effective since the beginning of 2014 and
according to this document, GC-C-IRMS analysis shall be applied on suspicious sample only
in confirmation step after the evaluation of the steroid profile through the adaptive model of
the ABP. In the case where the adaptive model cannot be used, IRMS shall be performed in
specific conditions such as a T/E ratio greater than 4 or a T or E concentration (adjusted for
the specific gravity) greater than 200 ng/mL in males or greater than 50 ng/mL in females.12
More details about IRMS analyses and interpretation in WADA-accredited laboratories are
expected in the upcoming new technical document dedicating to this analytical technique. In
summary, GC-C-IRMS represents a complementary but necessary information source for the
steroid profile evaluation [14450].
The administration of anabolic steroids is one of the most important issues in doping control
and is detectable through a change in the carbon isotopic composition of testosterone and/or
its metabolites. Gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-
IRMS), however, remains a very laborious and expensive technique and substantial amounts
of urine are needed to meet the sensitivity requirements of the IRMS. This can be
problematic because only a limited amount of urine is available for anti-doping analysis on a
broad spectrum of substances. In this work we introduce a new type of injection that
increases the sensitivity of GC-C-IRMS by a factor of 13 and reduces the limit of detection,
simply by using solvent vent injections instead of splitless injection. This drastically reduces
the amount of urine required. On top of that, by only changing the injection technique, the
detection parameters of the IRMS are not affected and there is no loss in linearity [13170].
The analysis of urinary metabolites of testosterone-related steroids through the

measurement of their carbon isotopic signature (delta13C) by gas chromatography
989
combustion/mass spectrometry (GC/C/IRMS) is a confirmation method employed in doping
control analyses. Stringent analytical conditions are essential to an accurate and precise
analysis as well as the proper selection of the metabolites, which forms the basis of the
refined method presented in one paper. In a simplified approach, following enzymatic
hydrolysis and extraction from a relatively low volume of urine sample, a one-step high-
performance liquid chromatography (HPLC) purification was developed for seven diagnostic
urinary metabolites (TS) including testosterone itself, dehydroepiandrosterone, 5alpha- and
5beta-androstanediol, epitestosterone, androsterone, etiocholanolone and two endogenous
reference compounds (ERC), 5beta-pregnanediol and 5alpha-androst-16-en-3beta-ol. These
steroids were pooled in three fractions and analyzed as such. With regards to the
GC/C/IRMS analysis, a multi-level isotopic calibration using the 'identical treatment' principle
was created. The proposed isotopic calibration yielded results for purified reference steroids
with a precision ≤0.15 and accuracy of ≤0.030 percent (between-assay, n=26). Compared to
other common endogenous reference compounds, those selected in this study had delta13C
values close to the target metabolites which, along with the proposed isotopic calibration,
produced narrow reference intervals within ± 0.3 percent for most diagnostic TS-ERC pairs,
in compliance with the requirements of the World Anti-Doping Agency. These carefully
controlled analytical conditions are compatible with routine operations, affording accurate
and precise results for the more diagnostically relevant metabolites such as testosterone
itself and the 5alpha- and 5beta-androstanediols. The values of the TS-ERC pairs measured
in reference populations are described and the results from the routine testing of several
hundreds of athletes' samples are discussed. Robust, this technique permitted the detection
of adverse findings that would have been missed had these low level metabolites not been
analyzed [13172].
Gas chromatographic/time-of-flight mass-spectrometric
The method of high sensitive gas chromatographic/time-of-flight mass-spectrometric

(GC/TOF-MS) analysis of steroids was developed. Low-resolution TOF-MS instrument (with
fast spectral acquisition rate) was used. This method is based on the formation of the silyl
derivatives of steroids; exchange of the reagent mixture (pyridine and N,O-
bis(trimethylsilyl)trifluoroacetamide (BSTFA)) for tert-butylmethylether; offline large sample
volume injection of this solution based on sorption concentration of the respective derivatives
from the vapour-gas mixture flow formed from the solution and inert gas flows; and entire
analytes solvent-free concentrate transfer into the injector of the gas chromatograph.
Detection limits for 100 microL sample solution volume were 0.5-2 pg/µL (depending on the
component). Application of TOF-MS model 'TruTOFcoupled with gas chromatograph and
ChromaTOF software (Leco, St Joseph, MO, USA) allowed extraction of the full mass
spectra and resolving coeluted peaks. Due to use of the proposed method (10 microL
sample aliquot) and GC/TOF-MS, two times more steroid-like compounds were registered in
the urine extract in comparison with the injection of 1 microl of the same sample solution
[11074].
HPLC-MS/MS
Improvements in doping analysis can be effected by speeding up analysis time and

extending the detection time. Therefore, direct detection of phase II conjugates of doping
agents, especially anabolic androgenic steroids (AAS), is proposed. Besides direct detection
of conjugates with glucuronic acid, the analysis of sulfate conjugates, which are usually not
part of the routine doping control analysis, can be of high interest. Sulfate conjugates of
methandienone and methyltestosterone metabolites have already been identified as long-
term metabolites. This study presents the synthesis of sulfate conjugates of six commonly
990
used AAS and their metabolites: trenbolone, nandrolone, boldenone, methenolone,
mesterolone, and drostanolone. In the following these sulfate conjugates were used for
development of a fast and easy analysis method based on sample preparation using solid
phase extraction with a mixed-mode sorbent and detection by high performance liquid
chromatography coupled to tandem mass spectrometry (HPLC-MS/MS). Validation
demonstrated the suitability of the method with regard to the criteria given by the technical
documents of the World Anti-Doping Agency (WADA). In addition, suitability has been proven
by successful detection of the synthesized sulfate conjugates in excretion urines and routine
doping control samples [150161].
HPLC on a porous graphitized carbon column coupled to an Orbitrap

The presence in a urinary matrix of a large number of endogenous steroids and
corticosteroids with similar structures can hamper the detection of specific exogenous
steroids using liquid chromatography/mass spectrometry (LC/MS) with reversed-phase
columns. Therefore, the development of LC/MS methods using alternative columns is of
great interest. Porous graphitized carbon is a unique stationary phase for high-performance
liquid chromatography (HPLC), with properties differing from traditional silica-based and
polymeric stationary phases. The new method involves enzymatic hydrolysis, liquid-liquid
extraction, and determination by high-temperature HPLC/Orbitrap mass spectrometry
(HTLC/Orbitrap MS) with atmospheric pressure photoionization (APPI). To achieve APPI of
doping substances, the mobile phase consisted of 0.1 percent CF3COOH (A) and a mixture
of acetonitrile/2-propanol (25:75 v/v), containing 0.1 percent CF3COOH (B), which was used
as an effective proton source. A screening method for the detection of 57 exogenous steroids
has been developed. The method was validated by spiking 10 different blank urine samples
at different concentration levels. Validation parameters included limit of detection (LOD),
selectivity, ion suppression, extraction recovery, and repeatability. All studied compounds
had an LOD lower than the minimum required performance level. Of the 57 steroids studied,
55 showed recovery better than 70percent. For all of the analytes, the relative retention times
proved to be stable between days, with relative standard deviations (RSDs) smaller than 0.3
percent. In addition, the interday RSDs of the peak area ratios ranged between 0.7 percent
and 14.5 percent. It was concluded that the proposed method matches the basic
requirements of all methods used to analyze drugs or metabolites in an antidoping
laboratory, i.e. sensitivity, selectivity, and specificity. The acquisition of full-scan mass
spectra with accurate masses can be a valuable tool in the retrospective evaluation of
analyzed samples for anabolic steroids recently added to the prohibited list [150163].
Enzymatic hydrolysis of conjugated steroid metabolites
The optimum conditions for hydrolysing conjugated metabolites of steroid hormones in

bovine urine were performed with Helix pomatia juice, beta-glucuronidase from bovine liver
and preparations of limpets and abalone entrails using response surface methodology. The
experimental design and empirical modelling used allowed us to assess the main effects of
factors (time, temperature, pH and enzyme quantity) and to predict the optimum conditions
for each enzyme preparation. Confirmatory experiments were applied to check the predicted
values and to validate the model. The comparison of the enzyme preparation efficiency for
various conjugate steroids and the study of possible by-product synthesis led us to select
abalone entrails to hydrolyse natural dehydroepiandrosterone, etiocholanolone,
epitestosterone; 17 alpha-estradiol and estrone in bovine urine. The optimum conditions
were found to be 20 h at 42 degrees C with the pH adjusted to 5.2 and using 12,000 units of
enzyme preparation [00042].
HPLC using atmospheric pressure chemical ionisation
991
A specific and sensitive method based on tandem mass spectrometry with on-line high-
performance liquid chromatography using atmospheric pressure chemical ionisation (LC-
APCI-MS-MS) for the quantitation of anabolic hormone residues (17beta-19-nortestosterone,
17beta-testosterone and progesterone) and their major metabolites (17alpha-19-
nortestosterone and 17alpha-testosterone) in bovine serum and urine is reported.
[2H2]17Beta-testosterone was used as internal standard. The analytes were extracted from
urine (following enzymatic hydrolysis) and serum samples by liquid-liquid extraction and
purified by C18 solid-phase extraction. Ionisation was performed in a heated nebulizer
interface operating in the positive ion mode, where only the protonated molecule, [M+H]+,
was generated for each analyte. This served as precursor ion for collision-induced
dissociation and two diagnostic product ions for each analyte were identified for the
unambiguous hormone confirmation by selected reaction monitoring LC-MS-MS. The overall
inter-day precision (relative standard deviation) ranged from 6.37 to 2.10 percent and from
6.25 to 2.01 percent, for the bovine serum and urine samples, respectively, while the inter-
day accuracy (relative error) ranged from -5.90 to -3.18 percent and from -6.40 to -2.97
percent, for the bovine serum and urine samples, respectively. The limit of quantitation of the
method was 0.1 ng/mL for all the hormones in bovine serum and urine. On account of its
high sensitivity and specificity the method has been successfully used to confirm illegal
hormone administration for regulatory purposes [00043].
Quantification of neurosteroids in rat plasma and brain
A simplified method for the quantitative analysis of neurosteroids in rat plasma and brain is
described. The method uses negative chemical ionization gas chromatography/mass
spectrometry and involves the synthesis of pentafluorobenzyloxime/trimethylsilyl ether
derivatives with excellent chromatographic and electron-capturing properties. Deuterium-
labeled analogs of the steroids of interest were synthesized and used as internal standards.
The steroids (allopregnanolone, epiallopregnanolone, pregnenolone, testosterone, and
dehydroepiandrosterone) were isolated from the plasma or brain matrix by a rapid and
straightforward solid-phase extraction procedure. The mass spectrometer was operated in a
selective ion monitoring mode, allowing for picograms of neurosteroids to be quantified from
biological extracts. The method was linear over the concentration range (100 to 8000 pg from
0.3 ml plasma and 250 to 8000 pg from 100 mg brain tissue) with good precision and
accuracy. In experimental protocols, the procedure was suitable for measuring
concentrations of endogenous neurosteroids in rat plasma and brain. Significant elevations
were observed in the frontal cortex for allopregnanolone and pregnenolone following a swim
stress and for allopregnanolone and epiallopregnanolone following allopregnanolone
injection (8 mg/kg, sc). The present method allows accurate determination of neurosteroids
and will be helpful in elucidating the role of neurosteroids in health and disease [00044].
This study proposes a new analytical methodology for the determination of trace levels of
testosterone (T) and epitestosterone (E) in urine matrices using bar adsorptive
microextraction combined with liquid desorption followed by high-performance liquid
chromatography with diode array detection (BAmyE-LD/HPLC-DAD). The comparison of
different sorbent coatings (five activated carbons, one styrene-divinylbenzene, two modified
pyrrolidone, one ciano and one n-vinylpyrrolidone polymers) through BAmyE showed that the
latter phase presented much higher selectivity and capacity offering multiple mechanisms of
interaction. Assays using this phase were performed on 25 mL of water samples spiked at
the 8.0 microg/L level, yielded average recoveries of 92.1 and 93.4 percent for T and E,
992
respectively, under optimized experimental conditions; BAmyE (n-vinylpyrrolidone): 16h
(1000 rpm), pH 5.5; LD: acetonitrile, 30 min under sonication treatment. From the developed
analytical methodology, suitable detection limits were achieved (0.4 micrg/L) and good linear
dynamic ranges (1.4-16. microg/L) with remarkable determination coefficients. By using the
standard addition methodology, the application of the present analytical approach on urine
samples revealed good sensitivity. The proposed method, which operated under the floating
sampling technology, proved to be a suitable sorption-based static microextraction
alternative for screening T, E and the T/E ratio in urine samples for doping control purposes.
The methodology showed to be easy to implement, demonstrating good reproducibility,
sensitivity and robustness, allowing the possibility to choose the most selective sorbent
coating according to the compounds of interest [14747].
LC-silver ion coordination ionspray/triple-quadrupole mass spectrometry
Metal ion coordination ionspray (M(+) CIS) ionization is a powerful technique to enhance
ionization efficiency and sensitivity. In this study, we developed and validated an analytical
method for simultaneous ionization and analysis of 84 anabolic androgenic steroids (65
exogenous and 19 endogenous) using liquid chromatography-silver ion coordination
ionspray/triple-quadrupole mass spectrometry (LC-Ag(+) CIS/MS/MS). The concentrations of
silver ions and organic solvents have been optimized to increase the amount of silver ion
coordinated complexes. A combination of 25 μM of silver ions and methanol showed the best
sensitivity. The validation results showed the intra- (0.8-9.2 %) and inter-day (2.5-14.9 %)
precisions, limits of detection (0.0005-5.0 ng/mL), and matrix effect (71.8-100.3 %) for the
screening analysis. No significant ion suppression was observed. In addition, this method
was successfully applied to analysis of positive samples from suspected abusers and useful
for the detection of the trace levels of anabolic steroids in human urine samples [14748].
Anabolic androgenic steroids (AAS) are an important class of doping agents. The
metabolism of these substances is generally very extensive and includes phase-I and phase-
II pathways. In this work, a comprehensive detection of these metabolites is described using
a 2-fold dilution of urine and subsequent analysis by liquid chromatography-high resolution
mass spectrometry (LC-HRMS). The method was applied to study 32 different metabolites,
excreted free or conjugated (glucuronide or sulfate), which permit the detection of misuse of
at least 21 anabolic steroids. The method has been fully validated for 21 target compounds
(8 glucuronide, 1 sulfate and 12 free steroids) and 18 out of 21 compounds had detection
limits in the range of 1-10 ng/mL in urine. For the conjugated compounds, for which no
reference standards are available, metabolites were synthesized in vitro or excretion studies
were investigated. The detection limits for these compounds ranged between 0.5 and 18
ng/mL in urine. The simple and straightforward methodology complements the traditional
methods based on hydrolysis, liquid-liquid extraction, derivatization and analysis by gas
chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry
(LC-MS) [150164].
Carbon isotope ratio (CIR)
Carbon isotope ratio (CIR) analysis has been routinely and successfully used in sports drug
testing for many years to uncover the misuse of endogenous steroids. One limitation of the
method is the availability of steroid preparations exhibiting CIRs equal to endogenous
steroids. To overcome this problem, hydrogen isotope ratios (HIR) of endogenous urinary
steroids were investigated as a potential complement; results obtained from a reference
993
population of 67 individuals are presented herein. An established sample preparation method
was modified and improved to enable separate measurements of each analyte of interest
where possible. From the fraction of glucuronidated steroids; pregnanediol, 16-androstenol,
11-ketoetiocholanolone, androsterone (A), etiocholanolone (E), dehydroepiandrosterone (D),
5alpa- and 5beta-androstanediol, testosterone and epitestosterone were included. In
addition, sulfate conjugates of A, E, D, epiandrosterone and 17alpa- and 17beta-
androstenediol were considered and analyzed after acidic solvolysis. The obtained results
enabled the calculation of the first reference-population-based thresholds for HIR of urinary
steroids that can readily be applied to routine doping control samples. Proof-of-concept was
accomplished by investigating urine specimens collected after a single oral application of
testosterone-undecanoate. The HIR of most testosterone metabolites were found to be
significantly influenced by the exogenous steroid beyond the established threshold values.
Additionally, one regular doping control sample with an extraordinary testosterone/
epitestosterone ratio of 100 without suspicious CIR was subjected to the complementary
methodology of HIR analysis. The HIR data eventually provided evidence for the exogenous
origin of urinary testosterone metabolites. Despite further investigations on HIR being
advisable to corroborate the presented reference-population-based thresholds, the
developed method proved to be a new tool supporting modern sports drug testing
procedures [12155].
Isotope ratio mass spectrometry (IRMS) testing is performed to determine if an atypical

steroid profile is due to administration of an endogenous steroid. Androsterone (Andro) and
etiocholanolone (Etio), and/or the androstanediols (5alpha- and 5beta-androstane-
3alpha,17beta-diol) are typically analyzed by IRMS to determine the 13C/12C ratio. The ratios
of these target compounds are compared to the 13C/12C ratio of an endogenous reference
compound (ERC) such as 5beta-pregnane-3alpha,20alpha-diol (Pdiol). Concentrations of
Andro and Etio are high so 13C/12C ratios can easily be measured in most urine samples.
Despite the potentially improved sensitivity of the androstanediols for detecting the use of
some testosterone formulations, additional processing steps are often required that increase
labour costs and turnaround times. Since this can be problematic when performing large
numbers of IRMS measurements, we established thresholds for Andro and Etio that can be
used to determine the need for additional androstanediol testing. Using these criteria, 105
out of 2639 urine samples exceeded the Andro and/or Etio thresholds, with 52 of these
samples being positive based on Andro and Etio IRMS testing alone. The remaining 53 urine
samples had androstanediol IRMS testing performed and 3 samples were positive based on
the androstanediol results. A similar strategy was used to establish a threshold for Pdiol to
identify athletes with relatively 13C-depleted values so that an alternative ERC can be used to
confirm or establish a true endogenous reference value. Adoption of a similar strategy by
other laboratories can significantly reduce IRMS sample processing and analysis times,
thereby increasing testing capacity [13095].
Carbon isotope ratio combined with hydrogen isotope ratio

Carbon isotope ratio (CIR) analysis has been routinely and successfully applied to doping
control analysis for many years to uncover the misuse of endogenous steroids such as
testosterone. Over the years, several challenges and limitations of this approach became
apparent, e.g., the influence of inadequate chromatographic separation on CIR values or the
emergence of steroid preparations comprising identical CIRs as endogenous steroids. While
the latter has been addressed recently by the implementation of hydrogen isotope ratios
(HIR), an improved sample preparation for CIR avoiding co-eluting compounds is presented
herein together with newly established reference values of those endogenous steroids being
relevant for doping controls. From the fraction of glucuronidated steroids 5beta-pregnane-
3alpha,20alpha-diol, 5alpha-androst-16-en-3alpha-ol, 3alpha-Hydroxy-5beta-androstane-
11,17-dione, 3alpha-hydroxy-5alpha-androstan-17-one (ANDRO), 3alpha-hydroxy-5beta-
994
androstan-17-one (ETIO), 3beta-hydroxy-androst-5-en-17-one (DHEA), 5alpha- and 5beta-
androstane-3alpha,17beta-diol (5aDIOL and 5bDIOL), 17beta-hydroxy-androst-4-en-3-one
and 17alpha-hydroxy-androst-4-en-3-one were included. In addition, sulfate conjugates of
ANDRO, ETIO, DHEA, 3beta-hydroxy-5alpha-androstan-17-one plus 17alpha- and androst-
5-ene-3beta,17beta-diol were considered and analyzed after acidic solvolysis. The results
obtained for the reference population encompassing 67 males and females confirmed earlier
findings regarding factors influencing endogenous CIR. Variations in sample preparation
influenced CIR measurements especially for 5alphaDIOL and 5betaDIOL, the most valuable
steroidal analytes for the detection of testosterone misuse. Earlier investigations on the HIR
of the same reference population enabled the evaluation of combined measurements of CIR
and HIR and its usefulness regarding both steroid metabolism studies and doping control
analysis. The combination of both stable isotopes would allow for lower reference limits
providing the same statistical power and certainty to distinguish between the endo- or
exogenous origin of a urinary steroid [13165].
One report described a rapid, simple, and highly selective approach to perform testosterone
competitive immunoassay by capillary electrophoresis (CE) and LIF detection. The method
involves using synthesized fluorescence-labeled testosterone as a tracer to compete with
testosterone. Two polyclonal antibodies arised and their respective tracers have been
optimized and a system is used for the quantification of testosterone by CE-based
immunoassay. The method is developed with a wide working range of 3.70-2000 ng/mL and
a limit of detection at 1.11 ng/mL. Tests for normal and positive urine samples show that this
method has the potential to be applied in testosterone doping control [08185].
Prediction of metabolic pattern of new derivatives of AAS
The aim of one work was to develop a flexible in vitro synthesis procedure, which can be
applied in order to study and predict the metabolic patterns of new derivatives of anabolic
androgenic steroids with respect to most prominent target compounds for doping control
purposes. Microsomal and S9 fraction of human liver preparations were used as a source of
metabolising enzymes and the co-substrates of the synthesis mixture were selected to favour
phase-I metabolic reactions and glucuronidation as phase-II conjugation reactions. Model
compounds within the study were 4,9,11-trien-3-one steroids, structural derivatives of
gestrinone and trenbolone, which both are included in the list of prohibited compounds in
sports by the World Anti-Doping Agency (WADA). The correlation between in vitro
metabolism of human microsomes and in vivo excretion studies in human was compared
with gestrinone and subsequently, the applicability of the in vitro model for prediction of AAS
metabolic pathways for new doping agents was evaluated. All the AAS examined within this
study were successfully metabolised using the developed in vitro model, hydroxylation,
reduction and glucuronide conjugation being the most prominent reaction pathways.
Hydroxylated and glucuronide-conjugated metabolites of in vivo experiment with gestrinone
were the same metabolites formed in the enzyme-driven process, thus showing good in vitro-
in vivo correlation. Liquid chromatographic-mass spectrometric and tandem mass
spectrometric methods were developed, relying on the positive polarity of electrospray
ionisation, which also allowed the direct detection of intact glucuronide-conjugated AAS
metabolites. Due to charge delocalisation and high proton affinity, the developed method was
proven effective in the analysis of anabolic steroids metabolites bearing extensive
conjugated double bond systems in their structures [08187].
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Mass spectometry
The metabolism of two anabolic steroids – oxymetholone and desoxymethyltestosterone –

was reinvestigated to identify new targets potentially valuable for the antidoping analysis.
Excretion urine samples from the laboratory reference collection were used in this study.
Following fractionation of the urinary extract by means of high performance liquid
chromatography (HPLC), each fraction was subjected to gas chromatography-mass
spectrometry (GC-MS) and gas chromatography-tandem mass spectrometry (GC-MS/MS)
analysis after trimethylsilylation. About 20 metabolites were found for desoxymethyl-
testosterone and more than 40 for oxymetholone, with many of them being isomeric
compounds. In addition to the well-known reduced and hydroxylated metabolites, 18-nor-
17,17-dimethyl and 18-nor-17-hydroxymethyl-17-methyl steroids were also identified. Having
evaluated all the metabolites in terms of how long they could be detected, it was suggested
that 18-nor-2epsilon,17beta-hydroxymethyl-17alpha-methyl-5alpha-androst-13-en-3alpha-ol
is an important marker of oxymetholone abuse. In case of desoxymethyltestosterone, better
detectability could be achieved if 18-nor-17,17-dimethyl-5alpha-androst-13-en-2epsilon,
3alpha-diol is monitored. These novel metabolites could be detected using GC-MS/MS at
least for 14 days after administration of these anabolic steroids compared to 5-7 days for
previously reported metabolites [12157].
Mass spectrometric identification of anabolic androgenic steroids challenges standard

doping-control methods. To reveal a doping offence the presence of prohibited anabolic
androgenic steroids at trace levels in the picogram-per-millilitre range must be confirmed as
reliable. Human urine samples containing epitrenbolone, metandienone metabolite (17beta -
hydroxymethyl-17alpha-methyl-18-norandrost-1,4,13-trien-3-one), stanozolol, 16beta-
hydroxystanozolol and 4beta-hydroxystanozolol were analysed using LC-FAIMS-MS/MS.
These substances are prohibited in sport according to World Anti-Doping Agency (WADA)
regulations. Glucuronides were hydrolysed and prepared by liquid-liquid extraction. Excellent
recovery and precision were obtained for all compounds. Linear calibration results for
epitrenbolone and metandienone metabolite were obtained and concentration information
could be determined in the ranges of reliable response between 750-1200 and 100-600
pg/mL, respectively. Limits of detection were estimated at 25 pg/mL (stanozolol), 50 pg/mL
(metandienone metabolite, 16beta-hydroxystanozolol), 100 pg/mL (4beta-hydroxystanozolol)
and 500 pg/mL (epitrenbolone). The assay was applied to doping-control samples. For all
analytes, LC-FAIMS-MS/MS resulted in excellent interference removal, which effectively
extends the post-dose detection time [09068].
Regularly new anabolic steroids appear on the black market. In most cases these
substances are marketed on websites or are confiscated during inspections. 1,(5alpha)-
Androstene-17beta-ol-3-one, also known as 1-testosterone, is one of these substances
presented to body-builders as a nutritional supplement or a pro-hormone. 1-Testosterone
closely resembles the natural hormone testosterone except for a 1,2-double bound instead of
a 4,5-double bound. 1-Androstene-3beta,17beta-diol is transformed into 1-testosterone after
oral administration. 1-Testosterone, 1-androstene-3beta,17beta-diol and some other related
“new” anabolic steroids were studied with gas chromatography coupled to mass
spectrometry (GC-MS) and Liquid chromatography coupled to tandem mass spectrometry
(LC-MS2) methods. Similarities in spectra to known analytes, which may lead to pitfalls in the
interpretation of the derivatised analytes, are discussed [06100].
Electrospray ionization tandem mass spectrometry
Anabolic steroids are structurally similar compounds, and their product-ion spectra obtained
996
by tandem mass spectrometry under electrospray ionization conditions are quite difficult to
interpret because of poly-ring structures and lack of a charge-retaining center in their
chemical structures. In the present study, the fragmentation of nine anabolic steroids of
interest to the racing industry was investigated by using triple quadrupole mass
spectrometer, Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer, and a
linear ion trap instrument. With the aid of an expert system software (Mass Frontier version
3.0), accurate mass measurements, and multiple stage tandem mass spectrometric (MS(n))
experiments, fragmentation pathways were elucidated for boldenone, methandrostenolone,
tetrahydrogestrinone (THG), trenbolone, normethandrolone and mibolerone. Small
differences in the chemical structures of the steroids, such as an additional double-bond or a
methyl group, result in significantly different fragmentation pathways. The fragmentation
pathways proposed in this paper allow interpretation of major product ions of other anabolic
steroids reported by other researchers in a recent publication. The proposed fragmentation
pathways are helpful for characterization of new steroids. The approach used in this study for
elucidation of the fragmentation pathways is helpful in interpretation of complicated product-
ion spectra of other compounds, drugs and their metabolites [06101].
Electrospray ionization time-of-flight mass spectrometry (ESI-TOFMS)

Findings of illegal hormone preparations such as syringes, bottles, cocktails, and so on, are
an important information source for the nature of the current abuse of anabolic steroids and
related compounds as growth-promoting agents in cattle. A new screening method for
steroids in cocktails is presented based on liquid chromatography (LC) with diode-array UV-
absorbance detection and electrospray ionization time-of-flight mass spectrometry (ESI-
TOFMS). Accurate mass measurements were performed at a mass resolution of 4000 using
continuous introduction of a lock mass through a second (electro)sprayer. Similar
experiments were carried out using dual-sprayer quadrupole time-of-flight mass spectrometry
(ESI-QTOFMS/MS) at a mass resolution of 10 000 with data-dependent MS/MS acquisition;
i.e. beyond an intensity threshold for the [M + H](+) ions, MS/MS spectra were automatically
acquired at three different collision energies. Elemental compositions were calculated for
precursor and product ions and it is shown that the combined information from LC retention
behavior, UV spectra, elemental compositions, and accurate mass MS/MS spectra yield a
fast impression of the steroids present in the complex mixture. Using a new software tool for
structure elucidation of MS/MS spectra, an additional non-steroidal additive was identified as
well [01045].
GCxGC-TOFMS
The application of comprehensive two-dimensional gas chromatography coupled to time-of-

flight mass spectrometry (GCxGC-TOFMS) for the analysis of six anabolic agents (AAs) in
doping control is investigated in this work. A non-polar-polar column configuration with
0.2microm film thickness second dimension (2D) column was employed, offering much better
spread of the components on 2D when compared to the alternative 0.1microm 2D column.
The proposed method was tested on the "key" AA that the World Anti-Doping Agency
(WADA) had listed at the low ng/mL levels (clenbuterol, 19-norandrosterone, epimethendiol,
17alpha-methyl-5alpha-androstane-3alpha,17beta-diol, 17alpha-methyl-5beta-androstane
3alpha,17beta-diol and 3'-OH-stanozolol). The compounds were spiked in a blank urine
extract obtained by solid-phase extraction, hydrolysis and liquid-liquid extraction; prior to
analysis they were converted to the corresponding trimethylsilyl (TMS) derivatives. The limit
of detection (LOD) was below or equal to the minimum required performance limit (MRPL) of
2ng/mL defined by WADA, and the correlation coefficient was in the range from 0.995 to
0.999. The method allows choosing an ion from the full mass spectra which shows the least
interference from the matrix and/or the best sensitivity; this can only be done if full scan mass
spectral data are available. The advantage of GCxGC over classical one-dimensional GC
997
(1D GC), in terms of separation efficiency and sensitivity, is demonstrated on a positive urine
control sample at a concentration of 5ng/mL. The obtained similarity to the in-house created
TOFMS spectra library at this level of concentration was in the range from 822 to 932 (on the
scale from 0 to 999). Since full mass spectral information are recorded, the method allows
the retro-search of non-target compounds or new "designer steroids", which cannot be
detected with established GC-MS methods that use (SIM) mode [09066].
UHPLC-HRMS
Aiming at the identification of new, complementary biomarkers for endogenous steroid

abuse, the utility of a steroidomic approach using UHPLC-HRMS was assessed. In a
controlled elimination study with orally administered testosterone undecanoate (80 mg), urine
samples were subjected to a holistic steroid analysis followed by chemometric/statistical data
evaluation. Here, numerous glucuronidated or sulfated steroids, the deconjugated analogs,
of which mostly constitute the established steroid profile, were found to support the
discrimination of the groups having received either placebo or testosterone undecanoate.
The study demonstrated the principle of modern analytical approaches commonly referred to
as “-omics” strategies and its potential application to issues of doping controls; in order to
consider the whole (holistic) picture of such approaches, complementary analyses (e.g. by
means of GC-HRMS) might be required to strengthen the outcome and value [13012].
Single-stage-Orbitrap-MS
A prominent trend which has been observed in recent years in the analysis of veterinary
drugs and growth-promoting agents is the shift from target-oriented procedures, mainly
based on liquid chromatography coupled to triple-quadrupole mass spectrometry (LC-QqQ-
MS), towards accurate mass full scan MS (such as time of flight (ToF) and Fourier Transform
(FT) Orbitrap MS). In this study the applicability of high resolution single-stage-Orbitrap-MS
for confirmatory analysis of growth-promoting agents in meat was compared to that of a
QqQ-MS. Validation according to CD 2002/657/EC demonstrated that steroid analysis based
on Orbitrap MS, operating at a resolution of 50,000 FWHM, is indeed capable to compete
with QqQ-MS in terms of selectivity/specificity, while providing excellent linearity (for most
compounds >0.99) but somewhat inferior sensitivity. Indeed, CCalphas reached from 0.04 to
0.88 microg/kg for the 34 anabolic steroids upon MS/MS detection, while upon Orbitrap MS
detection a range of 0.07-2.50 microg/kg was observed. Using QqQ-MS adequate precision
was obtained since relative standard deviations, associated with the repeatability and intra-
laboratory reproducibility, were below 20%. In the case of Orbitrap MS, for some compounds
(i.e. some estrogens) this threshold was exceeded and thus poor precision was observed,
which is possibly caused by the lack in sensitivity. Overall, it may be concluded that Orbitrap-
MS offers an adequate performance in terms of linearity and precision but lacks in sensitivity
for some of the compounds [13166].
Full-capillary sample injection combined with a sweeping CE stacking method
One study describes an on-line stacking CE approach by sweeping with whole capillary
sample filling for analyzing five anabolic androgenic steroids in urine samples. The five
anabolic steroids for detection were androstenedione, testosterone, epitestosterone,
boldenone, and clostebol. Anabolic androgenic steroids are abused in sport doping because
they can promote muscle growth. Therefore, a sensitive detection method is imperatively
required for monitoring the urine samples of athletes. In this research, an interesting and
reliable stacking capillary electrophoresis method was established for analysis of anabolic
steroids in urine. After liquid-liquid extraction by n-hexane, the supernatant was dried and
998
reconstituted with 30 mM phosphate buffer (pH 5.00) and loaded into the capillary by
hydrodynamic injection (10 psi, 99.9 s). The stacking and separation were simultaneously
accomplished at -20 kV in phosphate buffer (30 mM, pH 5.0) containing 100 mM sodium
dodecyl sulfate and 40 % methanol. During the method validation, calibration curves were
linear over a range of 50-1,000 ng/mL for the five analytes. In the evaluation of precision and
accuracy for this method, the absolute values of the RSD and the RE in the intra-day (n=3)
and inter-day (n=5) analyses were all less than 6.6 percent. The limit of detection for the five
analytes was 30 ng/mL. Compared with simple MECK, this stacking method possessed a
108- to 175-fold increase in sensitivity. This simple and sensitive stacking method could be
used as a powerful tool for monitoring the illegal use of doping [13167].
Oxidizing adulterants’ effect on the steroid profile of human urine
Steroid profiling is the most versatile and informative technique adapted by doping control
laboratories for detection of steroid abuse. The absolute concentrations and ratios of
endogenous steroids including testosterone, epitestosterone, androsterone, etiocholanolone,
5alpha-androstane-3alpha,17beta-diol and 5beta-androstane-3alpha,17beta-diol constitute
the significant characteristics of a steroid profile. In the present study we report the influence
of various oxidizing adulterants on the steroid profile of human urine. Gas chromatography-
mass spectrometry analysis was carried out to develop the steroid profile of human male and
female urine. Oxidants potassium nitrite, sodium hypochlorite, potassium permanganate,
cerium ammonium nitrate, sodium metaperiodate, pyridinium chlorochromate, potassium
dichromate and potassium perchlorate were reacted with urine at various concentrations and
conditions and the effect of these oxidants on the steroid profile were analyzed. Most of the
oxidizing chemicals led to significant changes in endogenous steroid profile parameters
which were considered stable under normal conditions. These oxidizing chemicals can cause
serious problems regarding the interpretation of steroid profiles and have the potential to act
as masking agents that can complicate or prevent the detection of the steroid abuse [13168].
Electrospray ionization tandem mass spectrometry (ESI-MS/MS)
Electrospray ionization tandem mass spectrometry (ESI-MS/MS) was used to investigate the
effect of different substitutions introduced during metabolism on fragmentation patterns of
four anabolic steroids including methyltestosterone, methandrostenolone, cis-androsterone
and adrenosterone, along with their metabolites. Collision-induced dissociation (CID)
analysis was performed to correlate the major product ions of 19 steroids with structural
features. The analysis is done to portray metabolic alteration, such as incorporation or
reduction of double bonds, hydroxylations, and/or oxidation of hydroxyl moieties to keto
functional group on steroidal skeleton which leads to drastically changed product ion spectra
from the respective classes of steroids, therefore, making them difficult to identify. The
comparative ESI-MS/MS study also revealed some characteristic peaks to differentiate
different steroidal metabolites and can be useful for the unambiguous identification of
anabolic steroids in biological fluid. Moreover, LC-ESI-MS/MS analysis of fermented extract
of methyltestosterone, obtained by Macrophomina phaseolina was also investigated [13169].
Glucuronoconjugated metabolites
In humans, conjugation with glucuronic acid is the most important phase II metabolic reaction
of steroidal compounds. Glucuronoconjugated metabolites have been conventionally studied
by using beta-glucuronidase enzymes to release the phase I metabolites. It is well-known
that hydrolysis with beta-glucuronidase presents some limitations that may result in the
underestimation of some conjugates. The aim of the present work was to develop and to
999
evaluate liquid chromatography-tandem mass spectrometry (LC-MS/MS) scan methods for
the open detection of steroid glucuronides in urine samples. The mass spectrometric
behavior of thirteen representative steroid glucuronides, used as model compounds, was
studied. Characteristic ionization and collision induced dissociation behaviors were observed
depending on the steroid glucuronide structure. Neutral loss (NL of 176, 194, 211, and 229
Da) and precursor ion (PI of m/z 141, 159, and 177, in positive mode and m/z 75, 85, and
113, in negative mode) scan methods were evaluated. The NL scan method was chosen for
the open detection of glucuronoconjugated steroids due to its sensitivity and the structural
information provided by this method. The application of the NL scan method to urine samples
collected after testosterone (T) undecanoate administration revealed the presence of two T
metabolites which remain conjugated as glucuronides after an enzymatic hydrolysis of the
urine. 3alpha,6beta-Dihydroxy-5alpha-androstan-17-one (6beta-hydroxyandrosterone) glucu-
ronide and 3alpha,6beta-dihydroxy-5beta-androstan-17-one (6beta-hydroxyetiocholanolone)
glucuronide were established as the structures for these metabolites, by comparing the
structure of the steroids released after chemical hydrolysis with reference materials. An
increase of 50-300-fold of these metabolites after oral administration of T undecanoate was
observed, proving that their determination can be useful in the doping control field. Moreover,
these results exemplify that significant information might be missed, unless direct methods
for the determination of steroid glucuronides are employed [13164].
Relative retention times
A quantitative structure-retention relationship (QSRR) study has been performed to correlate

relative retention times (RRTs) of trimethylsilylated (TMS) anabolic androgenic steroids
(AAS) with their molecular characteristics, encoded by the respective descriptors, for the
prediction of RRTs of novel molecules, using gas chromatography time-of-flight mass
spectrometry (GC-TOF-MS). The elucidation of similarities and dissimilarities among the data
structures was carried out using principal component analysis (PCA). Successful models
were established using multiple linear regression (MLR) and partial least squares (PLS)
techniques as a function of topological, three-dimensional (3D) and physicochemical
descriptors. The models are useful for the estimation of RRTs of designer steroids for which
no analytical data is available [09069].
Variability in the 13C/12C ratios
The determination of the carbon isotope ratio in androgen metabolites has been previously
shown to be a reliable, direct method to detect testosterone misuse in the context of
antidoping testing. Here, the variability in the 13C/12C ratios in urinary steroids in a widely
heterogeneous cohort of professional soccer players residing in different countries
(Argentina, Italy, Japan, South Africa, Switzerland and Uganda) was examined. Carbon
isotope ratios of selected androgens in urine specimens were determined using gas
chromatography/combustion/isotope ratio mass spectrometry (GC-C-IRMS). Urinary steroids
in Italian and Swiss populations were found to be enriched in 13C relative to other groups,
reflecting higher consumption of C3 plants in these two countries. Importantly, detection
criteria based on the difference in the carbon isotope ratio of androsterone and pregnanediol
for each population were found to be well below the established threshold value for positive
cases. The results obtained with the tested diet groups highlight the importance of adapting
the criteria if one wishes to increase the sensitivity of exogenous testosterone detection. In
addition, confirmatory tests might be rendered more efficient by combining isotope ratio mass
spectrometry with refined interpretation criteria for positivity and subject-based profiling of
steroids [09070].
1000
Principal components analysis
Principal components analysis was applied to anabolic androgenic steroids molecules

referred in the WADA list of prohibited substances, resulting to their classification into six
distinct groups related to structure features where metabolic alterations usually occur. The
metabolites of the steroids participating to these six groups were treated using the Excel(c)
classification filters showing that common metabolism routes are derived for each of the
above principal components analysis classes, leading to the proposed metabolism schemes
of the study. This rule-based approach is proposed for the prediction of the metabolism of
unknown, chemically modified steroids, otherwise named as designer steroids. The
metabolites of three known, in the literature, anabolic androgenic steroids are estimated
using the proposed metabolism schemes, confirming that their use could be a useful tool for
the prediction of metabolic pathways of unknown anabolic androgenic steroids [09071].
Androgen receptors assay
The utility of androgen receptor-based bioassays to probe for the presence of AAS and other
non-steroidal anabolic agents in dietary products (with and without additional mass
spectrometric measurement) has been demonstrated with various applications and reports in
the past. One of the main advantages of this approach is the assay's capability to indicate
the presence of one or more substances able to bind to the androgen receptor, even if the
structures and compositions of the substrates are unknown to the analyst. Moreover, the
bioassay will provide information on the sum of androgen receptor activation. Hence, if two or
more anabolic agents are present at low concentration, their detection is facilitated compared
to methods that are designed to measure each analyte individually. Once suspicious
bioassay results are obtained, products can be scrutinized for known as well as possibly
unknown anabolic agents as recently shown in a study using a combined bioaffinity mass
spectrometry methodology employing a competitive inhibition binding assay interfaced to a
UHPLC-MS/MS system. In terms of doping controls, particularly the first-mentioned feature
of measuring the combined androgen receptor binding of multiple analytes was evaluated as
a potential means to tackle the issue of testosterone doping. The androgenic activity in urine
as well as serum was measured prior to and after intramuscular testosterone enanthate
administration, demonstrating that the readout of the bioassay was elevated independent
from UGT2B17 ins/ins, ins/del, and del/del genotypes, suggesting that this approach might
complement traditional steroid profile measurements [14009].
Anabolic androgenic steroids (AAS) share the activation of the androgen receptor (AR) as
common mechanism of action. The mammalian androgen responsive reporter gene assay
(AR CALUX bioassay), measuring compounds interacting with the AR can be used for the
analysis of AAS without the necessity of knowing their chemical structure beforehand,
whereas current chemical-analytical approaches may have difficulty in detecting compounds
with unknown structures, such as designer steroids. One study demonstrated that AAS
prohibited in sports and potential designer AAS can be detected with this AR reporter gene
assay, but that also additional steroid activities of AAS could be found using additional
mammalian bioassays for other types of steroid hormones. Mixtures of AAS were found to
behave additively in the AR reporter gene assay showing that it is possible to use this
method for complex mixtures as are found in doping control samples, including mixtures that
are a result of multi drug use. To test if mammalian reporter gene assays could be used for
the detection of AAS in urine samples, background steroidal activities were measured. AAS-
spiked urine samples, mimicking doping positive samples, showed significantly higher
androgenic activities than unspiked samples. GC-MS analysis of endogenous androgens and
AR reporter gene assay analysis of urine samples showed how a combined chemical-
analytical and bioassay approach can be used to identify samples containing AAS. The
1001
results indicate that the AR reporter gene assay, in addition to chemical-analytical methods,
can be a valuable tool for the analysis of AAS for doping control purposes [09072].
The utility of androgen receptor-based bioassays to probe for the presence of AAS and other
non-steroidal anabolic agents in dietary products (with and without additional mass
spectrometric measurement) has been demonstrated with various applications and reports in
the past. One of the main advantages of this approach is the assay's capability to indicate
the presence of one or more substances able to bind to the androgen receptor, even if the
structures and compositions of the substrates are unknown to the analyst. Moreover, the
bioassay will provide information on the sum of androgen receptor activation. Hence, if two or
more anabolic agents are present at low concentration, their detection is facilitated compared
to methods that are designed to measure each analyte individually. Once suspicious
bioassay results are obtained, products can be scrutinized for known as well as possibly
unknown anabolic agents as recently shown in a study using a combined bioaffinity mass
spectrometry methodology employing a competitive inhibition binding assay interfaced to a
UHPLC-MS/MS system. In terms of doping controls, particularly the first-mentioned feature
of measuring the combined androgen receptor binding of multiple analytes was evaluated as
a potential means to tackle the issue of testosterone doping. The androgenic activity in urine
as well as serum was measured prior to and after intramuscular testosterone enanthate
administration, demonstrating that the readout of the bioassay was elevated independent
from UGT2B17 ins/ins, ins/del, and del/del genotypes, suggesting that this approach might
complement traditional steroid profile measurements [13009].
Although the specificity and unambiguous nature of mass spectrometry-based methods is

undisputed, the search for complementary approaches, especially for initial test methods, is
unbowed. Here the utility of effect-based test methods such as those utilizing bioassays with
androgen-receptors have been extensively reviewed. These assays can indicate the
presence of agents stimulating the human androgen receptor without detailed knowledge of
the substrate; however, proof of the misuse of anabolic agents remains to be provided, most
likely by structural identification of the banned substance, for example, by mass
spectrometry. In addition, immunological methodologies have been proposed to support the
detection of AAS from human serum in a recent communication. By means of three different
polyclonal antibodies (raised against boldenone, stanozolol, and tetrahydrogestrinone) and
their respective cross reactivities, a total of 11 AAS is described to be detectable in less than
3 h. The authors highlight the sensitivity of the assay as being in agreement with WADAs
MRPL; however, for serum samples no MRPL is given as to AAS concentrations and urine
specimens have to be taken into account where metabolic processes and other potential
[13012].
Receptor binding (competitive) assays

Receptor binding assays are based on the binding affinity of a ligand for its receptor. For this
assay, purified receptor is immobilized on a column or suspended in a homogenate and to
this, radiolabeled testosterone of known concentration is added. For the test, the suspect
molecule is added to the radiolabeled testosterone, and it is measured whether the unknown
molecule displaces the binding of the testosterone. If there is displacement then the unknown
molecule has AR binding affinity. This type of assay only measures binding to AR and
therefore is not able to differentiate between agonist and antagonist activity, or if any activity
per se. Receptor binding assays require the use of radiolabelling, which presents a hazard if
used as a routine screening test. These assays can be developed as high-throughput and
are relatively easy to perform. To date, they have been used in a number of applications,
including screening animal feed for growth hormones and screening for potential endocrine
disrupting chemicals (EDCs). They can also be used to investigate the potential potency of
anabolic steroids by assessing the ability to bind to AR [13084].
1002
Solvent and solid-phase extraction of natural and synthetic anabolic
Liquid-liquid (using dichloromethane) and liquid-solid extraction processes (using disposable

C18 cartridges) were applied to human urine samples spiked with 15 androgenic anabolic
steroids (natural and synthetic). The extraction recoveries were assessed from different
HPLC separations of anabolic steroids using water-acetonitrile mobile phase, and using
calibration graphs obtained by injection into HPLC of standard samples of these compounds
before and after extraction. The procedures, including sample preconcentration, showed
extraction efficiencies over 90 percent which were independent on a wide range of
concentrations tested. Solid phase extraction yielded poor results for oximetolone, danazol
and dehydroepiandrosterone. For real urine samples, hydrolysis using beta-glucuronidase
and washing using sodium hydroxide before and after solvent extraction, respectively, is
recommended [01046].
High-performance liquid chromatography-ion trap mass spectroscopy
The measurement of androgen steroids has been utilized as a clinical indicator of adrenal
function, androgen abuse, and as a prediction of general health or biological aging. An
improved high-performance liquid chromatography-ion trap mass spectroscopic method with
sonic spray ionization (SSI) technology for the quantification of individual urinary 17-
ketosteroid sulfates and glucuronides was developed and validated. Sample preparation was
simplified using a C18 cartridge followed by direct injection onto a reversed-phase HPLC
column. Individual 17-ketosteroid from 63 urinary specimens collected in a 24-h period was
measured. 17-Ketosteroid conjugates, total 17-KS-S and the ratio of total 17-KS-S to
creatinine referred to herein as the Anabolic/Catabolic Index (ACI) showed statistically
significant negative correlations with age [01047].
Sensor chip preparation and assay construction for immunobiosensor
Immuno-sensing with optical biosensors is a new technique that is currently being exploited
for detection of residues in foods. One study explored the possibilities of obtaining fast
screening assays for a number of illegal veterinary drug residues at low concentrations.
Analyte specific sensor surfaces were prepared and used to construct inhibition assays with
various antibody reagents. Assay sensitivities for calibration curves in buffer solutions around
0.5 ng/mL in terms of IC50 (concentration of the inhibitor) values were achieved for the beta-
agonist clenbuterol and the hormone analogues ethinylestradiol and trenbolone. Assay
performance was optimised by evaluating factors such as sensor surface ligand density,
active antibody concentration, biosensor flow rate, etc [01048].
Androgen bioassays
Androgen bioassays used for detection differ from the techniques described above because
they mimic AR function and are not dependent on chemical structure. There are a number of
different bioassays ranging from those based on whole animals to those based on
mammalian or yeast cells. The Hershberger assay is an example of an in vivo androgen
bioassay. The endpoint of this assay is a measured increase in the weight of androgen-
dependent tissues. It is based on orchidectomised animals that produce little endogenous
sex steroid hormones. These animals are treated with the test compound. If the test
compound is androgenic, it will promote growth of androgen-dependent tissues. As this is an
in vivo assay, metabolism of test molecules can also be tested by analyzing metabolites
present in the blood stream and/or urine. As metabolism occurs upon treatment, this assay
1003
cannot be used to screen for activation or inactivation of androgens but it does allow the
dissection of anabolic and androgenic effects of the test molecule or its metabolites. An
assay based in animals is not feasible for routine sports doping screening in WADA
laboratories. This has led to the development of in vitro cell-based androgen bioassays to
screen for androgenic compounds. In vitro cell-based bioassays are widely used to detect
androgenic molecules. They were first developed to test environmental pollutants (endocrine
disrupting chemicals, EDCs) for their ability to alter normal hormonal function. Many
substances including detergents (nonylphenol and other alkylphenols), plastics (bisphenol
A), pesticides, insecticides, and even pharmaceutical wastes such as birth control tablets
(ethinylestradiol) are now classified as EDCs. In vitro yeast androgen bioassays can be used
in combination with other detection methods such as ultra high performance liquid
chromatography combined with time-of-flight-tandem mass spectrometry (UHPLC/TOFMS)
or liquid chromatography screening method. Moreover, bioassays can detect androgens in
samples where LC-MS/MS could not, highlighting that bioassays have a valuable role in the
Cell-based androgen bioassays

Cell proliferation assays can be used to measure the hormonal activity of a suspected
agonist (or antagonist) in a sample because hormones, via their specific receptors, stimulate
cell growth. In these assays, radioactive-labeled nucleotides are included in the culture
media that become incorporated into DNA as cells proliferate. The radiolabel that
incorporates into cells is a direct measurement of cell proliferation. These assays can
measure both agonist and antagonist activity, as an agonist for the receptor of interest can
stimulate cell growth, whereas an antagonist will block cell growth in the presence of an
agonist. To date, this type of bioassay has not been extensively used to screen for
androgens, however, it is the basis of the E-screen, which uses the human breast-cancer cell
line (MCF-7) to screen for xenoestrogens. This assay is relatively simple to perform and is
amenable to high-throughput readouts. However, results can be confounded by cell
expression of other receptors (such as AR and glucocorticoid receptor) that induce non-
specific cell proliferation. The assay relies on cell growth and, therefore, it can take days to
produce results. Thus, it is not really feasible for use in sport doping laboratories [13084].
Sensitivities of various androgen bioassays.

The detection of prohormones or an understanding of potential metabolism of test extracts is
central for the analysis of nutritional sport supplements. Mammalian cell lines may be limited
in their metabolic capacity, compared to in vivo metabolism. To address this, a liver tissue
metabolism step has been combined with the yeast AR/ARE/EGFP-based bioassay. In this
two-step assay, test extracts are first incubated with a bovine liver S9 fraction, the extract
recovered, and then exposed to the yeast AR bioassay. As the liver tissue is whole, it is
expected that this ex vivo approach will mimic the in vivo capacity for enzymatic conversions
of steroids and therefore detect both prohormones and/or strong androgenic metabolites. In
another example of introducing a pre-metabolism step prior to testing with a yeast AR
bioassay to allow for prohormone or androgen metabolite detection, samples were pre-
treated with a Helix pomatia enzyme mix to activate inactive hormone conjugates including
sulphates, glucuronides and glycosides. This example was in the setting of feed
supplementation, rather than nutraceutical supplements, however, it is possible that a similar
approach could be used to detect such conjugates if they were components of nutraceuticals
[13084].
A conjugated hapten microarray based on miniature immunoassay for fast and multiplex
detection of anabolic steroids is reported for the first time. This preliminary study investigated
1004
the possibility of using a microarray technology as a multisteroid detection assay. The
microarray system used eight monoclonal antibodies raised against three steroid conjugates,
4-androsten-4-chloro-17beta-ol-3-one, 1,5alpha-androsten-1beta-methyl-17beta-ol-3-one,
and 5beta-androsten-1-en-17beta-ol-3-one, which were conjugated to BSA by the active
ester method. In addition to 4 commercial conjugated haptens, 18 steroid-BSA conjugates
were synthesized and from all these a conjugated hapten microarray was fabricated. The
analyzed substances included 42 types of anabolic steroid reference materials and 28
positive urine samples. Of these, 24 anabolic steroids and 12 positive urines were
successfully detected [04068].
Protein assay
The purpose of one study was to develop a rapid and sensitive method utilizing the state-of-
the-art protein arrays technique to detect urinary anabolic steroids (ASs) in athletes. Three
experiments were designed to investigate the feasibility of the protein arrays for ASs testing.
Firstly, androgen receptor (AR) and estrogen receptor (ER) protein arrays were prepared on
polysaccharide-coated slides to investigate whether they can bind to ASs (affinity tests).
Secondly, in comparison to adrenergic receptor (the receptor of beta-blockers) and opioid
receptor (the receptor of narcotic analgesics) arrays, AR and ER protein arrays were used to
test whether they can determine the ASs positive urine sample specifically (specific binding
tests). At last protein arrays were used to estimate qualitatively the ASs in positive urine
samples (qualitative tests). From the results of the affinity tests the shape of the dose-
dependence curve suggested a positive cooperative binding of ASs with the protein arrays.
The AR and ER protein arrays showed affinities for fluorescence labelled testosterone and
estradiol that were similar to those of literatures (0.65 vs 0.89 nM, 5.96 vs 10.3 nM,
respectively). Based on the data, the sensitivity of testing can reach 0.1 nM that was much
better than the World Anti-Doping Code (WADA) standard. Specific binding tests showed
that the prohibited substance in positive urine samples belonged to the anabolic estrogenic
inhibitor of ASs. From the results of qualitative tests, it could be estimated that there were
anabolic androgenic steroids in the positive urine samples and their concentration was lower
than 50 microM methyltestosterone. The total time of the test process for ASs in urine
needed less than 1 h. In summary, the present study showed that the protein arrays method
provided a highly sensitive and rapid alternative to screen urine samples for the detection of
the misuse of ASs in athletes and was suitable for testing in both weekly training sessions as
well as large-scale competition events [06102].
Enzyme-immunoassay kit
One study assessed an enzyme-immunoassay (EIA) kit for measuring the salivary
testosterone and cortisol concentrations of weightlifters. Saliva samples (n=64) were
collected from male and female weightlifters during normal training procedures and analysed
for testosterone and cortisol using a commercial EIA kit and a criterion radioimmunoassay
(RIA) method. Significant correlations were demonstrated between the EIA and RIA
measurements of salivary testosterone and cortisol concentrations. Further examination by
sample and gender revealed similar relationships. The EIA concentrations of salivary
testosterone and cortisol were found to be slightly greater (10-13 %) than the RIA values.
Similar discrepancies were noted when gender comparisons were made, although the
relative information on testosterone (males > females) and cortisol (males=females) were
consistent for both assay methods. In conclusion, a commercially available EIA kit provided
valid measures of the salivary testosterone and cortisol concentrations of male and female
weightlifters. Factors to consider when using an EIA kit include the hormone(s) of interest,
the magnitude of the correlations, as well as the descriptive information gained (e.g.
1005
absolute, relative) and its uses within sport [10341].
Triptorelin test
In a case report a 34-year-old man had a single dose (100 mug) of triptorelin (triptorelin test).
Within 1 month, the patient's serum testosterone was in the normal range, and he reported a
return to normal energy and libido. The World Anti-Doping Code has proved to be a very
powerful and effective tool in the harmonization of antidoping efforts worldwide, but it is
insufficient to combat this illegal phenomenon. To tackle the serious side effects caused by
doping we believe that it is necessary to increase monitoring and adopt severe sanctions,
particularly with regard to Internet sites [10074].
Serum inhibin B as a potential marker of testosterone doping
The aim of one study was to explore effectors of the pituitary-testicular axis suitable as
potential biochemical markers to screen for testosterone doping. A pilot study with male
bodybuilding athletes with a self-reported history of testosterone doping (repeated
intramuscular administration of testosterone preparations, last injection 8 weeks or less ago)
were compared with an equal sized control group matched for sex, age, and body mass
index. Fifteen healthy young men of white background were tested for inhibin B,
testosterone, luteinizing hormone (LH), follicle-stimulating hormone (FSH). Although the
levels of testosterone, LH, and FSH did not differ between the 2 groups, the serum
concentrations of inhibin B in individuals with a history of testosterone doping were
exclusively at or below the lower limit of the normal range for adult men (100-400 pg/mL).
Inhibin B was significantly lower in those men who used testosterone for weight lifting (76 +
36 ng/L) than in controls (182 + 35 ng/L). It was concluded that a low concentration of serum
inhibin B may reflect the application of exogenous testosterone and appears to be a potential
marker associated with anabolic androgenic steroid doping [10075].
This work presents the validation study of the comprehensive two-dimensional gas
chromatography (GC x GC)-time-of-flight mass spectrometry method performance in the
analysis of the key World Anti-Doping Agency anabolic agents in doping control. The relative
abundance ratio, retention time, identification and other method performance criteria have
been tested in the GC x GC format to confirm that they comply with those set by WADA.
Furthermore, tens of other components were identified with an average similarity of >920 (on
the 0-999 scale), including 10 other endogenous sterols, and full mass spectra of 5,000+
compounds were retained. The testosterone/epitestosterone ratio was obtained from the
same run. A new dimension in doping analysis has been implemented by addressing
separation improvement. Instead of increasing the method sensitivity, which is accompanied
by making the detector increasingly "blind" to the matrix (as represented by selected ion
monitoring mode, high-resolution mass spectrometry (MS) and tandem MS), the method
capabilities have been improved by adding a new "separation" dimension while retaining full
mass spectral scan information. Apart from the requirement for the mass spectral domain
that a minimum of three diagnostic ions with relative abundance of 5 percent or higher in the
MS spectra, all other WADA criteria are satisfied by GC x GC operation. The minimum of
three diagnostic ions arises from the need to add some degree of specificity to the acquired
mass spectrometry data; however, under the proposed full MS scan method, the high MS
similarity to the reference compounds offers more than the required three diagnostic ions for
an unambiguous identification. This should be viewed as an extension of the present criteria
to a full-scan MS method [10076].
1006
Designer drugs
Effective detection of the abuse of androgenic-anabolic steroids in human and animal sports
often requires knowledge of the drug's metabolism in order to target appropriate urinary
metabolites. “Designer” steroids are problematic since it is difficult to obtain ethical approval
for in vivo metabolism studies due to a lack of a toxicological profile. In one study, the in vitro
metabolism of estra-4,9-diene-3,17-dione is reported for the first time. This is also the first
study comparing the metabolism of a designer steroid in the three major species subject to
sport's doping control; namely the equine, canine and human. In order to allow the
retrospective analysis of sample testing data, the use of a high-resolution (HR) accurate-
mass Thermo LTQ-Orbitrap LC-MS instrument was employed for metabolite identification of
underivatised sample extracts. The full scan HR-LC-MS Orbitrap data was complimented by
several further experiments targeted at elucidating more detailed structural information for
the most abundant metabolites. These included; HR-LC-MS/MS of the underivatised
metabolites, functional group selective chemical derivatisation followed by full scan HR-LC-
MS, enzyme inhibition experiments and full scan electron ionization GC-MS analysis of
methoxyamine-trimethylsilyl derivatives. The major metabolite detected in all species, and
therefore the most suitable candidate for screening of estra-4,9-diene-3,17-dione abuse, was
proposed to be an isomer of 17-hydroxy-estra-4,9-dien-3-one. Less significant metabolic
pathways in all species included hydroxylation and reduction followed by hydroxylation.
Reductive metabolism in the canine was less significant than in the other two species, while
the equine was unique in producing a di-reduced metabolite (proposed to be an isomer of
estra-4,9-diene-3,17-diol) and also relatively large quantities of d-ring hydroxy and hydroxy-
reduced metabolites [10077].
Adequate detection of designer steroids in the urine of athletes is still a challenge in doping
control analysis and requires knowledge of steroid metabolism. In one study we investigated
whether uPA(+/+)-SCID mice carrying functional primary human hepatocytes in their liver
would provide a suitable alternative small animal model for the investigation of human steroid
metabolism in vivo. A quantitative method based on liquid chromatography-tandem mass
spectrometry (LC-MS/MS) was developed and validated for the urinary detection of 7 known
methandienone metabolites. Application of this method to urine samples from humanized
mice after methandienone administration allowed for comparison with data from in vivo
human samples and with reported methandienone data from in vitro hepatocyte cultures. The
LC-MS/MS method validation in mouse and human urine indicated good linearity, precision,
and recovery. Using this method it was quantified 6 of 7 known human methandienone
metabolites in the urine of chimeric mice, whereas in control nonchimeric mice we detected
only 2 metabolites. These results correlated very well with methandienone metabolism in
humans. In addition, it was detected 4 isomers of methandienone metabolites in both human
and chimeric mouse urine. One of these isomers has never been reported before. The
results of this proof-of-concept study indicate that the human liver-uPA(+/+)-SCID mouse
appears to be a suitable small animal model for the investigation of human-type metabolism
of anabolic steroids and possibly also for other types of drugs and medications [09073].
Molecularly imprinted polymer filaments (MIPFs)
An online system that can perform dynamic microextraction, on-coating derivatization and
desorption, and subsequent GC-MS analysis with a large-volume injection was developed. A
derivatization cell as the conjunction of the online system was developed for the online
extraction and derivatization. To evaluate the feasibility of the online system,
methyltestosterone molecularly imprinted polymer filaments (MIPFs) were prepared for the
1007
selective online extraction of five androgenic steroids, namely, methyltestosterone,
testosterone, epitestosterone, nandrolone, and metandienone. Under the optimized
conditions, the detection limits of testosterone and epitestosterone were 0.09 and 0.12 μg/L,
respectively, which were under the minimum required performance limits between 2 and 10
μg/L from the World Anti-Doping Agency. The detection limits of the other three androgenic
steroids were varied from 0.04 to 0.18 microg/L. Finally, the MIPFs-GC-MS method was
applied for the determination of androgenic steroids in urine, and satisfactory recovery (78-97
%) and reproducibility (3.2-8.9 %) were obtained. The proposed online coupling system
offers an attractive alternative for hyphenation to GC instruments and could also be extended
to other adsorptive materials [13176].
An online system that can perform dynamic microextraction, on-coating derivatization and
desorption, and subsequent GC-MS analysis with a large-volume injection was developed. A
derivatization cell as the conjunction of the online system was developed for the online
extraction and derivatization. To evaluate the feasibility of the online system,
methyltestosterone molecularly imprinted polymer filaments (MIPFs) were prepared for the
selective online extraction of five androgenic steroids, namely, methyltestosterone,
testosterone, epitestosterone, nandrolone, and metandienone. Under the optimized
conditions, the detection limits of testosterone and epitestosterone were 0.09 and 0.12
microg/L, respectively, which were under the minimum required performance limits between
2 and 10 microg/L from the World Anti-Doping Agency. The detection limits of the other three
androgenic steroids were varied from 0.04 to 0.18 microg/L. Finally, the MIPFs-GC-MS
method was applied for the determination of androgenic steroids in urine, and satisfactory
recovery (78.0-96.9 %) and reproducibility (3.2-8.9 %) were obtained. The proposed online
coupling system offers an attractive alternative for hyphenation to GC instruments and could
also be extended to other adsorptive materials [13177].
Leidenfrost phenomenon assisted thermal desorption
Rapid detection of trace level anabolic steroids in urine is highly desirable to monitor the
consumption of performance enhancing anabolic steroids by athletes. The present article
describes a novel strategy for identifying the trace anabolic steroids in urine using
Leidenfrost phenomenon assisted thermal desorption (LPTD) coupled to dielectric barrier
discharge (DBD) ionization mass spectrometry. Using this method the steroid molecules are
enriched within a liquid droplet during the thermal desorption process and desorbed all-
together at the last moment of droplet evaporation in a short time domain. The desorbed
molecules were ionized using a dielectric barrier discharge ion-source in front of the mass
spectrometer inlet at open atmosphere. This process facilitates the sensitivity enhancement
with several orders of magnitude compared to the thermal desorption at a lower temperature.
The limits of detection (LODs) of various steroid molecules were found to be in the range of
0.05-0.1 ng/mL for standard solutions and around two orders of magnitude higher for
synthetic urine samples. The detection limits of urinary anabolic steroids could be lowered by
using a simple and rapid dichloromethane extraction technique. The analytical figures of
merit of this technique were evaluated at open atmosphere using suitable internal standards.
The technique is simple and rapid for high sensitivity and high throughput screening of
anabolic steroids in urine [14258].
This study proposes a new analytical methodology for the determination of trace levels of
testosterone (T) and epitestosterone (E) in urine matrices using bar adsorptive
microextraction combined with liquid desorption followed by high-performance liquid
1008
chromatography with diode array detection (BAmyE-LD/HPLC-DAD). The comparison of
different sorbent coatings (five activated carbons, one styrene-divinylbenzene, two modified
pyrrolidone, one ciano and one n-vinylpyrrolidone polymers) through BAmyE showed that the
latter phase presented much higher selectivity and capacity offering multiple mechanisms of
interaction. Assays using this phase were performed on 25 mL of water samples spiked at
the 8.0 myg/L level, yielded average recoveries of 92.1 and 93.4 percent for T and E,
respectively, under optimized experimental conditions; BAmyE (n-vinylpyrrolidone): 16 h
(1000 rpm), pH 5.5; LD: acetonitrile, 30min under sonication treatment. From the developed
analytical methodology, suitable detection limits were achieved (0.4 myg/L) and good linear
dynamic ranges (1.4-16.0 myg/L) with remarkable determination coefficients (r2>0.9978). By
using the standard addition methodology, the application of the present analytical approach
on urine samples revealed good sensitivity. The proposed method, which operated under the
floating sampling technology, proved to be a suitable sorption-based static microextraction
alternative for screening T, E and the T/E ratio in urine samples for doping control purposes.
The methodology showed to be easy to implement, demonstrating good reproducibility,
sensitivity and robustness, allowing the possibility to choose the most selective sorbent
coating according to the compounds of interest [14642].
Alternative methodologies for steroid quantification
While GC-MS (and recently GC-MS/MS) has been the analytical reference technique for
steroid quantification in urine matrix for many years, some alternative approaches have been
considered. First, the use of immunological tests was investigated, as the main advantages
of this method is the possibility of automated processes (simple and rapid), the lower costs
and the routine ease-of-use for non-scientific staff. Although ELISA assays have shown good
total specificity and appropriate sensitivity for T, the main drawbacks of this biochemical
approach are the cross-reactivity that could lead to wrong estimation of T concentration and
the restricted application to a single compound (eg, T) which is not compatible with the
intention to establish a profile with several steroids. In addition to these limitations for steroid
quantification and identification, radioimmunoassay (RIA) tests in urine present other
drawbacks such as non-availability of RIA assay kits in the market for urine steroid detection
and the matrix effect being more significant for RIA kits than for ELISA kits. Considering
these disadvantages, anti-doping laboratories never deemed immunoassays as a useful tool
to establish a steroid profile [14450].
The determination of steroid concentrations by GC-MS technique requires essential steps

prior to analysing the urine samples. Solid phase extraction (SPE) and/or LLE, hydrolysis,
evaporation and derivatisation are necessary to obtain robust and reliable data but could also
be a source of variability and inaccuracy. Measuring the steroid compounds by LC-MS
instruments could overtake these steps. The first attempts were made about 30 years ago to
detect steroids produced in rat liver microsomes. More recently, some authors have
published LC-MS methods to quantify T and E in human urine, but hydrolysis and extraction
steps were still required to detect the free fraction of the steroids. In the meantime, it was
developed a LC-MS method for the quantification of T and E conjugates (sulfate and
glucuronide) which stimulated many other authors to investigate the LC-MS detection of the
steroids conjugated fraction. Whereas quantification of steroids conjugates by LC-MS was
first published in 1996 and in 2011, it was presented a method based on a high-resolution
MS strategy for the quantification of 11 steroids conjugates after a simple SPE step. Two
years later, the same group increased the number of targeted analytes to 13 and applied
their quantification method on samples collected after T administration. A comparison was
made between data obtained with traditional GC-MS and LC-MS techniques, and as a
conclusion, a good correlation was depicted showing the possibility of measuring urinary
steroid based on conjugated compounds and by LC-MS technique. Despite these promising
1009
LC-MS results, this analytical approach is not encouraged in the recently published technical
document, but initial testing analysis and confirmation should be based on GC separation
[14450].
Proteomics and steroidomics
using a tandem mass tags shotgun proteomics approach. It was defined 28 candidate protein
based on partial least square discriminant analysis. The findings confirm the effectiveness of
One review presents the evolution of steroid analytical techniques, including gas
chromatography coupled to mass spectrometry (GC-MS), immunoassay (IA) and targeted
liquid chromatography coupled to mass spectrometry (LC-MS), and it evaluates the potential
of extended steroid profiles by a metabolomics-based approach, namely steroidomics.
Steroids regulate essential biological functions including growth and reproduction, and
perturbations of the steroid homeostasis can generate serious physiological issues;
therefore, specific and sensitive methods have been developed to measure steroid
concentrations. GC-MS measuring several steroids simultaneously was considered the first
historical standard method for analysis. Steroids were then quantified by immunoassay,
allowing a higher throughput; however, major drawbacks included the measurement of a
single compound instead of a panel and cross-reactivity reactions. Targeted LC-MS methods
with selected reaction monitoring (SRM) were then introduced for quantifying a small steroid
subset without the problems of cross-reactivity. The next step was the integration of
metabolomic approaches in the context of steroid analyses. As metabolomics tends to
identify and quantify all the metabolites (i.e. the metabolome) in a specific system,
appropriate strategies were proposed for discovering new biomarkers. Steroidomics, defined
as the untargeted analysis of the steroid content in a sample, was implemented in several
fields, including doping analysis, clinical studies, in vivo or in vitro toxicology assays, and
more. This review discusses the current analytical methods for assessing steroid changes
and compares them to steroidomics. Steroids, their pathways, their implications in diseases
and the biological matrices in which they are analysed will first be described. Then, the
different analytical strategies will be presented with a focus on their ability to obtain relevant
information on the steroid pattern. The future technical requirements for improving steroid
analysis was also presented [150169].
Screening hybridomas antigen microarray
Currently, dozens of anabolic androgenic steroids (AAS) are forbidden in the World Anti-
Doping Agency Prohibited List, however, despite extensive investigation, there are still lots of
1010
AAS without corresponding monoclonal antibodies. A steroid analog antigen microarray
made up of ten AAS was fabricated to screen the hybridoma and it was found an original
unsuccessful clone turned out to be a candidate anti-boldenone antibody, without any cross-
reactions with endogenous AAS or 44 different AAS standard reference materials tested. The
findings suggested that steroid analog antigen microarray could be a promising tool to screen
and characterize new applications of antibodies for structure analogs, and this also exhibits
the potential to fast identify effective epitopes of hybridomas in a single assay [150170].
Urinary steroids
One work presented a novel database search engine – MLibrary – designed to assist the
user in the detection and identification of androgenic anabolic steroids (AAS) and its
metabolites by matrix assisted laser desorption/ionization (MALDI) and mass spectrometry-
based strategies. The detection of the AAS in the samples was accomplished by searching
the mass spectrometric (MS) spectra against the library developed to identify possible
positives and by comparison of the tandem mass spectrometric (MS/MS) spectra produced
after fragmentation of the possible positives with a complete set of spectra that have
previously been assigned to the software. The urinary screening for anabolic agents plays a
major role in anti-doping laboratories as they represent the most abused drug class in sports.
With the help of the MLibrary software application, the use of MALDI techniques for doping
control is simplified and the time for evaluation and interpretation of the results is reduced. To
do so, the search engine takes as input several MALDI-TOF-MS and MALDI-TOF-MS/MS
spectra. It aids the researcher in an automatic mode by identifying possible positives in a
single MS analysis and then confirming their presence in tandem MS analysis by comparing
the experimental tandem mass spectrometric data with the database. Furthermore, the
search engine can, potentially, be further expanded to other compounds in addition to AASs.
The applicability of the MLibrary tool is shown through the analysis of spiked urine samples
[13178].
Liquid chromatographic/mass spectrometric screening method
Anabolic androgenic steroids (AAS) are metabolized extensively in the human body,
resulting mainly in the formation of glucuronide conjugates. Current detection methods for
AAS are based on gas chromatographic/mass spectrometric (GC/MS) analysis of the
hydrolyzed steroid aglycones. These analyses require laborious sample preparation steps
and are therefore time consuming. Our interest was to develop a rapid and straightforward
method for intact steroid glucuronides in biological samples, using liquid-phase
microextraction (LPME) sample clean-up and concentration method combined with liquid
chromatographic/tandem mass spectrometric (LC/MS/MS) analysis. The applicability of
LPME was optimized for 13 steroid glucuronides, and compared with conventional liquid-
liquid extraction (LLE) and solid-phase extraction (SPE) procedures. An LC/MS/MS method
was developed for the quantitative detection of AAS glucuronides, using a deuterium-labeled
steroid glucuronide as the internal standard. LPME, owing to its high specificity, was shown
to be better suited than conventional LLE and SPE for the clean-up of urinary AAS
glucuronides. The LPME/LC/MS/MS method was fast and reliable, offering acceptable
reproducibility and linearity with detection limits in the range 2-20 ng/mL for most of the
selected AAS glucuronides. The method was successfully applied to in vitro metabolic
studies, and also tested with an authentic forensic urine sample. For a urine matrix the
method still has some unsolved problems with specificity, which should be overcome before
the method can be reliably used for doping analysis, but still offering additional and
complementary data for current GC/MS analyses [03073].
1011
Friedel-Crafts acylation
The rapid Friedel-Crafts chromogenic acylation of alkene groups at ambient temperatures

using a 25:1 mixture of 98 percent acetyl chloride and 70 percent perchloric acid is shown to
have all the properties needed to serve as a potential quality control reagent that can be
used to routinely discriminate among steroid analogs. Although ostensibly a non-selective
reagent, from these and prior applications in terpenes and polyunsaturated acid esters, it is
seen that the reaction is capable of discriminating bewteen geometric isomers and even
enantiomers. The selectivity towards acylation of the alpha- over the beta-position at C-17
makes the method adaptable to screening for anabolic steroids. Reactions at that position
produce the more unusual results, including a positive color reaction for alpha-
methyltestosterone even though there is no alkene functional group in the vicinity of C-17.
For molecules with more than one alkene, concurrent acylations are independent one from
the other and, in the absence of any interferences, their spectral properties are found to be
additive [03074].
Micellar liquid chromatography
A systematic optimization of the HPLC separation of a complex mixture containing natural

and synthetic anabolic steroids by micellar liquid chromatography using a Hypersil (150 mm
x 3.0 mm i.d., 5 microm) C18 column and UV detection at 245 nm (exception is made for
oxymetolone and danazol which were monitorized at 280 nm) has been carried out. The
isocratic micellar mobile phases (from binary to quaternary) consisted of sodium dodecyl
sulphate and organic modifiers such as acetonitrile, tetrahydrofuran, propanol, butanol or
pentanol. The effect of the organic modifiers, surfactant concentration, temperature, ionic
strength and flow-rate on the separation has been studied. A micellar mobile phase 5%
propanol and 40 mM surfactant allowed the separation of 12 steroids out of 14 tested in
about 20 min. A bivariant optimization method for the micellar mobile phase propanol-
surfactant corroborated the above results [03075].
Liquid-phase microextraction sample clean-up

Anabolic androgenic steroids (AAS) are metabolized extensively in the human body,
resulting mainly in the formation of glucuronide conjugates. Current detection methods for
AAS are based on gas chromatographic/mass spectrometric (GC/MS) analysis of the
hydrolyzed steroid aglycones. These analyses require laborious sample preparation steps
and are therefore time consuming. Our interest was to develop a rapid and straightforward
method for intact steroid glucuronides in biological samples, using liquid-phase
microextraction (LPME) sample clean-up and concentration method combined with liquid
chromatographic/tandem mass spectrometric (LC/MS/MS) analysis. The applicability of
LPME was optimized for 13 steroid glucuronides, and compared with conventional liquid-
liquid extraction (LLE) and solid-phase extraction (SPE) procedures. An LC/MS/MS method
was developed for the quantitative detection of AAS glucuronides, using a deuterium-labeled
steroid glucuronide as the internal standard. LPME, owing to its high specificity, was shown
to be better suited than conventional LLE and SPE for the clean-up of urinary AAS
glucuronides. The LPME/LC/MS/MS method was fast and reliable, offering acceptable
reproducibility and linearity with detection limits in the range 2-20 ng/mL for most of the
selected AAS glucuronides. The method was successfully applied to in vitro metabolic
studies, and also tested with an authentic forensic urine sample. For a urine matrix the
method still has some unsolved problems with specificity, which should be overcome before
the method can be reliably used for doping analysis, but still offering additional and
complementary data for current GC/MS analyses [03076].
1012
In saliva
To validate the testosterone (T) and cortisol (C) concentration measures in saliva in
response to short high-intensity exercise 9 healthy males provided matching saliva and
plasma samples before and after a 30-second Wingate cycle test. Saliva was assayed for T
(Sal-T) and C (Sal-C) concentrations, and plasma for total T and total C, sex hormone-
binding globulin, corticosteroid-binding globulin (CBG) and albumin concentrations. The
plasma free and bioavailable hormones were calculated. The Sal-T and plasma T
correlations were weak to moderate when examined between individuals (pooled data for all
participants), but these relationships improved within individuals (data for each participant on
average). The Sal-C and plasma C correlations were strong both between individuals and
within individuals. The peak relative increases in Sal-T (35 + 9 %) and Sal-C (63 + 29 %)
concentrations exceeded the plasma total and/or free hormones, but not the bioavailable
hormones. Albumin and CBG also increased with exercise, along with blood lactate. It was
concluded that the Sal-T and Sal-C concentration measures were validated in response to
short high-intensity exercise, especially for individuals. The hormonal changes in saliva were
also more sensitive to exercise (i.e. greater relative responses) than the plasma total and/or
free hormones, potentially arising from changes in the binding proteins and blood lactate.
These findings support the use of saliva as a medium for steroid determination in sport
[10072].
In hair
Although not yet fully recognised by international sporting committees, hair analysis in doping
control may be a useful adjunct to drug testing of urine. It may permit access to retrospective
information and the identification of banned substances, especially when exogenous abuse
has to be distinguished from other forms of involuntary exposure to identical substances.
Negative hair results coupled with positive urine samples may be used to draw conclusions
of involuntary doping in sports whenever athletes claim not to have ingested any drug,
identical substances are present in their environment or are normal constituents of food and
beverages served to them immediately before the competition. Two cases are well described
in the literature in which hair analyses were fundamental in documenting positive doping after
urinalysis. In Brazil, 2 cases of athletes testing positive for banned substances caught our
attention because of the possibility of involuntary doping; hair analysis, if performed, may
have helped to clarify the results of the urinalysis. Despite the fact that it cannot be used for
routine control and overrule positive urinalysis, hair analysis can detect long term exposure
as well as those substances which are not excreted in urine. In the current International
Olympic Committee (IOC) code, hair analysis is not yet considered useful even in special
cases of doping control [01049].
Detection of anabolic steroids in hair samples has been possible only in fatal cases or in
cases of high-continuous dosages. In order to verify the possibility of detecting an acute
administration, a sensitive and specific assay has been developed for the simultaneous
determination of testosterone, nandrolone and some of their esters in hair. The analytes
were extracted from finely cut hair with methanol-trifluoroacetic acid overnight. After the
incubation, the mixture was evaporated to dryness, redissolved and extracted with hexane.
The dried organic layer was silanised and analysed by GC-MS and GC-MS-MS. A sensitivity
of at least 20 pg injected was obtained for all the analytes. In guinea pigs treated with a
single intramuscular dose of 10 mg/kg nandrolone decanoate, neither nandrolone decanoate
nor nandrolone were found in hair collected after 13 days, while both compounds were
clearly detectable after four repeated doses (each dose every 3-4 days) of 20 mg/kg
nandrolone decanoate. Neither nandrolone decanoate nor nandrolone could be detected in
1013
hair from a male healthy volunteer 1 month after treatment with 50 mg nandrolone
decanoate, while his urine still tested highly positive for the main nandrolone metabolite (>
100 ng/mL). Testosterone esters could not be detected in hair of healthy subjects collected
respectively 3, 2 and 1 month after a single intramuscular administration of 250 mg
testosterone enanthate (five subjects), a single intramuscular coadministration of 25 mg
testosterone propionate plus 110 mg testosterone enanthate (one subject), or a single oral
administration of 120 mg testosterone undecanoate (three subjects). Otherwise, hair analysis
revealed an increase of testosterone concentration corresponding to the period of treatment.
Analysis of blood and urine samples confirmed the absorption of those compounds. At the
sensitivity achieved by the present method, no detection of nandrolone, nandrolone
decanoate nor testosteron esters in hair seems to be obvious after a single dose
administration [00045].
In France during a famous bicycle race, the newspapers documented the degree in which
doping seemed to be supervised in some teams by managers and doctors. Use of anabolic
steroids and other substances was officially banned in the mid-seventies by sports
authorities. This policy has been enforced through urine testing before competition. It is well
known, however, that a latency period is all that is necessary to defeat these tests.
Nevertheless, hair analysis could be a promising tool when testing for periods that are not
accessible to urinalysis any more. It was developed different sensitive methods for testing
hair for amphetamines, anabolic steroids and their esters and corticosteroids. For
amphetamines, 50 mg of hair were digested with 1 M NaOH, extracted with ethyl acetate,
derivatized with TFA and analyzed by gas chromatography positive chemical-ionization mass
spectrometry. For corticosteroids, 50 mg of powdered hair were treated with methanol in an
ultrasonic bath and subsequently purified using a C18 solid phase extraction column.
Analysis was realized by high performance liquid chromatography coupled to electrospray-
ionization tandem mass spectrometry. For anabolic steroids and their esters, 100 mg of
powdered hair were treated with methanol in an ultrasonic bath for extraction of esters, then
alkaline digested with 1 M NaOH for an optimum recovery of other drugs. The two liquid
preparations were subsequently extracted with ethyl acetate, pooled, then finally highly
purified using a twin solid phase extraction on aminopropyl and silica cartridges. Residue
was derivatized with MSTFA prior to injection. Analysis was conducted by gas
chromatography coupled to a triple quadrupole mass spectrometer. Thirty cyclists were
sampled and tested both in hair and in urine. Amphetamine was detected 10 times in hair
(out of 19 analyses) compared to 6 times in urine (out of 30 analyses). Corticosteroids were
detected 5 times in hair (methylprednisolone 1 case, triamcinolone acetonide 3 cases and
hydrocortisone acetate 1 case) in hair (out of 12 analyses) compared to 12 times
(triamcinolone acetonide 10 cases and betamethasone 2 cases) in urine (out of 30
analyses). Anabolic steroids were detected twice (nandrolone 1 case, and testosterone
undecanoate 1 case) in hair (out of 25 analyses) compared to none in urine (out of 30
analyses) [00046].
Lists of banned classes of doping agents are released by the International Olympic
Committee, adopted by other sports authorities and updated regularly, including the
substance classes stimulants, narcotics, diuretics, anabolic agents, peptide hormones, beta-
blockers etc. There are different classes of restriction: anabolic and masking agents
(anabolic steroids, diuretics etc.) are always banned for athletes regardless of their topical
activity (training or competition) several substances are permitted with certain restrictions
(caffeine below a cut-off value, or inhalation of some beta 2 agonists) beta-blockers are
prohibited in competitions of certain sports disciplines the majority of the substances
(stimulants, narcotics etc.) is prohibited during competitions, so that they do not have to be
analysed in out-of-competition samples. A differentiation between training and competition
period is impossible by means of hair analysis due to the uncertainty of (especially short-
1014
term) kinetic considerations related to hair growth. Therefore, the analytical identification of
doping relevant substances in hair is not always a sufficient criterion for a doping offence and
the identification of stimulants, beta-blockers etc. in hair would be entirely irrelevant. The
most interesting target substances are certainly the anabolic agents, because their desired
action (enhanced muscle strength) lasts longer than the excretion, leading to sophisticated
procedures to circumvent positive analytical results in competition control. Besides the
analysis of out-of-competition control samples, the long term detection of steroids in hair
could provide complementary information. An analytical approach to the identification of
exogenous steroids in hair requires consideration of the presence of many other steroids in
the hair matrix interfering the analysis at trace levels, and of a limited chemical stability. The
analysis of endogenous steroids in hair appears to be even more complicated, because the
possibility of many biotransformation reactions from (into) other precursors (metabolites) has
to be taken into account. Precursor substances of anabolic steroids (especially esters as
application forms) are very promising analytical targets of hair analysis, because they can
only be detected after an exogenous intake. The quantitative evaluation of active parent
compounds like testosterone (which is actively involved in physiological processes of hair
growth) in hair is still controversial. Clinical applications under reproducible conditions can be
useful, but the biovariability of these parameters will probably prevent the definition of
acceptable cut-off levels as a criterion of abuse [00047].
It is generally accepted that chemical testing of biological fluids is the most objective means
of diagnosis of drug use. The presence of a drug analyte in a biological specimen can be
used to document exposure. The standard in drug testing is the immunoassay screen,
followed by the gas chromatographic-mass spectrometric confirmation conducted on a urine
sample. In recent years, remarkable advances in sensitive analytical techniques have
enabled the analysis of drugs in unconventional biological specimens such as hair. The
advantages of this sample over traditional media like urine and blood are obvious: collection
is almost noninvasive, relatively easy to perform, and in forensic situations it may be
achieved under close supervision of law enforcement officers to prevent adulteration or
substitution. Moreover, the window of drug detection is dramatically extended to weeks,
months or even years. The aim of one review was to document the current detection of
anabolic steroids in hair [03077].
Several bodybuilders, all winners of international competitions, were arrested for trafficking of
a number of doping agents including anabolic steroids, ephedrine, beta-adrenergics, human
chorionic gonadotropin, antidepressants, and diuretics. In accordance with the recent French
law against doping, the judge asked to test seven bodybuilders to identify doping practices.
Hair and urine specimens were collected for analysis. After decontamination, a 100 mg hair
strand was pulverized in a ball mill, hydrolyzed, extracted, and derivatized to be tested by
GC/MS for anabolic steroids, beta-adrenergic compounds, ephedrine, and other doping
agents. Urine was analyzed for anabolic steroids and metabolites, beta-adrenergic
compounds, ephedrine, and human chorionic gonadotropin, in addition to a broad spectrum
screening with GC/MS. The following compounds were detected in urine: ephedrine (29 and
36 ng/mL, n=2), clenbuterol (0.2 to 0.3 ng/mL, n=3), norandrosterone (4.7 to 100.7 ng/mL,
n=7), norethiocholanolone (0.9 to 161.8 ng/mL, n=6), stanozolol (1 to 25.8 ng/mL, n=4),
methenolone (2.5 to 29.7 ng/mL, n=4), testosterone (3 to 59.6 ng/mL, n=7), epitestosterone
(1 to 20.4 ng/mL, n=7) and ratio testosterone/epitestosterone >6 for four subjects (18.5 to
59.6). The following drugs were detected in hair: ephedrine (0.67 and 10.70 ng/mg, n = 2),
salbutamol (15 to 31 pg/mg, n=3), clenbuterol (15 to 122 pg/mg, n=6), nandrolone (1 to 7.5
pg/mg, n=3), stanozolol (2 to 84 pg/mg, n=4), methenolone (17 and 34 ng/ml, n=2),
testosterone enanthate (0.6 to 18.8 ng/mg, n = 5), and testosterone cypionate (3.3 to 4.8
ng/mg, n=2). These results document the doping practice and demonstrate repetitive
exposure to anabolic compounds and confirm the value of hair analysis as a complement to
1015
urinalysis in the control of doping practice [02052].
The measurement of anabolic steroid levels in human hair is possible in order to distinguish
between pharmaceutical steroids and natural steroids. It was now presented the first
investigation into the physiological concentrations of anabolic steroids in human hair in
Chinese subjects. A gas chromatography-tandem mass spectrometry (GC/MS/MS) method
was developed for the simultaneous identification and quantitation of five endogenous
anabolic steroids (testosterone, epitestosterone, androsterone, etiocholanolone and
dehydroepiandrosterone) in hair. After basic hydrolysis, hair samples were extracted with
diethyl ether, derivatized and then detected using GC/MS/MS in the multiple-reaction
monitoring mode. The one precursor/two product ion transitions for each anabolic steroid
were monitored. The limits of detection for the five endogenous anabolic steroids were in the
0.1-0.2 pg/mg range. Within-day and between-day precisions were less than 20 percent. This
method was applied to the analysis of testosterone, epitestosterone, androsterone,
etiocholanolone, and dehydroepiandrosterone in human hair. Full-length hair samples were
taken at the skin surface from the vertex of 39 males, 30 females and 11 children from
China. None of the subjects were professional athletes. Testosterone and
dehydroepiandrosterone were detected in all the hair segments. The physiological
concentrations of testosterone were in the range 0.8-24.2 pg/mg, 0.1-16.8 pg/mg and 0.2-
11.5 pg/mg in males, females and children, respectively, however, the mean values of
dehydroepiandrosterone were much higher than the concentrations of testosterone. These
data are suitable reference values and are the basis for the interpretation of results from
investigations into the abuse of endogenous anabolic steroids [09074].
A simple and sensitive gas chromatography/tandem mass spectrometry (GC/MS/MS)

method is described for the detection of anabolic steroids, usually found in keratin matrix at
very low concentrations. Hair samples from seven athletes who spontaneously reported their
abuse of anabolic steroids, and in a single case cocaine, were analyzed for
methyltestosterone, nandrolone, boldenone, fluoxymesterolone, cocaine and its metabolite
benzoylecgonine. Anabolic steroids were determinate by digestion of hair samples in 1 m
NaOH for 15 min at 95 degrees C. After cooling, samples were purificated by solid-phase
and liquid-liquid extraction, then anabolic steroids were converted to their trimethylsilyl
derivative and finally analyzed by GC/MS/MS. For detection of cocaine and
benzoylecgonine, hair samples were extracted with methanol in an ultrasonic bath for 2 h at
56 degrees C then overnight in a thermostatic bath at the same temperature. After the
incubation, methanol was evaporated to dryness, and benzoylecgonine was converted to its
trimethylsilyl derivative prior of GC/MS/MS analysis. Results obtained are in agreement with
the athletes' reports, confirming that hair is a valid biological matrix to establish long-term
intake of drugs [07082].
New highly sensitive, specific, reliable, reproducible and robust LC-MS/MS methods were
developed to detect the anabolic steroids, nandrolone and stanozolol, in human hair for the
first time. Hair samples from 180 participants (108 males, 72 females, 62 % athletes) were
screened using ELISA which revealed 16 athletes as positive for stanozolol and 3 for
nandrolone. Positive samples were confirmed on LC-MS/MS in selective reaction monitoring
(SRM) mode. The assays for stanozolol and nandrolone showed good linearity in the range
1-400 pg/mg and 5-400 pg/mg, respectively. The methods were validated for LLOD, interday
precision, intraday precision, specificity, extraction recovery and accuracy. The assays were
capable of detecting 0.5pg stanozolol and 3.0 pg nandrolone per mg of hair, when
approximately 20 mg of hair were processed. Analysis using LC-MS/MS confirmed 11
athletes' positive for stanozolol (5.0 pg/mg to 86.3 pg/mg) and 1 for nandrolone (14.0 pg/mg)
thus avoiding false results from ELISA screening. The results obtained demonstrate the
application of these hair analysis methods to detect both steroids at low concentrations,
1016
hence reducing the amount of hair required significantly. The new methods complement
urinalysis or blood testing and facilitate improved doping testing regimes. Hair analysis
benefits from non-invasiveness, negligible risk of infection and facile sample storage and
collection, whilst reducing risks of tampering and cross-contamination. Owing to the wide
detection window, this approach may also offer an alternative approach for out-of-
competition testing [10073].
New highly sensitive, specific, reliable, reproducible and robust LC-MS/MS methods were
developed to detect the anabolic steroids, nandrolone and stanozolol, in human hair for the
first time. Hair samples from 180 participants (108 males, 72 females, 62 % athletes) were
screened using ELISA which revealed 16 athletes as positive for stanozolol and 3 for
nandrolone. Positive samples were confirmed on LC-MS/MS in selective reaction monitoring
(SRM) mode. The assays for stanozolol and nandrolone showed good linearity in the range
1-400pg/mg and 5-400pg/mg, respectively. The methods were validated for LLOD, interday
precision, intraday precision, specificity, extraction recovery and accuracy. The assays were
capable of detecting 0.5pg stanozolol and 3.0 pg nandrolone per mg of hair, when
approximately 20mg of hair were processed. Analysis using LC-MS/MS confirmed 11
athletes' positive for stanozolol (5.0 pg/mg to 86.3pg/mg) and 1 for nandrolone (14.0 pg/mg)
thus avoiding false results from ELISA screening. The results obtained demonstrate the
application of these hair analysis methods to detect both steroids at low concentrations,
hence reducing the amount of hair required significantly. The new methods complement
urinalysis or blood testing and facilitate improved doping testing regimes. Hair analysis
benefits from non-invasiveness, negligible risk of infection and facile sample storage and
collection, whilst reducing risks of tampering and cross-contamination. Owing to the wide
detection window, this approach may also offer an alternative approach for out-of-
competition testing [10453].
A method for the screening of various anabolic steroids and their esters in human hair, based
on liquid-chromatography-high resolution mass spectrometry using an Exactive benchtop
Orbitrap mass spectrometer, has been set up and validated. This method involved
methanolic incubation of 30 mg of hair and analysis of the relevant extract in HPLC using a
C18 column. The mass detector, with nominal resolving power of 100,000, operated in full
scan mode in APCI under positive ionization mode. Analytes were identified by exact mass,
correspondence of isotopic cluster and retention times. The limits of detection obtained
varied from 10 to 50 pg/mg, and limits of quantitation were 0.5 ng/mg for all compounds. The
method was linear for all analytes in the ranges from the LOQ to 6 ng/mg, giving correlation
coefficients >0.99 for all analytes. Also accuracy (intended as %E) and repeatability (%CV)
were always lower than 15 percent. Specificity was assessed by analysing ten blank samples
and fifteen samples from polidrug abusers. This method was applied to a real-life case,
resulting in the identification of testosterone undecanoate in the hair of a suspect. The
analyte identity was confirmed by the analysis of its in-source fragmentation and comparison
to a certified standard. Thanks to the scan acquisition, this method also enables
retrospective re-analysis of the acquired datafile in case a further analyte needs to be
screened [13179].
Faecal analyses
Faeces, which could be a potential alternative medium for doping control, have been used for
the detection of 1,4-androstadiene-3,17-dione administration to horses. Semi-quantitative
analyses of 1,4-androstadiene-3,17-dione, testosterone, 17alpha- and 17beta-boldenone
have been conducted in pre- and post-administration faeces, and in controls (untreated
stallions, geldings and mares). Sample preparation comprised diethyl ether extraction, lipid
removal, HPLC purification and derivatisation. 1,4-Androstadiene-3,17-dione, testosterone,
1017
17alpha- and 17beta-boldenone were analysed by GC-EI/MS/MS. Quantitative limits of
detection were 0.1 ng/g for 1,4-androstadiene-3,17-dione, and 0.025 ng/g for testosterone,
17alpha- and 17beta-testosterone. In post-administration samples from geldings and mares,
peak levels of 1,4-androstadiene-3,17-dione, 17alpha-, 17beta-boldenone and testosterone
were attained 24 h after administration. In untreated geldings and mares (in di- or anoestrus),
17alpha- and 17beta-boldenone and testosterone were not detected. Faeces from females in
oestrus had detectable levels of boldenone isomers and testosterone. 1,4-Androstadiene-
3,17-dione was undetectable in faeces collected from untreated horses, but the presence of
this androgen was recently reported in faeces from untreated swine and it would therefore be
advisable to check for its possible presence in a larger number of individual faecal samples
[08191].
In food
Anabolic steroids are banned from use in food-producing animals in the European Union
(Council Directive 96/22/EC). To control the zero-tolerance concept, an LC-MS/MS method
for the screening and confirmation of most of the relevant natural and synthetic estrogenic
and androgenic steroids in bovine and porcine blood plasma was developed and validated.
The method permits confirmation and quantification of all analytes above a concentration of
0.65 microg/L. The validation was carried out according to Commission Decision
2002/657/EC, Chapter 3.1.3 "Alternative Validation", by applying a matrix-comprehensive in-
house validation concept. Decision limit CCalpha, detection capability CCbeta, recovery,
repeatability, within-laboratory reproducibility and the uncertainty of measurement were
calculated. Furthermore, a factorial effect analysis was carried out to identify factors that
have a significant influence on the method. Factors considered to be relevant for the method
in routine analysis (e.g. operator, storage duration of the extracts before measurement and
different cartridge lots) were systematically varied on two levels [13182].
In egg
A cheap, reliable and practical high-performance liquid chromatography-tandem mass
spectrometric method was developed for the simultaneous determination of seven anabolic
steroids in eggs, including trenbolone, boldenone, nandrolone, stanozolol, methandienone,
testosterone and methyl testosterone. The analytes were extracted from the egg samples
using methanol. The extracts were subjected to the removal of fat by freezing-lipid filtration
and then further purified by liquid-liquid extraction using tert-butyl methyl ether. The analytes
were separated on a Luna C18 column by a gradient elution program with 0.1% formic acid
and acetonitrile. This method was validated over 1.00-100 ng/g for all steroids of interest.
The correlation coefficients (r) for each calibration curve are higher than 0.99 within the
experimental concentration range. The decision limits of the steroids in eggs ranged from
0.20 to 0.44 ng/g, and the detection capabilities were below 1.03 ng/g. The average
recoveries were between 66.3 and 82.8% in eggs at three spiked levels of 1.00, 1.50 and
2.00 ng/g for each analyte. The between-day and within-day relative standard deviations
were in the range of 2.4-11 percent. High matrix suppression effects were observed for all
compounds of interest [13183].
Musk extracts
The relevance of IRMS analyses in sports drug testing was highlighted in a recent case
report concerning the administration of musk pod extracts to female elite athletes. Numerous
steroidal components relevant to doping controls and steroid profiling were influenced by the
preparation commonly used in traditional Chinese medicine (TCM) regimens and triggered
GC/C/IRMS analyses that confirmed the non-human origin of the urinary steroid metabolites.
1018
In a follow-up study, the diversity of musk preparations concerning steroid content and
respective CIR was demonstrated. Four batches of musk grains were purchased including
two specimens from wild musk deer and two from domesticized musk deer, outlining
substantial differences in both amounts of steroids and isotopic signatures. In administration
studies with two preparations (100 mg of musk grains) however no significant change in
urinary steroid profiles and CIR were found [13009].
Musk is widely used as a traditional drug in Asia for the treatment of stroke, tumour, and
cardiopathy with an oral dosage of 0.03-0.1 g per day. Because of the potential anabolic
effect, musk preparations have been included in the list of medical products containing
prohibited substances employed for doping. The application of musk pod formulation was
regarded as the reason of some adverse analytical findings in the 2011 FIFA Women's World
Cup. In order to investigate the influence of musk administration on the doping test, we
executed a chemical analysis and excretion study. The gas chromatography/mass
spectrometry (GC-MS) analysis demonstrated the diversity of steroid concentrations in musk
samples. Furthermore, the delta-13C-values of steroids from wild deer musk showed more
depleted than those of domestic deer musk by gas chromatography/combustion/isotope ratio
mass spectrometry (GC/C/IRMS) analysis. Because the steroids from some musk had delta-
13
C-values in the range of naturally produced steroids in human body, the possible abuse of
this kind of musk is very hard to be detected by isotope ratio mass spectrometry (IRMS) in
doping control. Musk grains from wild and domestic deer were administrated for the excretion
study respectively. Spot urine samples were collected from two male volunteers before and
after 100 mg musk grains administration. The profiles and carbon isotope ratios of urinary
steroids were determined by GC-MS and GC/C/IRMS. The ingestion of either wild or
domestic deer musk did not lead to the adverse analytical finding of doping control in the
single dosage of 100 mg [13184].
Musk is the dried secretion from the preputial follicles of the male musk deer, which are
located in a small sac (resulting from an infolding of the skin) in close proximity to the
preputial orifice. This sac (or pod), which contains the brownish musk, comprises a small
canal debouching close to the preputial orifice that allows the controlled release of the
unctuous product by the animal. Upon removal of moisture, the material converts into small,
dark, reddish-brown musk grains. In comprehensive studies, the lipid constituents of musk
were elucidated and numerous steroidal components were characterized. The administration
of musk extract, that is, ingredients obtained by extraction of the liquid secreted from the
preputial gland or resulting grains of the male musk deer (e.g. Moschus moschiferus), has
been recommended in traditional chinese medicine (TCM) applications and was listed in the
Japanese pharmacopoeia for various indications requiring cardiovascular stimulation, anti-
inflammatory medication or androgenic hormone therapy. Numerous steroidal components
including cholesterol, 5alpha-androstane-3,17-dione, 5beta-androstane-3,17-dione, andro-
sterone, etiocholanolone, epiandrosterone, 3beta-hydroxy-androst-5-en-17-one, androst-4-
ene-3,17-dione and the corresponding urea adduct 3alpha-ureido-androst-4-en-17-one were
characterised as natural ingredients of musk over several decades, implicating an issue
concerning doping controls if used for the treatment of elite athletes. In the present study, the
impact of musk extract administration on sports drug testing results of five females
competing in an international sporting event is reported. In the course of routine doping
controls, adverse analytical findings concerning the athletes' steroid profile, corroborated by
isotope-ratio mass spectrometry (IRMS) data, were obtained. The athletes' medical advisors
admitted the prescription of TCM-based musk pod preparations and provided musk pod
samples for comparison purposes to clarify the antidoping rule violation. Steroid profiles,
IRMS results, literature data and a musk sample obtained from a living musk deer of a local
zoo conclusively demonstrated the use of musk pod extracts in all cases which, however,
1019
represented a doping offence as prohibited anabolic-androgenic steroids were administered
[13185].
A user-friendly library
One work presents a novel database search engine - MLibrary - designed to assist the user
in the detection and identification of androgenic anabolic steroids (AAS) and its metabolites
by matrix assisted laser desorption/ionization (MALDI) and mass spectrometry-based
strategies. The detection of the AAS in the samples was accomplished by searching the
mass spectrometric (MS) spectra against the library developed to identify possible positives
and by comparison of the tandem mass spectrometric (MS/MS) spectra produced after
fragmentation of the possible positives with a complete set of spectra that have previously
been assigned to the software. The urinary screening for anabolic agents plays a major role
in anti-doping laboratories as they represent the most abused drug class in sports. With the
help of the MLibrary software application, the use of MALDI techniques for doping control is
simplified and the time for evaluation and interpretation of the results is reduced. To do so,
the search engine takes as input several MALDI-TOF-MS and MALDI-TOF-MS/MS spectra.
It aids the researcher in an automatic mode by identifying possible positives in a single MS
analysis and then confirming their presence in tandem MS analysis by comparing the
experimental tandem mass spectrometric data with the database. Furthermore, the search
engine can, potentially, be further expanded to other compounds in addition to AASs. The
applicability of the MLibrary tool is shown through the analysis of spiked urine samples
[13180].
Reference values from South America
The urinary steroid profile has been used in clinical endocrinology for the early detection of
enzyme deficiencies. In the field of doping, its evaluation in urine samples is used to
diagnose the abuse of substances prohibited in sport. This profile is influenced by sex, age,
exercise, diet, and ethnicity, among others; laboratories own reference ranges might
compensate for ethnic differences among population and inter-laboratory biases. One paper
shows the reference ranges obtained in the Antidoping Laboratory of Havana for the
following steroid profile parameters: ten androgens (testosterone, epitestosterone,
androsterone, etiocholanolone, 5alpha-androstan-3alpha,17beta-diol, 5beta-androstan-
3alpha,17beta-diol, dehydroepiandrosterone, epiandrosterone, 11beta-hydroxyandrosterone
and 11beta-hydroxyetiocholanolone), three estrogens (estradiol, estriol and estrone), two
pregnanes (pregnanediol and pregnanetriol) and two corticosteroids (cortisol and
tetrahydrocortisol). The urine samples (male: n=2454 and female: n=1181) and data
obtained are representative of population from Latin-American countries like Cuba,
Venezuela, Mexico, Dominican Republic, Guatemala and Chile. Urine samples were
prepared by solid-phase extraction followed by enzymatic hydrolysis and liquid-liquid
extraction with an organic solvent in basic conditions. Trimethylsilyl derivatives were
analyzed by gas chromatography coupled to mass spectrometry. Reference ranges were
established for each sex, allowing the determination of abnormal profiles as a first diagnostic
tool for the detection of the abuse of androgenic anabolic steroids. The comparison with the
Caucasian population confirms that the urinary steroid profile is influenced by ethnicity
[13181].
Experimental
Contextual interaction
Seasonal changes in steroid hormones are known to have a major impact on social behavior,
1020
but often are quite sensitive to environmental context. In the bi-directionally sex changing
fish, Lythrypnus dalli, stable haremic groups exhibit baseline levels of interaction. Status
instability follows immediately after male removal, causing transiently elevated agonistic
interactions and increase in brain and systemic levels of a potent fish androgen, 11-
ketotestosterone (KT). Coupling KT implants with a socially inhibitory environment for
protogynous sex change induces rapid transition to male morphology, but no significant
change in social behavior and status, which could result from systemically administered
steroids not effectively penetrating into brain or other tissues. Here, it was first determined
the degree to which exogenously administered steroids affect the steroid load within tissues.
Second, it was examined whether coupling a social environment permissive to sex change
would influence KT effects on agonistic behavior. It was implanted cholesterol (Chol, control)
or KT in the dominant individual (alpha) undergoing sex change (on d0) and determined the
effects on behavior and the degree to which administered steroids altered the steroid load
within tissues. During the period of social instability, there were rapid (within 2h), but
transient effects of KT on agonistic behavior in alphas, and secondary effects on betas. On
d3 and d5, all KT, but no Chol, treated females had male typical genital papillae. Despite
elevated brain and systemic KT 5days after implant, overall rates of aggressive behavior
remained unaffected. These data highlight the importance of social context in mediating
complex hormone-behavior relationships [13186].
Prepuberal induction
Few data are available on adolescent users because most behavioral studies on anabolic-
androgenic steroids (AAS) abuse have been performed in adults. Studies evaluating the
impact of long-term effects of AAS abuse on the prepubertal phase are even more
uncommon. Accordingly, this study was developed to test the hypothesis that changes
induced by the use of AAS during the adolescent phase may be noted in the adult phase
even when the AAS treatment cycle is discontinued. Therefore, not only behavioral changes
but also possible autonomic and electrolyte disorders were evaluated. For this purpose, we
used male prepubertal, 26-day-old (P26) Wistar rats that were treated with vehicle (control,
n=10) or testosterone propionate (TP; 5 mg/kg intramuscular (IM) injection, AAS, n=10) five
times per week for 5 weeks, totaling 25 applications during the treatment. Aggression tests
were performed at the end of the cycle (P54-56), whereas open-field tests (OFTs), elevated
plus maze (EPM) behavioral tests and measurements of heart rate variability (HRV), fluid
intake and pathology were conducted in the adult phase (P87-92). The AAS group showed
greater aggressiveness in the pubertal phase and higher levels of horizontal and vertical
exploration and anxiety-related behavior in the adult phase than the control group. HRV tests
showed an increase in sympathetic autonomic modulation, and hydroelectrolytic assessment
showed lower basal intake levels of hypertonic saline than the control group, without
statistically significant changes in the basal intake of water. These data together suggest that
the use of AAS during the prepubertal phase induces behavioral, autonomic and
hydroelectrolytic changes that manifest in the adult phase even when treatment is
discontinued in late adolescence in rats [13187].
Effect on myosin heavy chain expression in hindlimb muscles of male rats

It was examined the effect of male sex hormones on the myosin heavy chain (MHC)
expression of the soleus and extensor digitorum longus (EDL) muscles. Young male
adult Wistar rats were treated over a 25-day period with either oil (CON, n=8),
nandrolone (nortestosterone decanoate, NAN, n=8), nandrolone combined with
endurance exercise (treadmill running, NAN+EXE, n=8), or were castrated (CAS,
n=8). The MHC composition of the soleus and EDL muscles was measured by
electrophoresis. Castration and treatment with nandrolone had no effect on the
relative levels of MHC in the soleus and EDL. In contrast, in NAN+EXE rats, the
1021
relative level of MHC-1 increased and MHC-2a decreased only in the soleus. In
conclusion, it appears that endogenous anabolic/androgenic steroids are not
essential for the maintenance of the MHC expression of fast- and slow-twitch
muscles in the young adult male rat. In addition, nandrolone combined with
endurance exercise induced a shift from a fast to a slower MHC phenotype of the
slow-twitch muscle [00048].
Morphological effects of an anabolic steroid on muscle fibres of the diaphragm in mice

Until recently, rats have been used for studies on the effects of anabolic steroids (AS) for
improving physical performance. However, no consistent results on the changes in muscle
fibre volume have ever been obtained. In this study we investigated the morphological effects
of nandrolone phenylpropionate, an AS, on the muscle fibres of the diaphragm in adult male
mice. AS (5 mg/kg/week) was injected intramuscularly in the experimental group (AS-
administration group), and peanut oil (0.08 mL/week) was injected in the control group. The
cross-sectional areas of Type I (red) and Type II (white and intermediate) muscle fibres after
4 weeks of AS administration were 135 and 139 percent larger than those in the control
group, respectively. The mean cross-sectional areas of mitochondria in the subsarcolemmal
(SS) region and interfibrillar (IF) region in Type I fibres and the SS region of the Type II
muscle fibres in the AS-administration group were 139, 135 and 124 percent larger than
those in the control group, respectively. No significant differences in the cross-sectional area
of mitochondria were noted between the group of mice administered with AS for 8 weeks and
the control group, showing that the effect of the AS administration attained a peak at 4
weeks, but that the effect of the 8 weeks of AS administration declined. It was concluded
from these results that the response of Type I muscle fibres to AS is stronger than that of
Type II muscle fibres, as is the response of SS mitochondria compared to IF mitochondria.
We also suggested that the two types of mitochondria may have different roles and that
administration of AS may cause a different response in the two types of mitochondria
[00049].
Non-linear effects in the retention of an avoidance task induced by anabolic steroids

The effects of pregnenolone sulfate and ethylestrenol on retention of a passive avoidance
task have been re-examined with special emphasis placed on the distributions of latencies
found in the passive avoidance task using rats. This study used two retention tests, one 24 h
after training the other at 48 h after training. In the first experiment in that study a range of
doses of two anabolic steroids, pregnenolone sulfate and ethylestrenol, were given s.c. just
after the footshock training trial. In experiment 2 a similar range of doses of both steroids was
given to the rats 1 h before the first retention test. Placing emphasis on the distributions
rather than measures of central tendencies revealed that, in contrast to the vehicle treated
animals, the anabolic steroid treated animals exhibited bimodal distributions of response
latencies. These differences between control and hormone treated animals were observed in
both experiments. The new information was interpreted in terms of non-linear dynamics
including some aspects of Chaos theory [00050].
Separation of natural and synthetic anabolic steroids

An HPLC separation of a complex mixture containing 14 androgenic anabolic steroids
(natural and synthetic) for screening purposes has been carried out. The applied optimization
method involved the use of binary, ternary and quaternary mobile phases containing
acetonitrile, methanol or tetrahydrofuran as organic modifiers. The effect of different
reversed-phase packings and temperature on the separation using acetonitrile as organic
modifier was studied. The optimum separation was achieved by using a water-acetonitrile
(55:45, v:v) mobile phase and a Hypersil ODS (250 mm x 4.6 mm) 5 microm column (30
degrees C) in about 38 min, allowing the separation of 14 out of 14 compounds tested (when
1022
danazol is excluded, 13 out of 14 were separated in 23 min). Calibration graphs were
obtained using bolasterone, methyltestosterone and canrenone as internal standards.
Detection limits were in the range 0.012-0.11 microg/mL. The optimized separation was
applied for monitoring the norethindrone acetate hydrolysis from tablets and to the analysis,
after liquid-liquid extraction, of urine samples spiked with steroids [00051].
Electrospray and atmospheric pressure chemical ionization tandem mass spectrometry

Mass spectrometric and tandem mass spectrometric behavior of eight anabolic steroid
glucuronides were examined using electrospray (ESI) and atmospheric pressure chemical
ionization (APCI) in negative and positive ion mode. The objective was to elucidate the most
suitable ionization method to produce intense structure specific product ions and to examine
the possibilities of distinguishing between isomeric steroid glucuronides. The analytes were
glucuronide conjugates of testosterone (TG), epitestosterone (ETG), nandrolone (NG),
androsterone (AG), 5alpha-estran-3alpha-ol-17-one (5alpha-NG), 5beta-estran-3alpha-ol-17-
one (5beta-NG), 17alpha-methyl-5alpha-androstane-3alpha,17beta-diol (5alpha-MTG), and
17alpha-methyl-5beta-androstane-3alpha,17beta-diol (5beta-MTG), the last four being new
compounds synthesized with enzyme-assisted method in our laboratory. High proton affinity
of the 4-ene-3-one system in the steroid structure favored the formation of protonated
molecule [M + H]+ in positive ion mode mass spectrometry (MS), whereas the steroid
glucuronides with lower proton affinities were detected mainly as ammonium adducts [M +
NH4]+. The only ion produced in negative ion mode mass spectrometry was a very intense
and stable deprotonated molecule [M - H]- . Positive ion ESI and APCI MS/MS spectra
showed abundant and structure specific product ions [M + H - Glu]+, [M + H - Glu - H2O]+,
and [M + H - Glu - 2H2O]+ of protonated molecules and corresponding ions of the
ammonium adduct ions. The ratio of the relative abundances of these ions and the stability of
the precursor ion provided distinction of 5alpha-NG and 5beta-NG isomers and TG and ETG
isomers. Corresponding diagnostic ions were only minor peaks in negative ion MS/MS
spectra. It was shown that positive ion ESI MS/MS is the most promising method for further
development of LC-MS methods for anabolic steroid glucuronides [00052].
Clinical guidelines for detection of exposure of doping with anabolic steroids
Evaluation of potential, suspected, or known AAS users should include a specific history,
physical examination, and laboratory testing. Young men who participate in weight training,
bodybuilding, or sports that require strength and power are at highest risk for AAS use. A
high index of suspicion is warranted during the clinical evaluation of these individuals.
Fearing the possible legal consequences or a competitive ban, individuals may not admit to
using these drugs [04018].
History
The drug history should be taken in a systematic manner. Begin by inquiring about the use of
nutritional supplements and over-the-counter ergogenic aids. The use of ephedra, creatine,
and pro-hormones like androstenedione commonly precedes or accompanies AAS use.
Then, ask if the patient knows other people who use AAS because athletes at high risk of
using AAS are more likely to know other users than low-risk nonusers are. Next, the clinician
should ask whether the patient has ever tried AAS. If there is a positive history of current or
previous AAS use, a detailed drug history should ensue. It is important to establish the
athlete’s self-administered drug regimen, documenting the quantity of AAS, weekly dosages,
relative durations of the AAS cycles and off-cycles, and approximate date when the athlete
first began using AAS. It is important to distinguish the hypogonadal or aging male receiving
1023
low-dose pharmacologic testosterone replacement from the athlete abusing higher
suprapharmacologic doses of AAS. The latter individual is at greater risk of AAS-related
complications. Finally, because the majority of AAS users have a palate for poly-pharmacy,
ask about the use of other performance-enhancing drugs, such as growth hormone. The
clinician should also undertake a systematic inquiry regarding the common subjective side
effects of AAS use, such as acne, gynecomastia, and so forth 04[018].
Physical examination
When a physician suspects chemical enhancement in an athlete with pronounced skeletal

muscle hypertrophy, there are several physical signs that point a finger toward AAS use. A
simple, strategic physical evaluation is all that is required to detect an anabolic steroid user.
In the well-muscled athlete, the physician should look for acne, gynaecomastia, and
cutaneous striae in the deltopectoral area. Four out of every 5 steroid users exhibit at least
one of these physical side effects of AAS. If, in addition, the physician discovers needle-stick
marks (in the buttocks, thighs, or deltoid) and testicular atrophy, the diagnosis of AAS use is
a slam-dunk. The female AAS user may exhibit muscular hypertrophy, hirsutism, male-
pattern baldness, voice deepening, breast tissue atrophy, or clitoral hypertrophy [04018].
Management
AAS users may present to an orthopaedic sports physician with symptoms relating directly to
their drug abuse or with unrelated sporting injuries or trauma. Common AAS-related
problems manifest as dermatologic (acne, gynecomastia, injection related), endocrine
(testicular atrophy, decreased libido, infertility), or psychiatric symptoms (mania, withdrawal,
depression). During preoperative evaluation, a suspected or positive history of AAS use has
special relevance. An apparently healthy AAS user may be at increased risk of complications
during the perioperative and postoperative period, and a high index of suspicion is key. It is
advisable for athletes with a positive history of AAS use to undergo medical clearance before
surgery. These individuals may exhibit cardiac abnormalities such as hypertension, left
ventricular hypertrophy, impaired diastolic filling, and rhythm irregularities. A raised
hematocrit and potential for hypercoagulopathy places AAS users at risk of adverse
circulatory events. Altered lipoprotein profiles and liver enzyme changes may also be
relevant. It is also important to be aware of the high rate of poly-pharmacy among AAS
users. Nine out of tem AAS users are likely to be taking other drugs in addition to AAS,
including stimulants (ephedra, amphetamine, cocaine), anabolic agents (GH, insulin, IGF),
recreational drugs (methylenedioxymethamphetamine, opiates), and other miscellaneous
drugs (diuretics, thyroxine). Patients should be advised to stop all performance-enhancing
drugs, herbal supplements, and nonprescribed medications prior to elective surgery. To
identify potential perioperative risks, the preoperative workup should include a detailed drug
history; a complete physical examination; blood work, including CBC and liver function; and
an EKG. Abnormal findings may require further investigation and rectification prior to elective
surgery under general anesthesia. Discussing the concerns with the patient will provide an
incentive for the individual to discontinue their drug use. Counseling the patient regarding the
risks of AAS use is a valuable health education tool during the physician-patient consultation.
The physician should make the athlete aware of the high risk of short-term subjective side
effects that affect four out of five AAS users. Common subjective symptoms such as acne,
gynecomastia, decreased libido, and alopecia may serve as a more potent deterrent to drug
use than the less common, subclinical long-term risks. AAS use by adolescents and females
should be strongly discouraged because of the high risk of irreversible complications even
with short-term use. Several other suggestions may be of benefit to adult male AAS users.
For instance, reducing the dose and duration of AAS use can help minimize the risk of
1024
complications. Weekly doses of 600 mg of testosterone or its equivalent for cycles lasting
less than 12 weeks appear to cause few side effects during scientific studies. Furthermore,
esters of testosterone that possess powerful androgenic properties are more likely to induce
a potent insult to the hypothalamic-pituitary axis than other less androgenic formulations.
Both the physician and patient should also recognize the risk of withdrawal symptoms on
cessation of AAS use and how this leads to a physical dependence, habituation, and long-
term AAS usage. A useful axiom is, the bigger the dose, the bigger the muscle, the bigger
the problem [04018].
Laboratory abnormalities in anabolic-androgenic steroid users
A laboratory investigation is useful in patients with open or suspected anabolic steroids. I can
then be found [04018]:
Complete blood count Increased hemoglobin and hematocrit

Cholesterol levels Increased HDL-C
Liver function tests Increased ALT, AST
Hormone levels Decreased luteinizing hormone, folliclestimulating
hormone
Increased testosterone (using AAS)
Decreased testosterone (during withdrawal)
Electrocardiogram Left ventricular hypertrophy, QT dispersion
Echocardiogram Impaired diastolic function
Urine analysis Positive for AAS and other drugs of abuse
Markers for anabolic steroids
The screening of testosterone misuse in the doping control field is normally performed by the
measurement of the ratio between the concentrations of testosterone and epitestosterone
excreted as glucuronides (T/E). Despite the satisfactory results obtained with this approach,
the measurement of T/E presents some limitations like the long-term detection of oral
testosterone administration. Recently, several testosterone metabolites released after basic
treatment of the urine have been reported (androsta-1,4-dien-3,17-dione, androsta-4,6-dien-
3,17-dione, 17beta-hydroxy-androsta-4,6-dien-3-one and 15-androsten-3,17-dione). In one
work, the usefulness of these metabolites for the detection of oral testosterone misuse were
evaluated and compared with the conventional T/E measurement. For this purpose, 173
urine samples collected from healthy volunteers were analysed in order to obtain reference
concentrations for the four metabolites released after alkaline treatment. On the other hand,
urine samples collected from five volunteers before and after testosterone undecanoate
administration were also analysed. Concentrations of androsta-4,6-dien-3,17-dione and
17beta-hydroxy-androsta-4,6-dien-3-one showed a similar behaviour as the T/E, allowing the
detection of the misuse for several hours after administration. More promising results were
obtained by quantifying androsta-1,4-dien-3,17-dione and 15-androsten-3,17-dione. The time
in which the concentrations of these analytes could be differentiated from the basal level was
between 3 and 6 times longer than the obtained with T/E, as a result, an improvement in the
detection of testosterone abuse can be achieved. Moreover, several ratios between these
compounds were evaluated. Some of them improved the detection of testosterone misuse
when comparing with T/E. The best results were obtained with those ratios involving
androsta-1,4-dien-3,17-dione [11576].
The aim of one study was to explore effectors of the pituitary-testicular axis suitable as
1025
potential biochemical markers to screen for testosterone doping. It was a pilot study with
male bodybuilding athletes with a self-reported history of testosterone doping (repeated
intramuscular administration of testosterone preparations, last injection 8 weeks or less ago)
compared with an equal sized control group matched for sex, age, and body mass index.
Fifteen healthy young men of white background had inhibin B, testosterone, luteinizing
hormone (LH), follicle-stimulating hormone (FSH) tested. Although the levels of testosterone,
LH, and FSH did not differ between the 2 groups, the serum concentrations of inhibin B in
individuals with a history of testosterone doping were exclusively at or below the lower limit of
the normal range for adult men (100-400 pg/mL). Inhibin B was significantly lower in those
men who used testosterone for weight lifting. It was concluded that low concentration of
serum inhibin B may reflect the application of exogenous testosterone and appears to be a
potential marker associated with anabolic androgenic steroid doping [10095].
AICAR
Influencing the endurance in elite sports is one of the key points in modern sports science.
Recently, a new class of prohibited substances reached in the focus of doping control
laboratories and their misuse was classified as gene doping. The adenosine monophosphate
activated protein kinase activator 5-amino-4-imidazolecarboxyamide ribonucleoside (AICAR)
was found to significantly enhance the endurance even in sedentary mice after treatment.
Due to endogenous production of AICAR in healthy humans, considerable amounts were
present in the circulation and, thus, were excreted into urine. Considering these facts, the
present study was initiated to fix reference values of renally cleared AICAR in elite athletes.
Therefore a quantitative analytical method by means of isotope-dilution liquid
chromatography (analytical column: C6-phenyl) coupled to tandem mass spectrometry, after
a sample preparation consisting of a gentle dilution of native urine, was developed. Doping
control samples of 499 athletes were analysed, and AICAR concentrations in urine were
determined. The mean AICAR value for all samples was 2,186 ng/mL with a standard
deviation of 1,655 ng/mL. Concentrations were found to differ depending on gender, type of
sport and type of sample collection (in competition/out of competition). The method was fully
validated for quantitative purposes considering the parameters linearity, inter- (12 %, 7 %
and 10 %) and intraday precision (14 %, 9 % and 12 %) at low, mid and high concentration,
robustness, accuracy (approx. 100 %), limit of quantification (100 ng/mL), stability and ion
suppression effects, employing an in-house synthesised 13C5-labelled AICAR as internal
standard [10096].
Activity on carboanhydrases
The in vitro effects of the anabolic compounds, zeranol, 17beta-estradiol, diethylstilbestrol

(DES), and trenbolone, on the activity of purified human carbonic anhydrase I and II were
evaluated. In vitro carboanhydrase enzyme activity was determined colorimetrically using the
CO2 hydration method of Maren. IC50 values of the compounds that caused inhibition were
determined by means of activity percentage diagrams. The IC50 concentrations of zeranol, 17
beta-estradiol, DES and trenbolone on humanmcarbonic anhydrase I were 94, 55, 10, 898
microM and for human carboanhydrase II 89, 159, 439 and 101 microM, respectively
[11110].
Purchase over the Internet
A case of hypogonadotropic hypogonadism due to the chronic abuse of anabolic steroids

1026
purchased over the Internet was reported. It was decribed the clinical symptoms, androgen
normalization, levels of serum testosterone, follicle-stimulating hormone, and luteinizing
hormone, and after withdrawal, within 1 month, the patient's serum testosterone was in the
normal range, and he reported a return to normal energy and libido [10454].
Anabolic steroid use and condome use
Previous research has revealed a significant bivariate relationship between anabolic-

androgenic steroid (AAS) use and reduced condom use among adolescent boys. However,
to date, no known studies have explored the psychological mechanisms that may explain this
relationship. Thus, the current study sought to examine two possible mediators in the
association between AAS and condom use: depressive symptoms and substance use. Data
were extracted from a nationally representative sample of US adolescents. Participants were
3,780 U.S. high school boys who responded to self-report items assessing a number of
health behaviors, including symptoms of depression, substance use, AAS use, and use of
condoms during their most recent act of intercourse. Both depression and substance use
were significant mediators in the relationship between AAS and condom use. However, when
these effects were contrasted, the indirect effect of substance use was significantly stronger
in magnitude than the effect of depression. Although AAS use was associated with sexual
risk behaviors among adolescent boys, significant variance in this relationship was
accounted for by elevated levels of depression and substance use, with substance use
demonstrating a particularly salient pathway [13161].
Case reports
Most users of anabolic androgenic steroids (AAS) are male, but the prevalence of such use
appears to be increasing in females. It was presented a sudden unexpected death in a
female fitness athlete with a possible connection to use of doping agents [08192].
It was presented a case of a 19-year-old male athlete with protein C deficiency who
developed proximal deep venous thrombosis and pulmonary embolism while abusing
anabolic-androgenic steroids. Anabolic-androgenic steroids have been reported to have
anticoagulatory and profibrinolytic effects in patients with protein C deficiency. Despite these
antithrombotic effects, the patient developed repeated venous thromboembolism during
treatment with low-molecular-weight heparin. The net effect of anabolic-androgenic steroids
on the haemostatic system may change from antithrombotic to prothrombotic in male
abusers of anabolic steroids with protein C deficiency [08193].
An increase in the use of anabolic and amino acid supplements has been linked to a diverse
array of pathologies. It was reported multi-organ damage resulting from the abuse and
uncontrolled use of anabolic steroid supplements, mainly testosterone. A 24-year-old white
man presented with abdominal pain concomitant with nausea and vomiting. Laboratory
analysis revealed hypercalcemia, elevated liver enzymes and high levels of amylase, lipase
and creatine protein kinase [08194].
1027
Experimental
GABA type A receptors
Anabolic androgenic steroids (AAS) can promote detrimental effects on social behaviors for
which GABA type A (GABAA) receptor-mediated circuits in the forebrain play a critical role.
While all AAS bind to androgen receptors (AR), they may also be aromatized to estrogens
and thus potentially impart effects via estrogen receptors (ER). Chronic exposure of wild-type
male mice to a combination of chemically distinct AAS increased action potential frequency,
selective GABAA receptor subunit mRNAs, and GABAergic synaptic current decay in the
medial preoptic area. Experiments performed with pharmacological agents and in AR-
deficient mutant mice suggest that the AAS-dependent enhancement of GABAergic
transmission in wild-type mice is AR-mediated. In AR-deficient mice, the AAS elicited
dramatically different effects, decreasing AP frequency, spontaneous IPSC amplitude and
frequency and the expression of selective GABAA receptor subunit mRNAs. Surprisingly, in
the absence of AR signaling, the data indicate that the AAS do not act as ER agonists, but
rather suggest a novel in vivo action in which the AAS inhibit aromatase and impair
endogenous ER signaling. These results show that the AAS have the capacity to alter
neuronal function in the forebrain via multiple steroid signaling mechanisms and suggest that
effects of these steroids in the brain will depend not only on the balance of AR- versus ER-
mediated regulation for different target genes, but also on the ability of these drugs to alter
steroid metabolism and thus the endogenous steroid milieu [09075].
Effect of subcutaneous testosterone on emotionality
The androgenic steroid testosterone is well known for its function in reproduction, sexual
differentiation and sexual behavior. A growing number of human and animal studies suggest
a modulatory role of testosterone in the regulation of emotionality and associated psychiatric
disorders, including depressive-like disorders. However, most of the studies have been
carried out in subjects deficient in androgenic steroid levels. Here, it was tested potential
beneficial effects of subcutaneously applied testosterone on emotionality and depressive-like
behavior in healthy male rats. For this purpose, male Wistar rats (3-4 months) received either
vehicle or testosterone (1.0, 2.0, 4.0mg/kg) subcutaneously and were tested for potential
effects on motor activity and anxiety-like behavior in a novel open field and elevated plus-
maze. The forced swim test was used for assessing potential beneficial effects of
testosterone on depressive-like behavior. The results show, that, while subcutaneous
application of testosterone failed to influence spontaneous motor activity as well as anxiety-
like behavior in the open field, a trend for an increase in the time spent on the open arms in
the elevated plus-maze with the highest dose was found. Furthermore, in the forced swim
test, testosterone application induced a dose-dependent reduction of immobility behaviour,
indicating antidepressant-like action of testosterone in healthy animals [09076].
Effect of testosterone in castrated guinea pigs
Anabolic steroids are widely used to increase skeletal muscle mass and improve physical
performance. Some dietary supplements also include potent steroid precursors or active
steroid analogs such as nandrolone. One previous study reported the anabolic steroid effects
on skeletal muscle mass in a castrated guinea pig model with muscle measured using a
highly quantitative magnetic resonance imaging (MRI) protocol. The aim of one study was to
apply this animal model and in vivo MRI protocol to evaluate the growth effects of four widely
1028
used over-the-counter testosterone and nandrolone precursors: 4-androstene-3 17-dione
(androstenedione), 4-androstene-3beta 17beta-diol (4-androsdiol), 19-nor-4-androstene-
3beta-17beta-diol (bolandiol) and 19-nor-4-androstene-3 17-dione (19-norandrostenedione).
The results showed that providing precursor to castrated male guinea pigs led to plasma
steroid levels sufficient to maintain normal skeletal muscle growth. The anabolic growth
effects of these specific precursors on individual and total muscle volumes, sexual organs,
and total adipose tissue over a 10-week treatment period, in comparison with those in the
respective positive control testosterone and nandrolone groups, were documented
quantitatively by MRI [09077].
Apoptosis and NOS2 (nitric-oxide synthase 2)
Anabolic-androgenic steroids (AAS) are synthetic derivatives of testosterone (T)

predominantly taken as drugs of abuse. Using in vivo treatment of adult male rats we
investigated the effects of testosterone enanthate (TE) a widely abused AAS, on apoptosis of
Leydig cells. Increased T and decreased luteinizing hormone levels in serum and decreased
intra-testicular T values were found in 2 and 10 weeks treated groups. Two weeks of TE-
treatment stimulated the expression of inducible nitric oxide synthase (NOS2) followed by
increased NO production, decreased mitochondrial membrane potential and increased
prevalence of Leydig cell apoptosis. This was prevented by in vivo administration of
androgen receptor blocker. The induced NOS2 level and apoptosis returned to control levels
after 10 weeks of TE-treatment but testes contained fewer Leydig cells. Overall, AAS in
addition to reduced steroidogenesis induce transient increase of Leydig cells apoptotic rate
through mechanism associated with androgen receptor, most likely involving NOS2 induction
[12158].
Androgen-induced cardiac autonomic dysfunction
One study aimed to evaluate the combined effects of exercise and antagonists of the
angiotensin II and aldosterone receptors on cardiac autonomic regulation and ventricular
repolarization in rats chronically treated with nandrolone decanoate (ND), a synthetic
androgen. Thirty male Wistar rats were divided into six groups: sedentary, trained, ND-
treated, trained and ND-treated, trained and treated with both ND and spironolactone, and
trained and treated with both ND and losartan. ND (10 mg/kg weekly) and the antagonists
(20 mg/kg daily) of the angiotensin II AT(1) (losartan) and aldosterone (spironolactone)
receptors were administered for 8 weeks. Exercise training was performed using a treadmill
five times each week for 8 weeks. Following this 8-week training and treatment period,
electrocardiogram recordings were obtained to determine the time and frequency domains of
heart rate variability (HRV) and corrected QT interval (QTc). Nandrolone decanoate
treatment increased the QTc interval and reduced the parasympathetic indexes of HRV
(RMSSD, pNN5 and high-frequency power) in sedentary and trained rats. The ratio between
low- and high-frequency power (LF/HF) was higher in ND-treated groups. Both losartan and
spironolactone treatments prevented the effects of ND on the QTc interval and the HRV
parameters (RMSSD, pNN5, high-frequency power, and the LF/HF ratio). The results show
that chronic treatment with a high dose of ND induces cardiac parasympathetic dysfunction
and disturbances in ventricular repolarization in both sedentary and exercised rats.
Furthermore, inhibiting the renin-angiotensin-aldosterone system using losartan, or
spironolactone, prevented these deleterious effects [12159].
Interaction of testosterone with cocaine
1029
Abuse of cocaine and androgenic-anabolic steroids (AASs) has become a serious public
health problem. Despite reports of an increase in the incidence of simultaneous abuse of
these substances, potential toxic interactions between cocaine and AASs are poorly known.
In one study, it was investigated the effects of either single or combined administration of
testosterone and cocaine for one or 10 consecutive days on autonomic (arterial pressure,
heart rate and tail cutaneous temperature) and neuroendocrine (plasma corticosterone)
responses induced by acute restraint stress in rats. Combined administration of testosterone
and cocaine for 10 days reduced the increase in heart rate and plasma corticosterone level,
as well as the fall in tail skin temperature induced by restraint stress. Furthermore, repeated
administration of cocaine inhibited the increase in arterial pressure observed during restraint,
and this effect was not affected by coadministration of testosterone. Ten-day combined
administration of testosterone and cocaine increased basal values of arterial pressure.
Moreover, chronic administration of testosterone induced rest bradycardia and elevated
basal level of plasma corticosterone. One-day single or combined administration of the drugs
did not affect any parameter investigated. In conclusion, the present study demonstrated that
combined administration of testosterone and cocaine changed the autonomic and
neuroendocrine responses to acute restraint stress. These findings suggest that interaction
between AASs and cocaine may affect the ability to cope with stressful events [12160].
An opioide-like dependence
Anabolic steroids are drugs of abuse. However, the potential for steroid reward and addiction
remains largely unexplored. This study used intracerebroventricular (ICV) testosterone self-
administration and controlled infusions of testosterone or vehicle in hamsters to explore
central mechanisms of androgen overdose. Forty-two hamsters used nose-pokes to self-
administer 1 microg/microl testosterone ICV 4 h/day in an operant chamber. During 1-56
days of androgen self-administration, 10 (24 %) hamsters died. Deaths correlated with peak
daily intake of testosterone. Of the hamsters that self-administered a peak intake of <20
microg/day, there was 100 percent survival (10/10). Survival decreased to 86 percent (19/22)
when daily testosterone intake peaked at 20-60 microg/day. Only 30 percent (three of 10)
survived when daily testosterone intake exceeded 60 microg/day. Deaths are not due to
volume or vehicle because ICV infusions of 80 muL vehicle had no effect. Testosterone
overdose resembles opiate intoxication. When male hamsters received infusions of 40
microg testosterone, locomotion (25 grid-crossings/10 min), respiration (73 breaths/min) and
body temperature (34 degrees C) were significantly reduced, compared with males receiving
vehicle infusions (186 crossings/10 min, 118 breaths/min, 36 degrees C). However, males
developed tolerance to continued daily testosterone infusion. After 15 days, locomotion,
respiration, and body temperature in testosterone-infused males were equivalent to that in
vehicle controls. The depressive effects of testosterone infusion are blocked by the opioid
antagonist, naltrexone. With naltrexone pre-treatment (10 mg/kg s.c.), locomotion,
respiration, and body temperature during testosterone infusion were equivalent to vehicle
controls. Likewise, naltrexone prevents the reinforcing effects of ICV testosterone self-
administration. These results indicate that testosterone at high doses causes central
autonomic depression, which may be a factor in deaths during self-administration. As well,
the depressive effects of large quantities of testosterone may be mediated, at least in part, by
an opioidergic mechanism [05068].
Effect on tissue glycogen
To increase tissue glycogen content many athletes use anabolic androgenic steroids (AAS).
However, the literature concerning the effects of androgens on glycogen metabolism is
conflicting. One study aimed to determine the influence of training and AAS on body weight
(bw), triglycerides, glucose, tissue glycogen and transaminases levels. Male Wistar rats,
1030
randomized into four groups (sedentary vehicle (SV), sedentary AAS (SA), trained vehicle
(TV) and trained AAS (TA)), were treated with nadrolone (5 mg/Kg, 2x/week, i.m.) or vehicle.
Trained rats performed jumps into water (4 sets, 10 repetitions, 30 sec rest) carrying a 50-
70% body wt-load strapped to the chest (5 days/week, 6 weeks). Two days after the last
session, the animals were killed. Trained animals presented lower bodyweight and
triglycerides levels and higher glycogen content in liver and in gastrocnemious than
sedentary ones. In the cardiac muscle, the association between training and AAS increased
glycogen content. In the soleus AAS increased glycogen. Exercise training and AAS had no
effect on blood glucose and transaminases levels. Training and AAS effects on glycogen
supercompensation are tissue-dependent and the effects of association between them were
only observed in the cardiac muscle. These data emphasize the necessity of more studies to
confirm greater effects of AAS than those promoted by physical exercise [05069].
Lack of effect on exploratory-based anxiety
Exposure to supraphysiological doses of androgens may disrupt affective components of

behavior. In this study, behavior of adult C57Bl/6 male mice was studied after exposure to
the anabolic androgenic steroid (AAS) 17alpha-methyltestosterone (17alpha-meT; 7.5
mg/kg) via a subcutaneous osmotic pump for 17 days. Controls received vehicle implants
(0.9 % NaCl + 30 % cyclodextrine). On day 15, experimental animals were challenged with
an ethanol (EtOH) injection (i.p.; 1 g/kg) while controls received saline injections. Five
minutes after the injection, animals were tested in an automated elevated plus maze (EPM)
or in automated activity chambers. In addition, injection-free animals were tested for ethanol
consumption on day 16 after an overnight water deprivation period. Whereas chronic
exposure to 17alpha-meT did not modulate open arm behavior, EtOH-exposed animals
made more entries into the open arms than controls. A significant reduction of risk
assessment behaviors (rearing, flat approach behavior, and stretch attended posture) over
the EPM was noted for EtOH-exposed animals whereas a reduction in stretch attended
postures was observed among 17alpha-meT-exposed animals. Locomotor activity, and light-
dark transitions in activity chambers remained unaltered. Exposure to AAS did not modulate
EtOH consumption. Our data suggest that exposure to a supraphysiological dose of 17alpha-
meT has minimal effects on exploratory-based anxiety [05070].
Submaximal training and anabolic androgenic steroids administration
The purpose of one study was to investigate the single and combined effects of submaximal
training and anabolic androgenic steroids (AAS) treatment on the activity of 3beta
hydroxysteroid dehydrogenase (3betaHSD) in rat Leydig cells (LC). Forty male Wistar rats
were distributed into 4 groups. Half of them exercised on treadmill. After 2 weeks half of the
trained and sedentary rats received weekly either 10 mg/kg. Nandrolone decanoate (ND) or
Placebo (Pl) i.m. for 6 weeks. The day after the last exercises all the groups: 1) sedentary +
Pl (SP); 2) sedentary + ND (SND); 3) trained + Pl (TP) and 4) trained + ND (TND) were
decapitated. On fresh cryostat sections of the testes of each animal enzymehistochemical
reaction for the activity of 3betaHSD was carried out. Our results demonstrate that in
sedentary rats ND treatment decreased the activity of 3betaHSD in the LC in comparison to
SP. Endurance training also decreased the activity of 3betaHSD in TP group compared to
SP. On sections of the testes of group TND a pronounced reduction in the enzyme activities
of 3betaHSD in the LC was found in comparison with the other groups. In conclusion we
suggest that submaximal endurance training and/or administration of AAS downregulate the
steroidogenic enzyme activity of rat Leydig cells [03071].
1031
Effect of anabolic steroids of rat cardiomyocytes and adipocytes
To study the effect of short-term and long-term treatment with retabolil, an androgenic
anabolic steroid, on the activity of the enzymes ATP and LPL in rat cardiomyocytes and
adipocytes male Wistar rats (mean weight 195-200 g.) were given retabolil 50 mg/kg once
subcutaneously, another six were treated with retabolil at the same dose subcutaneously
once a week for 6 weeks and another six were used as controls treating them with
physiological saline in the same way. After six weeks the animals were sacrificed. Fragments
of the left ventricle of the heart and of the subcutaneous tissue from the gluteal region were
resected and enzyme-histochemical reactions for ATP and LPL were performed on fresh
cryostat sections. The cardiomyocytes of the rats treated only once with retabolil showed no
changes in the ATP and LPL activity in comparison with the controls. In the rats given a long-
term treatment with retabolil, the enzyme-histochemical reaction for ATP was better
expressed while that for LPL was weak. The subcutaneous adipose tissue of the long-term
retabolil-treated animals contained some adipocytes that expressed positive LPL and ATP
activity. The data suggest that androgenic anabolic steroids exert an effect on the activity of
the enzymes ATP and LPL in rat cardiomyocytes and adipocytes which depends on the
duration of treatment [03072].
Modulate adolescent steroid-induced aggression in hamsters
In male Syrian hamsters (Mesocricetus auratus), exposure to anabolic/androgenic steroids

(AAS) during adolescent development produces a mature and escalated aggressive
phenotype. Relatedly, in a series of studies, we have shown that adolescent AAS exposure
alters several neurotransmitter systems implicated in the control of aggression within the
latero-anterior subdivision of the hypothalamus (LAH); namely the vasopressin (AVP),
serotonin (5HT), and gamma-aminobutyric acid (GABA) neural systems. Similarly, dopamine
(DA) neural signaling through several receptors has also been linked to aggression. In
particular the DA D2-like receptor has been implicated in aggressive responding in mice and
hamsters. Accordingly, tyrosine hydroxylase (TH) containing neurons, afferent fibers, and DA
D2 receptors are present within regions associated with aggressive behavior that encompass
the hypothalamic neural circuit that controls offensive aggression in hamsters (i.e. the bed
nucleus of the stria terminalis, lateral septum, medial amygdala, and ventrolateral
hypothalamus) suggesting that DA activity modulates aggression through DA D2 receptors in
these brain regions. Interestingly, hypothalamic DA neurons are responsive to androgens in
a fashion consistent with the generation of the aggressive phenotype, so alterations in this
neural system may play a role in adolescent AAS-induced offensive aggression. Recently, it
was shown that direct application of DA D2 antagonists to the LAH dose dependently
suppresses adolescent AAS-induced offensive aggression in hamsters. Relatedly, it was also
found that adolescent AAS administration increases TH afferent development and DA D2
receptor localization/expression in the LAH of male Syrian hamsters. Importantly, these
alterations were confined to the LAH brain region as similar adolescent AAS-induced
changes were not observed in any other nuclei comprising the hypothalamic neural circuit
that controls aggression in hamsters. In particular, adolescent AAS-treated hamsters have
more TH neurons and afferent fibers within the nucleus circularis (NC) and medial supraoptic
nucleus (mSON) subregions of the. Taken together, these data suggest that adolescent AAS
exposure increases DA development and DA D2 receptor density in the LAH, and this
increase in LAH DA activity facilitates aggression through activation of LAH DA D2 receptor
signaling. In pubertal male Syrian hamsters, exposure to anabolic/androgenic steroids (AAS)
during adolescence facilitates a high level of offensive aggression modulated by the
enhanced development and activity of the vasopressin (AVP) and dopamine (DA) neural
systems within the latero-anterior hypothalamus (LAH), that is, a brain region implicated in
1032
the control of aggression. The present studies provide a detailed report of the pharmacologic
interactions between AVP and DA D2 receptor signaling within the LAH in the control of
adolescent AAS-induced offensive aggression. Male Syrian hamsters were treated with AAS
throughout adolescence and tested for aggression after local infusion of the DA D2 receptor
antagonist eticlopride (ETIC) alone, or in combination with AVP in the LAH in an effort to
determine the influence of DA D2 receptors relative to AVP-receptor mediated aggression
mechanisms. As previously shown, ETIC infusion into the LAH suppressed adolescent AAS-
induced aggressive responding; however, the AAS-induced aggressive phenotype was
rescued by the coinfusion of AVP into the LAH. These behavioral data indicate that
interactions between AVP and DA neural systems within the LAH modulate the control of
aggression following adolescent exposure to AAS and that DA D2 receptor signaling
functions upstream of AVP in the LAH to control this behavioral response [150171].
Steroids impair set-shifting and reversal learning in male rats
Anabolic-androgenic steroid (AAS) abuse is prevalent not only among elite athletes, but is
increasingly common in high school and collegiate sports. AAS are implicated in maladaptive
behaviors such as increased aggression and risk taking, which may result from impaired
cognition. Because they affect dopamine function in prefrontal cortical (PFC)-striatal circuitry,
AAS may disrupt PFC-dependent processes such as behavioral flexibility. This was the focus
of the present study. Adolescent male Long-Evans rats were treated chronically with high-
dose testosterone (7.5 mg/kg in water with 13 % cyclodextrin) or vehicle sc, and tested for
set-shifting and reversal-learning. For set-shifting, rats were trained on a visual cue task
(VCT), then were shifted to a direction cue task (DCT), or vice-versa. For reversal learning,
rats were first trained on VCT and were then required to press the opposite lever. 2-cue set-
shifting introduced a novel paradigm in which rats shifted from a 1-Light Visual Task (1LVT)
to a tone cue task (TCT). Testosterone-treated rats were significantly impaired on the set-
shift from DCT to VCT compared to vehicle-treated controls (trials to criterion: vehicle 240.9
± 29.9, testosterone 388. 3± 59.3). However, on the set-shift from VCT to DCT, testosterone
did not affect performance. During reversal-learning, testosterone significantly increased
trials to criterion. In 2-cue set-shifting, testosterone diminished performance and the
difference showed borderline significance. The results show that testosterone impairs
behavioral flexibility and have implications for understanding cognitive and behavioral
changes in human AAS users [150172].
Androgen deficiency exacerbates high-fat diet-induced metabolic alterations in male

mice
Androgen deficiency is associated with obesity, metabolic syndrome, and type 2 diabetes
mellitus in men, but the mechanisms behind these associations remain unclear. In this study,
we investigated the combined effects of androgen deficiency and high-fat diet (HFD) on body
composition and glucose homeostasis in C57BL/6J male mice. Two models of androgen
deficiency were used: orchidectomy (ORX) and androgen receptor knockout mice. Both
models displayed higher adiposity and serum leptin levels upon HFD, whereas no
differences were seen on a regular diet. Fat accumulation in HFD ORX animals was
accompanied by increased sedentary behavior and occurred in spite of reduced food intake.
HFD ORX mice showed white adipocyte hypertrophy, correlated with decreased
mitochondrial content but not function as well as increased lipogenesis and decreased
lipolysis suggested by the up-regulation of fatty acid synthase and the down-regulation of
hormone-sensitive lipase. Both ORX and androgen receptor knockout exacerbated HFD-
induced glucose intolerance by impairing insulin action in liver and skeletal muscle, as
evidenced by the increased triglyceride and decreased glycogen content in these tissues. In
1033
addition, serum IL-1beta levels were elevated, and pancreatic insulin secretion was impaired
after ORX. Testosterone but not dihydrotestosterone supplementation restored the castration
effects on body composition and glucose homeostasis. It was concluded that sex steroid
deficiency in combination with HFD exacerbates adiposity, insulin resistance, and beta-cell
failure in 2 preclinical male mouse models. The findings stress the importance of a healthy
diet in a clinical context of androgen deficiency and may have implications for the prevention
of metabolic alterations in hypogonadal men [150173].
1034
SIDE EFFECTS OF TESTOSTERONE AND OTHER ANABOLIC
STEROIDS
Overviews
To document adverse effects of anabolic-androgenic steroid (AAS) use in community-based

users attending a medical clinic it was made a prospective recruitment, questionnaire-based
interview, physical examination and investigations, with follow-up, of people who attended,
anonymously, an inner-city hospital clinic established specifically to examine AAS use. Fifty-
eight men, comprising 27 past AAS users, 14 present users and 17 potential users (who
formed the control group) were included. Cyclical use of oral and intramuscular, human and
veterinary AASs was reported. The most commonly reported source of AASs was friends (59
%), gymnasiums (25 %) and doctors (14 %). The most common reported adverse effects
were alterations in libido (61 %), changes in mood (48 %), reduced testis volume (46 %) and
acne (43 %). Although mean systolic and diastolic blood pressure was not significantly
different between groups, five present (29 %), 10 past (37 %) and one potential user (8 %)
were hypertensive. Gynaecomastia was found in 10 past users (37 %), two present users (12
%) and no potential users. Mean testis volume was significantly smaller in present users (18
mL) than in the other groups. Twenty past users (83 %), eight present users (62 %) and five
potential users (71 %) had abnormal liver function test results. After discussion of test
results, only 11 participants (19 %) reported they would not use AASs in the future. It was
concluded that adverse effects were reported by or detected in most of the AAS users who
attended the clinic. Despite awareness of adverse consequences, most participants planned
future use of AASs [00053].
This is an area of controversy as the risk of significant major side effects may have been
overstated in the healthy population using anabolic steroids. However, as studies in this area
are notoriously difficult, and there is no reporting of side effects to a central body, whilst one
cannot predict universal harm from using anabolic steroids the potential risks should be
monitored. For women the obvious masculinising effects can be damaging [01015].
Reported complications associated with anabolic-androgenic steroids misuse included

impaired post-exercise heart rate recovery, acute hepatitis (secondary to 17-alkylated steroid
abuse), collagen dysplasia, and general adverse cardiovascular effects. Moreover, negative
effects on mental health were observed in a retrospective study with retired elite athletes,
and once more, the commonly reported organic lesions such as testicular atrophy, testicular
fibrosis, arrested spermatogenesis, and left ventricular hypertrophy, were substantiated by
the autopsy results, which were conducted in cases of sudden or unnatural deaths where
toxicology revealed the individuals' AAS use. All these facts become arguably irrelevant in
the light of potential benefits provided by testosterone and its synthetic derivatives to
selected athletes [14715]
Performance-enhancing drugs (PEDs) gained wide popularity not only among sportsmen but
also among specific subsets of population, such as adolescents. Apart from their claimed
effects on athletic performance, they are very appealing due to the body shaping effect
exerted on fat mass and fat-free mass. Besides the "underestimated" massive misuse of
PEDs, the short- as well as long-term consequences of such habits remain largely
unrecognized. They have been strictly associated with serious adverse effects, but molecular
mechanisms are yet to be elucidated. Here, it was analyzed the current understanding of the
molecular effects of supraphysiological doses of doping agents in healthy biological systems,
at genomic and proteomic levels, in order to define the molecular sensors of organ/tissue
impairment, determined by their misuse. The focus is put on the anabolic androgenic steroids
1035
(AASs), specifically testosterone (T) and its most potent derivative dihydrotestosterone
(DHT), and on the peptide hormones, specifically the growth hormone (GH) and the insulin-
like growth factor-1 (IGF-1). A map of molecular targets is defined and the risk incidence for
human health is taken into account [150174].
AASs – as all other APEDs – may have not only the desired effect, but also adverse side
effects, resulting from the combination of different AASs in extremely high doses with other
drugs and from duration of administration over periods ranging from months to many years.
Due to the secret nature of this drug abuse type, doses and duration are mostly unknown
and properly controlled clinical trials do not exist. Hence the scientific assessment of the
sequelae of AASs abuse relies on case reports and on a few retrospective investigations,
making a review of the field in the age of evidence-based medicine extremely difficult and
frustrating. Nevertheless, this review is intended to inform the endocrinologist about
symptoms and diseases caused by AASs which, without specific knowledge, may be
misinterpreted while searching for their origin. Proper diagnosis is further hindered by the
reluctance of the doped patient to admit the consumption of AASs and being ignorant about
their possible serious side effects [150001].
Since the 1970s anabolic androgenic steroids (AAS) have been abused at ever increasing
rates in competitive athletics, in recreational sports and in bodybuilding. Exceedingly high
doses are often consumed over long periods, in particular by bodybuilders, causing acute or
chronic adverse side effects frequently complicated by additional polypharmacy. This review
summarizes side effects on non-reproductive organs and functions; effects on male and
female reproduction have been recently reviewed in a parallel paper. Among the most
striking AAS side effects are increases in haematocrit and coagulation causing
thromboembolism, intracardiac thrombosis and stroke as well as other cardiac disturbances
including arrhythmias, cardiomyopathies and possibly sudden death. 17alpha-alkylated AAS
are liver toxic leading to cholestasis, peliosis, adenomas and carcinomas.
Hyperbilirubinaemia can cause cholemic nephrosis and kidney failure. AAS abuse may
induce exaggerated self-confidence, reckless behavior, aggressiveness and psychotic
symptoms. AAS withdrawal may be accompanied by depression and suicidal intentions.
Since AAS abuse is not or only reluctantly admitted physicians should be aware of the
multitude of serious side effects when confronted with unclear symptoms [150175].
Some reported adverse effects of anabolic steroids included virilization in female users,
truncal acne, weight gain, gynecomastia, polycythemia, congestive cardiac failure and
myocardial infarctions, dyslipidemia, mood alteration, and renal failure. Due to the
predominantly illicit nature of AAS abuse, studying side effects and complications of
supraphysiologic doses is inherently difficult. Most clinical studies evaluate low-dose AAS for
the purposes of hormone replacement, either due to androgen deficiency in hypogonadal
syndromes, cachexia (i.e. conditions such as human immunodeficiency virus, chronic
obstructive pulmonary disease) or age-related declines (i.e. testosterone deficiency),
although the last area remains controversial [150011].
The use of doping agents are evident within competitive sport in senior and junior age
groups, where they are taken by non-elite as well as elite participants. They are also taken in
non-sporting contexts by individuals seeking to 'improve' their physique through an increase
in muscle and/or decrease in fat mass. Whilst attaining accurate data on the prevalence of
their use has limitations, studies suggest the illicit use of doping agents by athletes and non-
athletes may be 1-5 percent in the population and greater than 50 percent in some groups;
with the prevalence being higher in males. There is conclusive evidence that some doping
agents are anabolic and ergogenic. There is also evidence that the use of doping agents
such as: anabolic androgenic steroids; growth hormone and other anabolic agents;
1036
erythropoietin; and stimulants conveys considerable health risks that include, but are not
limited to: cardiovascular disease, diabetes, cancer, mental health issues, virilisation in
women, and the suppression of naturally produced androgens in men. One review outlined
the anabolic, ergogenic and health impacts of selected doping agents and methods that may
be used in both the sporting and physique development contexts. It also provides a brief
tabulated overview of the history of doping and how doping agents may impact upon the
analyses of clinical samples [150003].
Anabolic androgenic steroids (AASs) are appearance and performance-enhancing drugs

(APEDs) used in competitive athletics, in recreational sports, and by body-builders. The
global lifetime prevalence of AASs abuse is 6.4 percent for males and 1.6 percent for
women. Many AASs, often obtained from the internet and dubious sources, have not
undergone proper testing and are consumed at extremely high doses and in irrational
combinations, also along with other drugs. Controlled clinical trials investigating undesired
side effects are lacking because ethical restrictions prevent exposing volunteers to potentially
toxic regimens, obscuring a causal relationship between AASs abuse and possible sequelae.
Because of the negative feedback in the regulation of the hypothalamic-pituitary-gonadal
axis, in men AASs cause reversible suppression of spermatogenesis, testicular atrophy,
infertility, and erectile dysfunction (anabolic steroid-induced hypogonadism). Should
spermatogenesis not recover after AASs abuse, a pre-existing fertility disorder may have
resurfaced. AASs frequently cause gynecomastia and acne. In women, AASs may disrupt
ovarian function. Chronic strenuous physical activity leads to menstrual irregularities and, in
severe cases, to the female athlete triad (low energy intake, menstrual disorders and low
bone mass), making it difficult to disentangle the effects of sports and AASs. Acne, hirsutism
and (irreversible) deepening of the voice are further consequences of AASs misuse. There is
no evidence that AASs cause breast carcinoma. Detecting AASs misuse through the control
network of the World Anti-Doping Agency (WADA) not only aims to guarantee fair conditions
for athletes, but also to protect them from medical sequelae of AASs abuse [150001].
Anabolic-androgenic steroids (AAS) are synthetic derivatives of testosterone originally

designed for therapeutic uses to provide enhanced anabolic potency with negligible
androgenic effects. Although AAS continue to be used clinically today, the medical benefits
of low therapeutic doses of AAS stand in sharp contrast to the potential health risks
associated with the excessive doses self-administered not only by elite athletes and body
builders, but by a growing number of recreational users, including adolescent boys and girls.
The deleterious effects of AAS on peripheral organs and the incidence of altered behaviors in
AAS abusers have been well documented in a number of excellent current reviews for
clinical populations. However, a comparable synthesis of nonclinical studies has not been
made. The purpose in one review is to summarize the literature for animal models of the
effects of supraphysiological doses of AAS (e.g. those that mimic human abuse regimes) on
behaviors and on the neural circuitry for these behaviors. In particular, it was focused on
studies in rodents that have examined how AAS alter aggression, sexual behaviors, anxiety,
reward, learning, and locomotion and how AAS alter the expression and function of
neurotransmitter systems and other signaling molecules that underlie these behaviors
[03044].
The attraction of anabolic agents apparently continues to be unconfined among cheating

athletes and recreational sportsmen and women despite numerous comprehensive and new
reports on health risks attributed to the abuse of AAS, ranging from acne fulminans over
cardiovascular issues to increased risk of breast and Leydig cell cancer as well as psychic
disorders and dependence [14009].
Depending on duration and dosage of administration, AAS can cause various adverse effects
1037
marked by virilization and hirsutism in women, deepening of voice (permanent) as well as
testicular atrophy (reversible) and gynaecomastia in men. Furthermore, ASS abuse in
adolescence may induce premature epiphyseal fusion resulting in stunted growth. Also, via
both genomic and non-genomic pathways, AAS can trigger aggressive behaviour and
hostility as well as mood disturbances such as depression and hypomania. However, the
development of side-effects induced by supraphysiological doses of AAS correlate, at least
as regards the psychiatric symptoms, to the dose and duration of abuse. Considerable
changes of lipid profile result in decrease in HDL and increase in LDL concentration, as well
as diabetes mellitus, arterial hypertension and cardiac morbidity have been associated with
AAS abuse. The most typical cardiac abnormality is left ventricular hypertrophy with fibrosis,
while rare cardiovascular substrate is eosinophilic myocarditis. Nevertheless, the cause–
effect relationship between AAS abusers and cardiac death has not been conclusively
established, though ventricular arrhythmias and congestive heart failure have often been
reported, and thrombotic complications (intracardial thrombosis, stroke, venous
thromboembolism, cerebral venous sinus thrombosis) have been markedly associated with
AAS abusers [12011].
Anabolic steroids have been linked to many adverse health effects, including
cardiomyopathy, cerebrovascular events, hypertension, aggression, prostatic hypertrophy,
and cholestatic liver injury. In one study, 96 percent of users of anabolic steroids reported at
least one side effect. However, the absolute risks have not been fully evaluated. Users
typically take them in “cycles” that vary in length (often 8-12 weeks) to minimise side effects,
with a similar amount of time “off cycle” [12025].
Anabolic-androgenic steroids (AAS) administration has been shown to be associated with

cardiovascular side effects, urogenital problems, that is, gynecomastia, impotency,
hepatotoxicity, hepatocellular carcinoma, and neuropsychiatric disorders, that is,
aggressiveness and depression. Cardiovascular adverse effects of AAS abuse have been
reported sporadically as case reports of hypertension, myocardial infarction (MI) and stroke,
dysrhythmia, cardiomyopathy, and sudden cardiac death in body builders with long-term AAS
abuse in the recent years. Case reports on hard atherosclerotic endpoints (sudden cardiac
death, MI or stroke) comprise young AAS abusers without preexistent cardiac risk factors,
suggesting that a high AAS dose imposes additional independent risk to conventional
cardiovascular risk factors. It has been reported a more than four times higher incidence of
early death in professional athletes abusing AAS compared to the age- and sex-matched
general population, in a 12-years prospective observation. In addition to the potential risk
associated with AAS abuse, it is notable that therapeutic treatment with AAS in hypogonadic
men has recently been shown to be linked with a higher cardiovascular event rate. This
important finding underscores that there is a delicate balance between benefits and risks
related to AAS use as a treatment option for patients suffering from androgen deficiency.
Adding up clinical AAS use and illegal AAS abuse rates represent a new wave of AAS-
associated cardiovascular adverse consequences, in which early diagnosis can reduce
health burden. The wave of now middle-aged ex-AAS abusers and the increasing group of
the elderly AAS users necessitates more detailed documentation of the underlying
pathophysiology to enhance insight into the delicate balance between benefit and harm.
Owing to the obvious ethical reasons, prospective double-blinded human studies are not
easily justified. Accordingly, retrospective case-control studies of cohorts and prospectively
follow-up of such cohorts seem to be the most feasible strategy for human studies to obtain
more conclusive epidemiologic data [12125].
There are limited data on the short-term complication of AAS use with most data involving
case reports. The long-term health effects of chronic AAS abuse are not well defined
because of the difficulty in studying illicit drug use and the highlyvariable dosages involved
1038
with AAS abuse. Clinical and laboratory studies indicate that the administration of AASs
causes physiologic changes, primarily in the liver, reproductive system, and serum lipids.
The use of AAS produces an unfavorable change in the blood lipid profile (i.e. elevated low-
density lipoprotein, reduced high-density lipoprotein) with potentially increased risk of
coronary heart disease; although conflicting, some data suggest left ventricular hypertrophy
may persist after cessation of AAS use as a result of elevated blood pressure during use.
Other potential adverse effects include glucose intolerance (i.e. increased peripheral insulin
resistance), hyperinsulinism, behavioral and mood changes, cerebrovascular accidents,
prostate abnormalities, edema, and immune dysfunction. Most physiologic changes
associated with AAS useare reversible within several months of the cessation of AAS use
[13003].
Anabolic-androgenic steroids (AAS) are used as ergogenic aids by athletes and non-athletes
to enhance performance by augmenting muscular development and strength. AAS
administration is often associated with various adverse effects that are generally dose
related. High and multi-doses of AAS used for athletic enhancement can lead to serious and
irreversible organ damage. Among the most common adverse effects of AAS are some
degree of reduced fertility and gynecomastia in males and masculinization in women and
children. Other adverse effects include hypertension and atherosclerosis, blood clotting,
jaundice, hepatic neoplasms and carcinoma, tendon damage, psychiatric and behavioral
disorders. More specifically, this article reviews the reproductive, hepatic, cardiovascular,
hematological, cerebrovascular, musculoskeletal, endocrine, renal, immunologic and
psychologic effects. Drug-prevention counseling to athletes is highlighted and the use of
anabolic steroids is must be avoided, emphasizing that sports goals may be met within the
framework of honest competition, free of doping substances [05042].
In addition to what is traditionally known about the potential ergogenic effects of AAS, a
much larger list can be formed concerning the adverse effects of the use of repeated and
high doses of AAS. These include development of acne, jaundice, increased aggression,
depression, gynecomastia, infertility, sexual impotence, masculinization of women and
children, hepatotoxicity, hepatocellular carcinoma, and structural damage in tendons.
Adverse cardiovascular effects have also been reported with the use of AAS, such as
dyslipidemia, high blood pressure, cardiovascular remodeling, and deaths from heart attacks
and cerebral vascular accident. These episodes have been attributed to high doses over
prolonged periods. However, ethical and methodological difficulties (i.e. identification of AAS
users, impossibility of conducting experimental human studies) make it very difficult to
precisely quantify the adverse effects of using AAS. Most studies are limited in their
generalizability because they are case reports or are of limited sample size. Thus, available
data in the literature so far do not allow an exact determination of the adverse cardiovascular
effects of using AAS, at short-term or longer-term levels. Although several studies have
observed dyslipidemia, pathological changes in cardiac structure, and reports of deaths,
there is a need for an evidence-informed, generalizable, study enabling abroad overview of
the cardiovascular effects of the use of AAS [14613].
To date, the data available in the literature point to possible adverse effects of AAS use on
the cardiovascular system. Dyslipidemia, increased blood pressure, oxidative stress,
myocardial remodeling, and even myocardial infarction and deaths are likely to occur as a
result of AAS use. However, because of ethical and methodological limitations, the studies
are very limited, especially in the aspect of sample size, which makes it difficult to generalize
the data. A caveat is warranted when drawing conclusions from animal studies to human
beings. There are a reasonable number of studies reporting potential adverse cardiovascular
effects, while studies indicating an absence of adverse effects are not reported. The adverse
effects reviewed in this paper may be used as a resource in anti-doping educational
1039
programs. Supplying information, whatever its validity, is not equivalent, however, to creating
and sustaining needed, useable knowledge to enable health-promoting awareness,
perceptions, expectations, judgments, decisions, which are implemented or not, and
necessary learning, which is integrated into daily adapting and functioning in a range of roles,
networks, contexts, situations, and environments – each with their own conditions and
“demands” [14613].
Historically, the side effects of AAS use have been overstated. Serious health problems are
rare, and the more common adverse effects are benign and reversible. The incidence of
complications associated with the nonmedical use of AAS as performance-enhancing drugs
is unclear because the denominator of drug use in athletes is not well defined. However, data
from larger observational studies suggest that the majority (88-96 %) of AAS users
experience at least 1 minor subjective side effect, including acne (40%-54%), testicular
atrophy (40-51 %), gynecomastia (10-34 %), cutaneous striae (34 %), and injection site pain
(36 %). Recent prospective clinical studies report a good safety profile for pharmacologic and
suprapharmacologic doses of AAS when used in the short term. With the exception of a few
reversible laboratory abnormalities – decreased HDL, elevated hemoglobin, and raised liver
enzymes – high doses of AAS administered for periods of up to 20 weeks failed to
demonstrate any significant systemic toxicity. The potential adverse effects of AAS can be
divided into several categories, including cardiovascular, hepatic, endocrine/reproductive,
behavioral, dermatologic, andinjection related [04018].
Since most well designed studies are not able to address the AAS issue of real life, it should
be emphasised that the untoward effects may be more pronounced than has been
demonstrated in laboratory studies. Also, the extent of side effects in AAS users will be much
larger than may be expected on the basis of the available scientific data. Side effects can be
divided in subjective and objective. First, we describe studies that focused on subjective
perceived side effects. Later we discuss the undesired health effects that are open to
objectification [04002].
Self-reported adverse effects
To date, only a few reports investigating the self-reported adverse effects in athletes using
AAS have been published. These reports employing questionnaires showed clearly that the
majority of athletes experienced undesired health effects not only when on AAS, but also
after drug withdrawal. These data are very valuable since they indicate the extent of self-
reported untoward effects when using high doses of AAS in stacking regimens reflecting real-
life AAS abuse. The side effects reported in at least 40 percent of the male subjects in these
studies included increased sexual drive, occurrence of acne,]increased body hair and an
increase in aggressive behaviour.Furthermore, many other side effects affecting several
body systems were mentioned by the steroid users. These include fluid retention, elevated
blood pressure (BP), sleeplessness, increased irritability, decreased libido, increased
appetite, enhanced transpiration, increased feeling of well-being, depressive mood states,
loss of head hair and the occurrence of gynaecomastia [04002].
Data relating to female athletes are very scanty. In one study it was interviewed ten females
who all reported lowering of the voice brought on by AAS use. Furthermore, nine of ten
females admitted increased growth of facial hair, enlargement of the clitoris and an increase
in aggressiveness and appetite. In another study nine of ten interviewed female athletes had
experienced side effects due to steroid use. The side effects reported were acne (50 %), fluid
retention (40 %) and alteration of libido (50 %). Other side effects were only mentioned by
<20 percent of the women. Of great concern is that athletes are not aware of many side
effects during steroid administration, since several unwanted health effects may be detected
1040
only after thorough medical examination, including blood analysis [04002].
Short-term side effects
The attraction of anabolic agents apparently continues to be unconfined among cheating

athletes and recreational sportsmen and women despite numerous comprehensive and new
reports on health risks attributed to the abuse of AAS, ranging from acne fulminans over
cardiovascular issues to increased risk of breast and Leydig cell cancer as well as psychic
disorders and dependence [13009].
Most of the adverse effects following the use of AASs result from the enhancement of normal
physiologic response to testosterone by either direct receptor agonist activity or suppression
of steroid biosynthes is. In general, toxic effects associated with AAS abuse involve the
following:
- anabolic side effects

- enhanced androgenic effects
- estrogenic side effects
- antiandrogenic effects from the suppression of the hypothalamus-pituitary-
adrenal/gonadal axes
- hepatotoxicity
- neuropsychiatric effects
Methodological issues limit the determination of the toxic effects of illicit AAS use including
the extraordinary doses and types of AAS sused by athletes compared with medical use,
reporting bias of self-reports, the paucity of well-documented pathologic findings, and the
lack of well-defined postmortem markers of AAS use. Most medical data on the toxic effects
of AAS abuse involve case reports rather than epidemiologic studies. Pathologic
abnormalities from AAS abuse are best-documented in the cardiovascular system,
reproductive system, liver, and serum lipids. Animal studies suggest that AAS can cause
dysplasia of collagen fibrils and decreased tensile strength, and potentially the use of these
drugs could cause disruption of connective tissue [13003].
One year of abuse
The purpose of one study was to evaluate anabolic-androgenic steroid (AAS) abusing adults
every 2 weeks with a comprehensive behavioral and clinical assessment battery. The study
was conducted at the University of Pennsylvania Treatment Research Center; 10 subjects
were enrolled and 7 completed the protocol. AASs and other drugs were obtained and self-
administered by subjects through their usual mechanisms. On-study evaluations included
medical, behavioral, and drug-use assessments. While a high incidence of mood disorders
and substance abuse was found, few clinically relevant changes in physiological parameters
or laboratory measures were noted throughout the study. Changes as measured by various
behavioral rating scales were observed across time; however, these changes were not
clearly related to periods of reported AAS use. Additional factors such as life events,
subjects' other drug use, and the extended duration of activity of some of the AAS
preparations probably influenced the results. Differences in subject-reported adverse effects
were seen with respect to periods of AAS use and nonuse. Cycles of AAS nonuse were
associated with a greater percentage of subject-reported increased testicular size, appetite,
frequency of sexual activity, and libido. The results provide the first long-term, prospective
evaluation of the effects of AASs, when these drugs are administered in a naturalistic pattern
of abuse [03045].
1041
Anesthesia risk
One review summarised the physiological and pharmacological effects of the anabolic
steroids used to enhance performance in sports. The anabolic steroids promote muscle
growth and protein synthesis. Side-effects of anabolic steroids include cardiomyopathy,
atherosclerosis, hypercoagulopathy, hepatic dysfunction, and psychiatric and behavioural
disturbances. It is therefore appropriate that the anaesthetist be familiar with the abuse of
anabolic steroids, their potential adverse effects, and the peri-operative risk associated with
the use of these drugs [05060].
A strong tendency toward body enhancement and body forming in western industrial
societies makes it more likely for each anesthesiologist to get involved in the care of
bodybuilders. These patients quite frequently consume androgenic anabolic steroids, human
growth hormone and other drugs or substances which are believed to accelerate muscle
gain. Cardiovascular, hepatic, psychiatric, hormonal and infectious side effects or
complications are common and rarely monitored by health care professionals. The
anesthesia risk is not exactly known but seems to be determined mainly by cardiovascular
events like myocardial ischemia and dysrhythmias [08143].
Different effects of different anabolic steroids
All synthetic AAS are derived from testosterone. They have a carbon skeleton with 4 fused
rings; most have 19 carbons. Modifications include hydroxylation at the C10 position to
increase receptor binding affinity (e.g. nandrolone) esterification to slow release into
circulation (e.g. testosterone cypionate), or alkylation at the C17 position to permit moral
delivery by reducing first-pass metabolism in the liver (e.g. oxymetholone). AAS can be
converted to highly-androgenic or estrogenic metabolites. For testosterone,
dihydrotestosterone is the principle androgenic product; estradiol is the major estrogenic
metabolite. Non-aromatizable AAS (e.g. drostanolone) have fewer estrogenic side-effects
such as gynecomastia. Non-reducible AAS (e.g. oxandrolone) have fewer androgenic side-
effects such as acne, baldness, and prostatic hypertrophy because they have lower binding
affinity for the androgen receptor. For athletes subject to drug testing, a key drawback of
synthetic AAS is that their use is easily detected, since their metabolites are not normally
present. An extended precompetition wash-out period is necessary to avoid a positive test.
This varies with the route of administration and the half-life of the individual AAS. However,
long-acting AAS such as nandrolone can be detected for at least 6 months. By contrast, the
urinary metabolites of exogenous and endogenous testosterone are virtually identical. Many
athletes take long-acting testosterone esters such as testosterone propionate. Although
esterification prolongs the halflife in circulation, the active steroid is still testosterone [12100].
Doping is becoming an everyday problem in sports medicine. Its main feature is its
universality: it concerns all sports, even the most unexpected, from cycling to billiards; all
countries are affected with certain continental preferences with regards to the substances
used; it is seen in all levels of competition, both in amateurs and professionals. Doping is
observed early on, even in childhood. Many substances are used and they are increasingly
available: all bodily functions are targeted: cerebral, metabolic, cardiovascular, respiratory,
haematological and, in the near future, genetic. Detection of doping is difficult and
unpredictable in a legislative environment which is gradually improving. The different modes
of action of the doping substances often target the cardiovascular system, especially with
regards to their potential complications: hypertension, arrhythmias, thrombosis, coronary
artery and peripheral artery diseases and also cardiomyopathies. Every cardiologist should
1042
therefore be aware of the problem, even outside the context of sport, as it may impact on
daily cardiological practice [06065].
Toxicokinetics
Most of the data on the kinetics of testosterone and AASs is derived from the
pharmacokinetics of these compounds in animals or in hypogonadal males receiving
therapeutic doses of AASs. There are few data on the toxicokinetics of AASs in individuals
abusing AASs at doses up to 10-100 times the therapeutic dose. Despite the rapid
absorption of testosterone, the systemic bioavailability of oral testosterone is low as aresult
of extensive first-pass hepatic metabolism. Structural modifications of testosterone produce
synthetic testosterone derivatives (anabolic-androgenic steroids), which increase
bioavailability and prolong the duration of action. Alkylation of the 17-alpha position of
testosterone produces oral AAS, whereas esterification of the 17-beta position results in
injectable AAS (e.g. lipid-soluble cypionate or enanthate). The duration of action of these
esters depends on the rate of absorption from th esite of administration as determined by the
chain length of the acid moiety and the formulation. Hydrolysis of these esters in vivo
prolongs the duration of action compared with testosterone. Anabolic- androgenic steroids
can diffuse a cross the skin and mucous membranes, allowing other delivery modes
including transdermal patches, nasal sprays, and buccal tablets. Following oral
administration of 120 mg testosterone undecanoate, volunteer studies in dicate that plasma
concentrations of testosterone are detectable for about 1-6 h after administration using gas
chromatography–tandem massspectrometry. There are dramatic individual variations (i.e.10-
fold) in the peak total plasma testosterone concentrations. In a study of 61 eugonadal men
receiving long-acting gonadotropin-releasing hormone agonist to suppress endogenous
testosterone secretion, the mean nadir testosterone concentrations ranged from 2.53 to 23.7
ng/mL following weekly injections of testosterone enanthate doses of 25-600 mg for 20
weeks [13003].
Anabolic-androgenic steroids are bound in the plasma to sex-hormone-binding globulins.

Although testosterone is highly protein bound (i.e. 98 %) in plasma, the binding of AAS to
sex-hormone-binding globulins is highly variable base done animal studies. The metabolism
of endogenous testosterone involves the conversion to the estrogenic compound, estradiol,
via steroid aromatase and the androgenic compound, 5alpha-dihydrotestosterone, via
5alpha–steroid-reductase. Comparatively, the biotransformation of AASs is quite complex.
The initial and rateliming step in testosterone metabolism is reduction of the C4-C5 double
bond on the A-ring with 5alpha-reductase and 5beta-reductase. Hydroxylation of
testosterone by CYP450 isoenzymes results in the formation of a variety of minor urinary
metabolites of testosterone. Single-dose human excretion studies indicate that 6-beta-
hydroxylation is also a minor pathway for the biotransformation of boldenone (17beta-
hydroxyandrosta-1,4-dien-3-one) and 17alpha-methyl-testosterone. However, 6beta-
hydroxylation of the B-ring is the major metabolic pathway for 4-chloro-1,2-dehydro-17-alpha-
methyltestosterone, methandienone, and fluoxymesterone because the presence of a C1-C2
double bond in the former 2steroids and the C9 alpha-fluorine atom in the latter compound
blocks A-ring reduction. Metabolic changes (e.g. 12-hydroxylation) of AASs at the C-ring are
minor. D-ring metabolism by the enzymatic oxidation of 17beta-hydroxysteroid-
dehydrogenase to form the corresponding 17-ketosteroid is a major metabolic pathway for
testosterone and all AAS swith secondary 17beta-hydroxy groups (e.g. boldenone, clostebol,
drostanolone, mesterolone, methenolone, nandrolone, norclostebol, and stenbolone). The
main urinary metabolites of testosterone are androsterone (3alpa-hydroxy-5alpa-androstan-
17-one), etiocholanolone (3alpha-hydroxy-5beta-androstan-17-one), epiandrosterone (3beta-
1043
hydroxy-5alpha-androstan-17-one), 5alpha-androstane-3alpha, 17beta-diol, and 5beta-
androstane-3alpha,17beta-diol [13003].
In individuals without AAS use, only small amounts (i.e. about 1 %) of endogenous
testosterone appear unchanged in the urine. Phase II conjugation reactions couple AASs
and associated metabolite swith glucuronic acid or sulfate before excretion in the urine. The
vast majority (i.e. about 90 %) of the absorbed dose of testosterone appears in the urine as
glucuronide or sulfate conjugates. In a study of 8 hypogonadal males, the terminal
elimination half-lives of 500 mg and 1000 mg intramuscular doses of testosterone
undecanoate were 18.3 and 2.3 days and 23.7 and 2.7 days, respectively. The mean
residence times were 21.7 and 1.1 days and 23.0 and 0.8 days, respectively. Not all anabolic
steroids undergo phase II reactions. Unconjugated AASs in human urine include
oxandrolone, fluoxymesterone, 4-chloro-1,2dehydro-17alpha-methyltestosterone, and
formebolone, along with metabolites of oxandrolone, methandienone, and stanozolol. There
is very limited (i.e, about 5 %) enterohepatic recirculation of testosterone. Anabolic-
androgenic steroids readily cross the placenta [13003].
Impurities in illicit samples of anabolic steroids
There are few data on the purity of illicit amples of AASs as a result of the lack of regulation.
Consequently, there are no assurances that the chronic AAS abuser knows the dose or type
of AAS. The difficulty determining doses used by AAS abusers limits the ability of studies to
elucidate the effect of AAS abuse. Frequently, illicit samples of AASs do not contain declared
ingredients or concentrations of ingredients. Analysis of 70 products confiscated from illegal
sources demonstrated 17 (35 %) of the 48 steroidal compounds did not contain labeled
ingredients as measured by liquid chromatography-tandem mass-spectrometry, gas
chromatography mass-spectrometry with nitrogen-phosphorus detection, gel-
electrophoresis, and immunological tests. Visual inspection did not distinguish original
products from counterfeits [13003].
Mortality
The side effects of AS are dependent on dose and their metabolism. Misuse of AS is claimed
to have serious side effects. The mortality in 62 male weightlifters placed 1st-5th in weight
series 82.5-125 Kg in Finland was compared with the mortality of population controls. The
mortality during the 12-year follow-up was 12.9 percent for the weightlifters compared to 3.1
percent in the control population; thus, the risk of death among the weightlifters was 4.6
times higher [03002].
Anabolic steroids’ impact on the cardiovascular system
Overview
Cardiovascular effects of doping drugs are numerous, with different mechanisms:

vasoconstriction of amphetamines, erythropoietin and cocaine; sodium water retention of
anabolic steroids and corticosteroids; elevation in blood viscosity of erythropoietin,
perflurocarbon emulsion, recombinant hemoglobin and anabolic steroids; sympathetic
nervous system activation of amphetamines, beta 2 agonists and clenbuterol; lipids profile
disorder of anabolic steroids. Physical activity consequences, particularly bradycardia and
dehydration, are worsening. Thrombosis and arrythmogenic effects, with possibility of
1044
sudden death, are the severe immediate events. Hypertension and coronary diseases are
medium-term effects; acute myocardial infarction is frequent. Heart failure can be secondary
to cardiac muscle direct fibrosis, like with anabolic steroids. These cardiovascular effects are
serious and it is necessary to early detect the doping drugs use in sporstmen; all prescribing
physician should be aware of existing drugs and their clinical events [01050].
Increased left-ventricular mass is an important cardiovascular risk factor for morbidity and
mortality. Apart from obvious differences in cardiac size, the changes in left-ventricular mass
in response to age and hypertrophic stimuli are very different in men and women. Whereas
left-ventricular mass increases with age in apparently healthy women, it remains constant in
men. Under increased cardiac loading conditions, such as hypertension or aortic stenosis,
this disparity between sexes is even more striking. Findings are especially pronounced in
people aged 50 years or older, in whom reproductive hormone concentrations have fallen.
Whether the differences in left-ventricular mass changes are related to endogenous sex-
hormone concentrations has never been shown. Androgens have anabolic effects on cardiac
cells, and oestrogens have antiproliferative properties, it was therefore postulated that the
normal decline in endogenous sex hormones with age has contrary effects on ventricular
mass in men and women in normal and pathological states [01051].
Recent reports have significantly halted the enthusiasm regarding androgen-boosting;

suggesting that testosterone supplementation (TS) increases cardiovascular (CV) events. In
order to overcome some of the limitations of the current evidence, the authors performed an
updated systematic review and meta-analysis of all placebo-controlled randomized clinical
trials (RCTs) on the effect of TS on CV-related problems. Out of 2747 retrieved articles, 75
were analyzed, including 3016 and 2448 patients in TS and placebo groups, respectively,
and a mean duration of 34 weeks. The analyses, performed on the largest number of studies
collected so far, indicate that TS is not related to any increase in CV risk, even when
composite or single adverse events were considered. In RCTs performed in subjects with
metabolic derangements a protective effect of TS on CV risk was observed. The present
systematic review and meta-analysis does not support a causal role between TS and
adverse CV events. The results are in agreement with a large body of literature from the last
20 years supporting TS of hypogonadal men as a valuable strategy in improving a patient's
metabolic profile, reducing body fat and increasing lean muscle mass, which would ultimately
reduce the risk of heart disease [14646].
The use of doping substances and methods is extensive not only among elite athletes, but
also among amateur and recreational athletes. Many types of drugs are used by athletes to
enhance performance, to reduce anxiety, to increase muscle mass, to reduce weight or to
mask the use of other drugs during testing. However, the abuse of doping substances and
methods has been associated with the occurrence of numerous health side-effects. The
adverse effects depend on the type of the consumed drug, as well as the amount and
duration of intake and the sensitivity of the body, since there is a large inter-individual
variability in responses to a drug. Usually the doses used in sports are much higher than
those used for therapeutic purposes and the use of several drugs in combination is frequent,
leading to higher risk of side-effects. Among biomedical side-effects of doping, the
cardiovascular ones are the most deleterious. Myocardial infarction, hyperlipidemia,
hypertension, thrombosis, arrythmogenesis, heart failure and sudden cardiac death have
been noted following drug abuse. This paper reviews the literature on the adverse
cardiovascular effects after abuse of prohibited substances and methods in athletes, aiming
to inform physicians, trainers and athletes and to discourage individuals from using drugs
during sports [06063].
The intake of anabolic-androgenic steroids (AAS) leads to an increase in skeletal muscle

1045
mass and is prohibited as a doping measure in sport. AAS abuse is not limited to competitive
athletes. It is also prevalent in subjects who do body building or resistance training for
cosmetic reasons only. Out of the numerous and partly serious side effects, the
cardiovascular ones maybe most important. An increase in left ventricular muscle mass is
well documented, and some researchers have even reported concentric hypertrophy. By
contrast, resistance training without AAS intake does not lead to increased ventricular wall
thickness. AAS do not affect the systolic function of the left ventricle, whereas diastolic
function might be impaired. Different ultrastructural myocardial alterations have been
documented in animal studies. In addition, AAS can induce arterial hypertension. Blood
clotting and fibrinolysis are negatively affected, and several case studies of thrombi exist in
young strength athletes. Changes in the concentration of blood lipoproteins, particularly a
reduction in vessel-protective HDL cholesterol, can lead to early atherosclerosis. Many case
reports exist about cardiac deaths in seemingly healthy subjects-most often body builders
and other strength athletes. In fatal and nonfatal myocardial infarctions patent coronary
arteries were proven frequently. Besides the prothrombotic effects of AAS, an impaired
endothelial function and vasospasms are discussed hypothetically as pathomechanisms.
Also, cardiomyopathies can occur due to AAS abuse. On the basis of the described possible
cardiovascular side effects, it can be concluded that in cases of sudden cardiac deaths in
young athletes, a misuse of AS should be excluded [06064].
Scientific data on the cardiac and metabolic complications of AAS abuse are divergent and
often conflicting. A total of 49 studies describing 1,467 athletes were reviewed to investigate
the cardiovascular effects of the abuse of AAS. Although studies were typically small and
retrospective, some associated AAS abuse with unfavorable effects. Otherwise healthy
young athletes abusing AAS may show elevated levels of low-density lipoprotein and low
levels of high-density lipoprotein. Although data are conflicting, AAS have also been linked
with elevated systolic and diastolic blood pressure and with left ventricular hypertrophy that
may persist after AAS cessation. Finally, in small case studies, AAS abuse has been linked
with acute myocardial infarction and fatal ventricular arrhythmias. In conclusion, recognition
of these adverse effects may improve the education of athletes and increase vigilance when
evaluating young athletes with cardiovascular abnormalities [10305].
Abuse of anabolic androgenic steroids (AAS) has been linked to a variety of different
cardiovascular side effects. In case reports, acute myocardial infarction is the most common
event presented, but other adverse cardiovascular effects such as left ventricular
hypertrophy, reduced left ventricular function, arterial thrombosis, pulmonary embolism and
several cases of sudden cardiac death have also been reported. However, to date there are
no prospective, randomized, interventional studies on the long-term cardiovascular effects of
abuse of AAS. In one review it was studied the relevant literature regarding several risk
factors for cardiovascular disease where the effects of AAS have been scrutinized:
- echocardiographic studies show that supraphysiologic doses of AAS lead to both

morphologic and functional changes of the heart. These include a tendency to
produce myocardial hypertrophy, a possible increase of heart chamber diameters,
unequivocal alterations of diastolic function and ventricular relaxation, and most likely
a subclinically compromised left ventricular contractile function
- AAS induce a mild, but transient increase of blood pressure. However, the clinical
significance of this effect remains modest
- AAS confer an enhanced pro-thrombotic state, most prominently through an
activation of platelet aggregability. The concomitant effects on the humoral
coagulation cascade are more complex and include activation of both pro-coagulatory
and fibrinolytic pathways
1046
- users of AAS often demonstrate unfavorable measurements of vascular reactivity
involving endothelial-dependent or endothelial-independent vasodilatation. A degree
of reversibility seems to be consistent, though
- there is a comprehensive body of evidence documenting that AAS induce various
alterations of lipid metabolism. The most prominent changes are concomitant
elevations of LDL and decreases of HDL, effects that increase the risk of coronary
artery disease
- the use of AAS appears to confer an increased risk of life-threatening arrhythmia
leading to sudden death, although the underlying mechanisms are still far from being
elucidated
Taken together, various lines of evidence involving a variety of pathophysiologic mechanisms

suggest an increased risk for cardiovascular disease in users of anabolic androgenic steroids
[10064].
Athletes use androgenic-anabolic steroids but it may lead to dilated cardiomyopathy. It was
report a case of a 41-year-old bodybuilder with severe systolic dysfunction and Class IV
heart failure despite maximal medical therapy. He used anabolic steroids and insulin growth
factor, and did not have any other risk factors for cardiomyopathy. It was briefly reviewed the
literature and summarize other reported cases with similar scenarios. In most of them
cardiomyopathy was at least partially reversible after discontinuation of anabolics. Abuse of
anabolic steroids may be an uncommon cause of cardiomyopathy in young and otherwise
healthy individuals [09057].
Chronic heart failure (CHF) involves derangements in multiple neurohormonal axes leading
to a procatabolic state and wasting syndrome associated with significant mortality. Catabolic
abnormalities include excess catecholamines and glucocorticoids. Anabolic defects include
deficiencies of sex steroids, insulin resistance, and growth hormone (GH) resistance. These
abnormalities are also correlated with increased morbidity and mortality in CHF. Anabolic
axes have been augmented in pilot studies in CHF with testosterone, GH, insulin-like growth
factor-1, and GH secretagogues. Results have been varied although some treatments have
been associated with improved surrogate endpoints. One review article explores the current
understanding of metabolic derangements in CHF and highlights potential neuroendocrine
treatment strategies [09058].
Recent surveys and reports suggest that many athletes and bodybuilders abuse anabolic-
androgenic steroids (AAS). However, scientific data on the cardiac and metabolic
complications of AAS abuse are divergent and often conflicting. A total of 49 studies
describing 1,467 athletes were reviewed to investigate the cardiovascular effects of the
abuse of AAS. Although studies were typically small and retrospective, some associated
AAS abuse with unfavorable effects. Otherwise healthy young athletes abusing AAS may
show elevated levels of low-density lipoprotein and low levels of high-density lipoprotein.
Although data are conflicting, AAS have also been linked with elevated systolic and diastolic
blood pressure and with left ventricular hypertrophy that may persist after AAS cessation.
Finally, in small case studies, AAS abuse has been linked with acute myocardial infarction
and fatal ventricular arrhythmias. In conclusion, recognition of these adverse effects may
improve the education of athletes and increase vigilance when evaluating young athletes
with cardiovascular abnormalities [10431].
The most common cardiovascular consequences of AAS include atherosclerosis (secondary

to changes in cholesterol metabolism and platelet function), hypertension, cardiac
hypertrophy, impaired cardiac function, and sudden death. AAS use causes metabolic
derangements that increase the risk for atherosclerosis and thrombus formation. Studies
1047
using animal models and various steroid regimens have demonstrated changes in serum
cholesterol levels with decreased high-density lipoprotein and increased low-density
lipoprotein, both promoting atherosclerotic and peripheral vascular disease. Cholesterol
alterations vary among different AASs; alkylated agents (e.g. stanozolol) cause greater
changes than testosterone [07058]
.
AAS use also increases platelet reactivity without an associated thrombocytosis; this has
been proposed as an etiology for some of the myocardial infarctions, strokes, and peripheral
vascular disease events reported in otherwise healthy individuals. AAS use also increases
serum C-reactive protein (CRP), reflecting an inflammatory state that may contribute to
atheroma formation and peripheral vascular disease. Conversely, changes in lipid
metabolism may be protective from atheroma formation because of a reduction in lipoprotein
A. Many studies show that AASs cause abnormal cholesterol profiles, increased CRP, and
increased platelet reactivity. It is difficult to quantify the change in risk, but one study
estimates AASs triple the cardiovascular risk [07058]
.
Recent evidence suggests that low, rather than high, testosterone (T) is associated with
increased male morbidity and mortality. It was reviewed relationships between
hypogonadism, metabolic syndrome (MetS) and cardiovascular (CV) disease (CVD), along
with erectile dysfunction (ED), a common condition in the three diseases. Although several
experimental data indicate that T exerts a protective effect on vascular function,
epidemiological studies do not support a link between hypogonadism and CVD and three
meta-analyses found no significant effect of testosterone replacement therapy (TRT) on CV
events. Low T is associated with increased risk of CV death in community-dwelling men, and
in men with ED. It is possible that both low T and CVD are associated with another, still
unknown (or not assessed) factor, thus explaining the association, in the absence of any
causal relationship. A meta-analysis on the effect of TRT in MetS-associated hypogonadism
demonstrated positive effects of T on some of the components of MetS. Large-scale
interventional studies with TRT are therefore advisable [11092].
Cardiovascular disorders are known to be the most common cause of sudden death during
exercise. In younger athletes (below 45 years) this is due in the majority of cases to
congenital heart diseases, while in older people atherosclerosis is the primary cause. There
is, however, a non-negligible percentage of disorders of the cardiovascular system, even
sudden cardiac death (SCD), that are attributable to the use of performance-enhancing
drugs, either prohibited (doping) or legal. The users can be athletes, professional or amateur,
or just people engaging in exercise in gyms or fitness and leisure centres, while both sexes
and all age groups are involved. Seeking to improve their performance, according to the
event in which they participate, most athletes use a combination of prohibited substances
and methods, or of prohibited and non-prohibited drugs, so as to alleviate the complications
and/or to avoid being detected by screening. The most common and serious consequences
of almost all illicit drugs in sport concern the cardiovascular system. These disorders, such
as hypertension, cardiac arrhythmias, and acute myocardial infarction, may be manifested
either directly, or as the result of long-term use. Frequent complications may also occur in
other organs. Specifically, anabolic steroids have been implicated in liver cirrhosis and liver
or kidney cancer, growth hormone in diabetes mellitus, erythropoietin in thromboembolic
episodes, central nervous system stimulants in psychotic syndromes, and so on. Apart from
the prohibited substances, however, cardiovascular disorders may be caused by other
substances commonly used in sports, such as dietary supplements [12126].
A search of the English-language scientific literature from 1976 through March 2012 was
performed primarily by searching the MEDLINE, Embase, and Google databases. The key
words used in the search included androgens, anabolic, androgenic, steroids, exercise,
1048
athlete, cardiovascular, effects. The bibliographies of articles from the above search also
were searched for relevant articles; links on Web sites containing published articles were
searched for pertinent information [12119].
Potential adverse effects of AAS on the cardiovascular system include atherogenesis,

thrombosis, vasospasm, myocarditis, concentric left ventricular hypertrophy, myocardial
fibrosis, hypertrophic cardiomyopathy with ventricular dysrhythmias, and direct myocardial
injury. However, the contribution of AAS use to these potentiall adverse cardiovascular
effects remains unclear. Chronic AAS use enhances hepatic triglyceride lipase activity,
resulting in reduction of high-density lipoproteins and elevation of low-density lipoproteins.
Although these changes are reversible within several months of cessation of AAS use,
chronic AAS use theoretically increases the risk of cardiac disease. Potentially, the chronic
abuse of AAS enhances coagulability and thrombosis, but the clinical importance of this
potential adverse effect also remains unclear. Studies of chronic AAS abuse in weight lifters
suggest that some anabolic-androgenic steroid using weight lifters have accelerated
activation of their hemostatic system as evidenced by increased generation of both thrombin
and plasmin. A study of AAS-positive steroid using weight lifters indicated that thes
eindividuals had a higher percentage of abnormally high plasma thrombin-antithrombin
complexes along with elevated plasma concentrations of prothrombin fragment 1,
antithrombin II, and protein S, when compared with non-AAS using controls. Additionally, the
plasma concentrations of tissue plasminogen activator and its inhibit or were lower in AAS
users than in controls. Clinical studies on body builders suggest that chronic AAS use
impairs vascular eactivity independent of the smooth muscle hypertrophy and vascular
stiffness associated with bodybuilding. Anabolic-androgenic steroids decrease the production
of cyclic guanosine monophosphate (cGMP) by inhibiting guanylyltransferase. As ar esult,
AAS spotentially inhibit the ability of nitric oxide store lax smooth muscles in the coronary
arteries resulting in coronary artery vasospasm and potentially sensitizing AAS users to
sudden death. Case reports associate the chronic use of anabolic steroids with sudden
death and contraction band necrosis in the myocardium. In these cases, no other cause of
death was apparent, but the role of chronic, high-dose anabolic steroid use in these deaths
remains unclear. Athletes with certain genetic mutations and structural abnormalities may be
particularly vulnerable to the use of anabolic steroids including athletes with accessory AV
pathways, latent structural heart diseases (dilated cardiomyopathy, arrhythmogenic right
ventricular dysplasia type II, myocarditis, segmental arrhythmogenic ventricular
cardiomyopathy, and coronary artery anomalies), latent Brugada syndrome, mutations of the
long QT syndrome genes, and other genetic mutations of ion channels (cardia cryanodine
receptor gene defects and calsequestrin gene defects). Pathologic evidence of some of
these abnormalities may not appear on postmort emexamination. The use of diuretics to
mask the use of anabolic steroids may predispose these athletes to serious ventricular
dysrhythmias from hypokalemia and dehydration [13003].
Athletes commonly use drugs and dietary supplements to improve athletic performance or to
assist with weight loss. Some of these substances are obtainable by prescription or by illegal
means; others are marketed as supplements, vitamins, or minerals. Nutritional supplements
are protected from Food and Drug Administration regulation by the 1994 US Dietary
Supplement Health and Education Act, and manufacturers are not required to demonstrate
proof of efficacy or safety. Furthermore, the Food and Drug Administration lacks a regulatory
body to evaluate such products for purity. Existing scientific data, which consist of case
reports and clinical observations, describe serious cardiovascular adverse effects from use of
performance-enhancing substances, including sudden death. Although mounting evidence
led to the recent ban of ephedra (ma huang), other performance-enhancing substances
continue to be used frequently at all levels, from elementary school children to professional
athletes. Thus, although the potential for cardiovascular injury is great, few appropriately
1049
designed studies have been conducted to assess the benefits and risks of using
performance-enhancing substances. It was performed an exhaustive OVID MEDLINE search
to Identify all existing scientific data, review articles, case reports, and clinical observations
that address this subject. In a review, it was examined the current evidence regarding
cardiovascular risk for persons using anabolic-androgenic steroids including 2 synthetic
substances, tetrahydrogestrinone and androstenedione (andro), stimulants such as ephedra,
and nonsteroidal agents such as recombinant human erythropoietin, human growth
hormone, creatine, and beta-hydroxy-beta-methylbutyrate [05044].
A 36 year old competitive bodybuilder presented with increasing dyspnoea on exertion over a
six week period. He gave a 10 year history of use of anabolic steroids, growth hormone,
ephedrine, and thyroxine. Echocardiography demonstrated severe left ventricular
hypertrophy and systolic dysfunction. Serum ferritin was normal and there was no serological
evidence of viral infection or connective tissue disease. Angiography revealed normal
coronary arteries and cardiac magnetic resonance imaging (CMR) was performed to further
investigate the cause of the cardiomyopathy. The left ventricle, shown here in end diastole
(panel A) was noted to be severely hypertrophied (myocardial mass 465 g; normal range 85–
181 g), dilated (end diastolic volume 319 ml; normal range 102–235 mL), and systolic
function was severely impaired (ejection fraction 20 %). Imaging post administration of
gadolinium-DTPA was negative for late enhancement (panel B), excluding both myocardial
infarction and macroscopic evidence of myocardial fibrosis. Initial treatment has been
commenced with a diuretic, angiotensin converting enzyme inhibitor, β blocker, and
anticoagulation. Growth hormone excess has been associated with left ventricular
hypertrophy while anabolic steroids have been associated both with myocardial hypertrophy,
focal myocardial fibrosis, and premature myocardial infarction. Thyroxine may cause high
output cardiac failure. CMR is the non-invasive investigation of choice in unexplained heart
failure. This case illustrates that severe heart failure can occur in patients taking these
performance enhancing drugs without CMR evidence of either myocardial infarction or
myocardial fibrosis [05045].
Anabolic-androgenic steroids are synthetic derivatives of testosterone that some athletes

have used to enhance muscle mass and improve their athletic performance. Ephedrine is a
potent sympathomimetic agent that can lead to cardiomyopathy similar to that seen with
catecholamine excess. Adverse cardiovascular events attributed to anabolic steroid and
ephedra use, such as arrhythmias, myocardial infarction, cardiomyopathy, and sudden
death, are rarely reported. Bodybuilders have used gamma-hydroxybutyrate, a potent
secretagogue of growth hormone, to promote muscle development. Although dilated
cardiomyopathy is a known complication of excess growth hormone levels, it has not been
associated with use of gamma-hydroxybutyrate. A healthy 40-year-old man was admitted to
our hospital for new-onset congestive heart failure and severe acute hepatitis that developed
several months after he began using anabolic-androgenic steroids, ephedra, and gamma-
hydroxybutyrate supplements. Analysis with an objective causality assessment scale
revealed a probable adverse drug reaction between the patient's use of anabolic steroids,
ephedra, and gamma-hydroxybutyrate and the development of his cardiomyopathy and
acute liver injury [05046].
Non-therapeutic use of androgenic anabolic steroids are administered in supraphysiological

doses to enhance the development of muscle mass and strength and to reduce the recovery
time after strenuous training bouts. These doses are however associated with pathologic
changes in numerous physiological systems. Studies using rats have shown that
supraphysiological doses of anabolic steroids cause pathophysiological myocardial
hypertrophy in this model. In the mouse it has been associated with inadequate
vascularisation of the hypertrophied myocardium, and in isolated rat ventricular myocytes it
1050
has been linked to increased apoptosis. When combined with exercise, anabolic steroid use
has been shown to change exercise-induced physiological cardiac hypertrophy to
pathophysiological cardiac hypertrophy. In one study using the exercising rat, the anabolic
steroid-induced changes in myocardial hypertrophy were associated with changes in the ratio
of the left ventricular wall thickness to internal radius. These changes are thought to lead to
detrimental increases in LV wall stress and to act as one of the stimuli for abnormal heart
growth and development. It was hypothesized that AAS use increases myocardial
susceptibility to ischaemia-reperfusion injury. Rats were trained (swimming) with or without
intramuscular injection of nandrolone laurate (0.375 mg/kg). Untrained rats with or without
nandrolone served as controls. Hearts were mounted on the Langendorff perfusion
apparatus and mechanical function was measured before and after 20-min normothermic
global ischaemia. Myocardial tissue samples were collected for determination of tissue cyclic
nucleotide and TNFalpha concentrations. Anabolic steroids significantly decreased the rate
pressure product (RPP) of the exercise-trained rat heart. Reperfusion RPP was lower in both
the sedentary, and the exercise-trained, steroid-treated hearts than in their concurrent
vehicle-treated controls. Myocardial TNFalpha and cAMP concentrations were elevated in
the steroid-treated hearts when compared with their untreated counterparts. It was concluded
that supraphysiological doses of anabolic steroids, whether taken during exercise training or
under sedentary conditions increase myocardial susceptibility to ischaemia/reperfusion injury
in our model. This increased susceptibility may be related to steroid-induced increases in the
pre-ischaemic myocardial cAMP concentrations and/or increases in both pre-ischaemic and
reperfusion TNFalpha concentrations [05047].
Testosterone deficiency is highly prevalent in men with cardiovascular disease (CVD) and is
associated with an increased mortality. Low testosterone also has an adverse effect on
several cardiovascular risk factors, which include insulin resistance, diabetes, dyslipidaemia,
central adiposity and endothelial dysfunction. Male gender is a well-recognised risk factor for
premature CVD and mortality. The question of whether or not testosterone deficiency is a
contributory factor to atherogenesis or merely a biomarker of ill health arises. Animal studies
and experiments on isolated cells indicate that many of the mechanisms intimate to the
atherosclerotic process are beneficially modulated by testosterone. Epidemiological studies
have shown that men with endogenous testosterone levels in the mid-upper normal range
have reduced cardiovascular events and mortality compared to those with low or lower
range, and with high range testosterone. Testosterone replacement in men diagnosed with
hypogonadism where mid-normal range levels are achieved have shown a beneficial effect
on several cardiovascular risk factors, cardiac ischaemia, functional exercise capacity and
improved mortality. Yet studies where patients were either undertreated or given high-dose
testosterone have been associated with an increased risk of cardiovascular-related events.
Clinical monitoring and titration of testosterone dose is therefore of paramount importance
[14247].
One review evaluated the documented cardiovascular functioning among anabolic-

androgenic steroid (AAS) users. AAS users manifest a reduction in HDL cholesterol,
increased inflammatory markers, and oxidative stress. Adverse cardiovascular effects have
thus been reported with the use of AAS, such as dyslipidemia, high blood pressure,
cardiovascular remodeling, and deaths from heart attacks and cerebral vascular accident
Strong evidence associating AAS use with blood pressure at hypertensive levels, as well as
hypertrophy and cardiac dysfunction has also been reported. Both epidemiological and
autopsy studies attest the relationship between AAS use and early mortality. There are a
reasonable number of studies reporting potential adverse cardiovascular effects, while
studies indicating an absence of adverse effects are not reported [14247].
For decades, individual case reports or small case series have described a variety of
1051
cardiovascular effects, including cardiomyopathy, myocardial infarction, cerebrovascular
accidents, conduction abnormalities, and coagulation abnormalities, in known or suspected
AAS users. More recently, larger controlled studies, using a variety of methodologies, have
supported these findings. In a recent postmortem pathologic study, comparing 87 deceased
men testing positive for AAS with 173 control men, AAS users exhibited significantly greater
cardiac mass even after adjusting for body mass, age, and history of trauma. Another
pathologic study found ventricular hypertrophy, associated with fibrosis and myocytolysis,
after cardiac death in 4 AAS users. Recent conduction studies have demonstrated
decreased cardiac electrical stability, abnormal tonic cardiac autonomic regulation, and
ventricular repolarization abnormalities in AAS users; the last finding has also been
demonstrated in rats that received AAS. Perhaps most importantly, numerous recent
controlled studies (using echocardiography or cardiac magnetic resonance imaging to
compare AAS users with non-AAS-using athletes and/or nonathletes) have demonstrated
cardiomyopathy in AAS users, characterized by decreased ventricular ejection fractions and
reduced diastolic tissue velocities. One study also found decreased aortic elasticity in AAS
users. These changes may be profound but may be at least partially reversible after AAS
abstinence. However, loss of tissue elasticity appears likely due at least in part to increased
fibrotic content resulting from direct AAS-induced cellular injury and hence may be
irreversible [14426].
In addition to their direct effects on cardiac tissue, AAS cause dyslipidemia, characterized by
decreased high-density lipoprotein cholesterol (HDL-C) and increased low-density lipoprotein
cholesterol (LDL-C) – an established risk profile for atherosclerotic disease. This effect is
particularly associated with orally administered 17alpha-alkylated AAS. One imaging study of
14 professional weightlifters with long-term AAS exposure found coronary-artery calcium
scores much higher than expected for men of comparable age. Atherosclerotic coronary
disease may contribute to many of the cases of myocardial or cerebral infarction reported in
young men with known or suspected AAS use [14426].
In recent years the abuse of AAS has been associated with the occurrence of serious
cardiovascular events in healthy young athletes, including the development of
cardiomyopathy, atrial fibrillation, QT dispersion, cerebrovascular accident, myocardial
infarction, disturbances of the haemostatic system, ventricular thrombosis and systemic
embolism, and acute heart failure.Moreover, several reports associated AAS abuse with
cardiac sudden death.[ Although these reports must be interpreted with caution, they teach
us to look thoroughly at the different mechanisms in which AAS abuse may affect the
cardiovascular system. However, again, it should be remembered that in case reports the
most dramatic side effects are often described and that they do not prove a causal
relationship between AAS abuse and the disease condition or cardiac death [04002].
Several AAS-induced adverse cardiovascular effects have been reported, including

hypertension, left ventricular hypertrophy (LVH), impaired diastolic filling, arrhythmia,
erythrocytosis, altered lipoprotein profile, and thrombosis. Although the incidence of AAS-
induced adverse cardiovascular events is unknown, surgeons should be aware of their
potential for increasing the perioperative risk in athletes using AAS who are undergoing
elective surgery [04018].
Among the numerous documented toxic and hormonal effects of AAS, attention has been
focused especially on the cardiovascular effects during recent years. Increases in blood
pressure and peripheral arterial resistance are known from experimental studies, but there
are also effects on the heart muscle, primarily left ventricular hypertrophy with restricted
diastolic function. Severe cardiac complications such as cardiac insufficiency, ventricular
fibrillation, ventricular thromboses, myocardial infarction, or sudden cardiac death in
1052
individual strength athletes with acute AAS abuse have also been reported. In almost all
studies, acute side effects were examined only during AAS intake or within weeks to a few
months of their discontinuance. However, the extent to which these effects are reversible
after discontinuing intake of these agents and the degree to which they leave permanent
impairment are still controversial matters. In former Finnish world class powerlifters
suspected of AAS intake during their sports career, a 4.6 times higher mortality was reported
compared with a control group of 1094 men. However, the very small number of deaths (only
four cardiovascular deaths in the 62 athletes) restricted the validity of this study in
determining whether there was a higher cardiac risk in AAS users. To investigate the
reversibility of adverse cardiovascular effects after chronic abuse of anabolic androgenic
steroids (AAS) in athletes Doppler echocardiography and cycle ergometry including
measurements of blood pressure at rest and during exercise were undertaken in 32
bodybuilders or powerlifters, including 15 athletes who had not been taking AAS for at least
12 months (ex-users) and 17 currently abusing AAS (users), as well as in 15 anabolic-free
weightlifters. The ergometric performance in our subjects was within the normal range for
untrained persons of the same age, even when considering the moderate exhaustion of the
weightlifters (with a lower maximum heart rate towards the end of exercise). The typical
training in bodybuilding and weightlifting, including the moderate endurance training on a
cycle ergometer done by most of the athletes, does not result in significant increases in cycle
ergometry performance. Systolic blood pressure was higher in users (140 + 10 mmHg) than
in ex-users (130 + mmHg) or weightlifters (125 + 10 mm Hg). The results suggest that the
increases in blood pressure with AAS use are rather small and transient. Increased blood
pressure values or a reduced fall in blood pressure during sleep have been described with
AAS use, but not in all studies. These discrepancies probably reflect different preparations,
dosages, and intake cycles. It has been reported that five months after discontinuing AAS
intake, systolic blood pressure remained higher by 6 mm Hg at rest compared with an
anabolic-free control group. In animal experiments an increase in peripheral resistance was
still detectable six weeks after AAS use. Left ventricular muscle mass related to fat-free body
mass and the ratio of mean left ventricular wall thickness to internal diameter were not
significantly higher in users than in ex-users, but were lower in weightlifters. Left ventricular
wall thickness related to fat-free body mass was also lower in weightlifters, but did not differ
between users and ex-users. Left ventricular wall thickness was correlated with a point score
estimating AAS abuse in users. In all groups, systolic left ventricular function was within the
normal range. The maximum late transmitral Doppler flow velocity (Amax) was higher in
users than in weightlifters. The reversible ST segment changes observed in one user after
discontinuation of AAS use could be related to AAS intake. Only individual case descriptions
of ECG changes with AAS abuse are found in published reports. In a previous investigation,
ECG abnormalities could not be established. It was concluded that several years after
discontinuation of anabolic steroid abuse, strength athletes still show a slight concentric left
ventricular hypertrophy in comparison with AAS-free strength athletes [04056].
Particularly in athletes it is important to consider differences of fat-free body mass when

comparing echocardiographic measures. The left ventricular muscle mass and wall
thicknesses values of ex-users relative to fat-free body mass were similar to those of users.
The weightlifters group, however, showed significantly lower values. This suggests not only a
disproportionate increase in left ventricular muscle mass with AAS, but also residual left
ventricular hypertrophy more than one year after discontinuing AAS intake. Increases in left
ventricular muscle mass with AAS are well documented in strength athletes (with case
reports of typical hypertrophic cardiomyopathy) as well as in animals, where an increased
protein synthesis has been shown. Androgen receptors are known to be present in human
myocardial tissue. Intracellular oedema and mitochondrial swelling in myocytes could also
play a role in hypertrophy. In case reports, reversibility of significantly increased left
ventricular muscle mass has been described after discontinuation of AAS. Other studies
1053
suggest that, relative to body dimensions, AAS have a long lasting disproportionately
hypertrophic effect on the myocardium, as former users still show an increase in left
ventricular muscle mass four to six weeks or nine months after discontinuing these agents.
The results suggest that this effect is maintained for an even longer time. The extent to which
increased left ventricular muscle mass caused by AAS abuse represents a long term risk for
cardiac complications is controversial. The correlation between left ventricular muscle mass
and cardiovascular mortality that is suggested by epidemiological evidence may be
transferable to athletes only with caution. An increased left ventricular muscle mass of up to
170 g/m2 can be found in healthy highly endurance trained athletes. Left ventricular wall
thicknesses of 13–16 mm have been described in individual athletes with large body
dimensions involved in combined strength-endurance sports, such as rowing. In contrast to
those athlete’s hearts, clinical hypertrophic cardiomyopathy – even with endurance training –
is always associated with a rather small internal left ventricular diameter of less than 48-50
mm. The higher ratio of wall thickness to internal diameter in ex-users and users underlines
the assumption that there is a slight degree of concentric left ventricular hypertrophy in AAS
users, even more than one year after discontinuing the intake of these agents. Today, most
investigators agree that strength training without AAS intake does not induce concentric left
ventricular hypertrophy. Previous echocardiographic data in athletes taking AAS are less
conclusive. Some investigators describe significant wall thickening compared with steroid-
free strength athletes, with regression after eight weeks off treatment or no change after nine
weeks. Other studies, however, report only non-significant differences or no differences
between strength athletes using or not using AAS. Ex-users lie between the non-users
(weightlifters) and users with respect to left ventricular muscle mass, wall thicknesses, and
hypertrophic index values. This could suggest that the hypertrophic effect of AAS decreases
over the years. In the study, left ventricular systolic function in the users was still within the
normal range, in agreement with other investigators’ findings. An impairment of systolic left
ventricular function in animals has been shown with AAS. Impairment of diastolic left
ventricular function with AAS use is not unequivocal. These discrepancies might be explained
by different methods used to assess the diastolic function as well as by variations in the dose
of AAS. In the present study there was a higher maximum late transmitral flow velocity and at
least a tendency towards a lower E/Amax ratio in ex-users and users compared with
weightlifters, which might suggest a relaxation disturbance. Fibrosis of the myocardium, as
described in the case of anabolic abuse, could be responsible for this. The greater age of ex-
users should be considered, because diastolic function is known to decline with age;
however, the differences found were greater than could be expected from a difference in age
of 10 years and persisted after correction for age. The E/Amax ratio on Doppler
echocardiography depends on many other factors, especially preload and afterload
conditions, and this makes it difficult to draw definitive conclusions about diastolic left
ventricular function from conventional transmitral echocardiography [04056].
Anabolic steroid abuse in athletes has been associated with a wide range of adverse
conditions, including hypogonadism, testicular atrophy, impaired spermatogenesis,
gynaecomastia, and psychiatric disturbance. But what effect does steroid abuse have on the
cardiovascular system? Left ventricular hypertrophy (LVH) independently predicts
cardiovascular mortality and morbidity across diverse disease states. While cardiac diastolic
or contractile failure might result directly from structural change within the ventricle (such as
altered capillary density or matrix deposition), the association of LVH with cardiovascular
disease is more likely dependent upon the increased activity of shared physiological
pathways driving both processes. The nature of these underlying mechanisms remains
poorly understood. In this regard, escalating attention has focused on the potential role of
steroid hormones on LV growth responses. Whether of local or systemic origin, endogenous
steroid hormones appear to drive LV growth. Systemic glucocorticoid excess is associated
with significant hypertrophy. This action is more likely to be direct, rather than mediated
1054
through an elevated pressor burden, with aldosterone having similar effects. Local
myocardial renin-angiotensin systems (RAS) play a role in regulating LV growth, and at least
part of the hypertrophic responses to steroid hormones may be mediated through
upregulation of local RAS expression. Anabolic/androgenic steroids are likely to share such
influences on the LV hypertrophic response through actions on the androgen receptor (AR),
a transcriptional regulator. Indeed, ARs are almost ubiquitously expressed, being found not
only in skeletal muscle cells, but also on cardiac myocytes. Several lines of evidence also
implicate endogenous androgenic pathways in the development of cardiac hypertrophy,
including the demonstration of raised 5alpha reductase, aromatase, and AR expression in
hypertrophic hearts of both humans and mice [04057].
Given these putative effects of steroid hormones (and AAS in particular) on LV growth, it
might be expected exposure to exogenously administered steroid hormones to be associated
with an exaggerated LV hypertrophic response to any other hypertrophic stimulus. Exercise
is just such a potent cardiac hypertrophic stimulus. Meanwhile, athletes are increasingly
exposing themselves to supra-physiological doses of AAS. These are known to increase
skeletal muscle mass and strength – effects which form the basis for their administration to
enhance athletic performance. A variety of AAS are often taken simultaneously (so called
“stacking”), and in doses which result in 10-100 fold increases in androgen concentrations.
Administration regimens usually involve a 6-12 week cycle and are often administered in a
“pyramidal” fashion, with doses tapering from low to high to low. Abused substances include
testosterone, its 17-beta esters, and those based on modified steroid rings (including 17-
alpha derivatives). The largest group to make such use of AAS are the very group whose
LVH response to exercise is likely to be the greatest – the strength or resistance training
(RT) athletes. One study from 1995 suggested that two thirds of elite US powerlifters have
self reported use of AAS to enhance performance; even “dope testing” may be
underestimating the true extent of such use. What evidence is there that AAS administration
enhances the LV hypertrophic response to resistance exercise? Male bodybuilders and
powerlifters currently using AAS or ex-users who had abstained from AAS exposure for over
12 months (U and ExU, n= 17 and 15, respectively) were compared to 15 weightlifters who
denied current or past use of AAS (WL). Left ventricular wall thickness and cavity dimensions
were assessed using echocardiography, and muscle mass (LVMM) calculated using the
Devereux equation. Absolute LVMM measures were significantly greater for U than ExU or
WL, with differences between ExU and WL only reaching significance after adjustment for
body surface area or fat-free mass. These results suggest that AAS use increases the LV
hypertrophic response to exercise, an effect which might last for well over a year [04057].
Such data must nonetheless be treated with caution. It is known, for example, that the
magnitude and pattern of hypertrophy is dependent on the nature, duration, and intensity of
exercise undertaken. Thus, strength trained athletes (such as weightlifters, powerlifters,
bodybuilders, and throwers) develop a greater increase in wall thickness, a more concentric
pattern of LV growth, and a lesser increase in LV chamber internal dimensions 8 in
comparison to those undergoing predominantly aerobic/endurance exercise. In the study
under discussion, training patterns will have varied. One might suspect that subjects taking
AAS were also the most motivated to train (whether by initial predisposition, or psychological
impact of the steroid use itself). However, this does not seem to be the case as the authors
report that the magnitude of training did not differ between U, ExU, and WL groups. Even so,
more subtle differences in training pattern may have existed between bodybuilders,
powerlifters, and weightlifters. Although all groups lift exceptionally heavy weights, the total
load and training pattern are likely to differ. Other factors may also have been of influence.
Diet (including the use of supplements) may have differed between groups, as might the use
of other agents. Abusers of AAS frequently also self administer other drugs including
stimulants, antioestrogens, human chorionic gonadotrophin (hCG), and human growth
1055
hormone (hGH). It is unclear to what extent these and other drugs might have driven LV
growth, and whether the ExU group were still taking any of these. Neither can mechanistic
inferences be drawn from the data: the putative effects of AAS on LV growth may have been
mediated directly, or through secondary phenotypes such as alterations in circulating volume
or blood pressure. Certainly, resting systolic blood pressure is higher in the U v ExU group, a
difference which persists as a trend for exercising blood pressure. The use of such drugs (as
well as differences in patterns of training) may also have influenced fat-free mass and body
surface area. The adjustment for such anthropometric measures may have contributed to the
significance of the comparison between ExU and WL [04057].
If AAS use is associated with an exaggerated LV hypertrophic response to training, what are
the likely health implications? They may be profound. In terms of non-cardiac morbidity, AAS
use is associated with hypogonadism, testicular atrophy, impaired spermatogenesis,
baldness, acne, gynaecomastia, and psychiatric disturbance. Such drugs also have toxic
effects on metabolic profile and hepatic structure and function, as well as potentially
promoting neoplastic growth. Indeed, Parsinnen reported the 12 year mortality to be 13 per
1000 among 62 male powerlifters suspected of AAS use, compared to 3.1 percent in a
control population. LVH is thus an independent risk factor for cardiovascular mortality and
(through whatever mechanism) one might anticipate an excess cardiovascular mortality
among AAS users in whom LVH occurs. In addition, the recognised association of AAS use
with hypertension and dislipidaemia (raised low density lipoprotein and reduced high density
lipoprotein cholesterol, and raised triglycerides), as well as influences on coagulation and
platelet aggregation, might increase such risk. While it is debatable whether ASS use is
indeed associated with an increased risk of premature cardiovascular death, 38 percent of
the deaths in Parssinen’s powerlifting group were attributed to “myocardial infarction”, while
several case reports have attributed myocardial infarction in athletes to ASS abuse. In some
cases, infarction has occurred without evident coronary thrombosis or atherosclerosis,
leading to the hypothesis that ASS may induce coronary vasospasm in susceptible
individuals. Similarly there are several case reports of increased thromboembolic risk. In a
recent postmortem series of 34 AAS abusers aged 20-45 years (comprising 12 homicides,
11 suicides, 12 “accidental” deaths, and two of indeterminate cause), 12 of the deceased
showed cardiac pathology. Findings included hypertrophy (7 cases), myocardial or
endocardial fibrosis (5), cardiac steatosis (1), myocardial coagulation necrosis (2), and
coronary atheroma (4). Cardiac changes were adjudged to have contributed to death by
poisoning in two cases. However mediated, such a morbid burden is likely to rise with time
[04057].
Power athletes abuse anabolic androgenic steroids (AASs) and growth hormone (GH) to
gain their muscular mass and strength. We wanted to determine how massive, self-
administered doses of AASs with or without GH affect the left ventricular (LV) dimensions in
power athletes. These substances are assumed to increase LVmass mainly by thickening
the ventricular walls. Anecdotal evidence suggests a higher risk of cardiovascular events in
AAS abusers. We were interested to see if LV dimensions and function in AAS abusers
would indicate this increased risk. Twenty healthy male power athletes using massive doses
of AAS without (n=16) or with (n=4) GH volunteered for the study. The controls were 15
sedentary male non-users of hormones. LV mass, geometry and filling were studied using
standard echocardiographic methods. It was found a significant association between LV
mass and AAS dose. In contrast to the controls, LV mass (274 g in the athletes, 167 g in the
controls) among the AAS abusers did not correlate with body weight or height. Concomitant
use of AAS and GH further increased LV mass and associated with concentric remodelling of
LV. Multiple regression analysis indicated that the mean AAS dose accounted for 29 percent,
age for 14 percent and systolic blood pressure for 17 percent of the variance in LV mass. It
was concluded that AAS abuse associates dose-dependently with myocardial hypertrophy
1056
and that concomitant use of GH associates with concentric remodelling of the LV. The
findings suggest that AASs and GH have a direct effect on the myocardium [03046].
Cardiac arrhythmias are among the most important causes of non-eligibility to sports
activities, and may be due to different causes (cardiomyopathies, myocarditis, coronary
abnormalities, valvular diseases, primary electrical disorders, abuse of illicit drugs). The list
of illicit drugs banned by the International Olympic Committee and yearly updated by the
World Anti-Doping Agency includes the following classes: stimulants, narcotics, anabolic
agents (androgenic steroids and others such as beta-2 stimulants), peptide hormones,
mimetics and analogues, diuretics, agents with an antiestrogenic activity, masking agents.
Almost all illicit drugs may cause, through a direct or indirect arrhythmogenic effect, in the
short, medium or long term, a wide range of cardiac arrhythmias (focal or reentry type,
supraventricular and/or ventricular), lethal or not, even in healthy subjects with no previous
history of cardiac diseases. Therefore, given the widespread abuse of illicit drugs among
athletes, in the management of arrhythmic athletes the cardiologist should always take into
consideration the possibility that the arrhythmias be due to the assumption of illicit drugs
(sometimes more than one type), especially if no signs of cardiac diseases are present. On
the other hand, in the presence of latent underlying arrhythmogenic heart disease including
some inherited cardiomyopathies at risk of sudden cardiac death, illicit drugs could induce
severe cardiac arrhythmic effects [03047].
Since the abuse of androgenic-anabolic steroids (AAS) has been associated with the
occurrence of serious cardiovascular disease in young athletes, we performed two studies to
investigate the effects of short-term AAS administration on heart structure and function in
experienced male strength athletes, with special reference to dose and duration of drug
abuse. In Study 1 the effects of AAS were assessed in 17 experienced male strength
athletes (age 31) who self-administered AAS for 8 or 12-16 weeks and in 15 non-using
strength athletes (age 33) in a non-blinded design. In Study 2 the effects of administration of
nandrolone decanoate (200 mg/wk i. m.) for eight weeks were investigated in 16
bodybuilders in a randomised double blind, placebo controlled design. In all subjects M-mode
and two-dimensional Doppler-echocardiography were performed at baseline and after 8
weeks AAS administration. In the athletes of Study 1 who used AAS for 12-16 weeks a third
echocardiogram was also made at the end of the AAS administration period.
Echocardiographic examinations included the determination of the aortic diameter (AD), left
atrium diameter (LA), left ventricular end diastolic diameter (LVEDD), interventricular septum
thickness (IVS), posterior wall end diastolic wall thickness (PWEDWT), left ventricular mass
(LVM), left ventricular mass index (LVMI), ejection fraction (EF) and right ventricular diameter
(RVD). For assessment of the diastolic function measurements of E and A peak velocities
and calculation of E/A ratio were used. In addition, acceleration and deceleration times of the
E-top (ATM and DT, respectively) were determined. For evaluation of factors associated with
stroke volume the aorta peak flow (AV) and left ventricular ejection times (LVET) were
determined. In Study 1 eight weeks AAS self-administration did not result in changes of
blood pressure or cardiac size and function. Additionally, duration of AAS self-administration
did not have any impact on these parameters. Study 2 revealed that eight weeks
administration of nandrolone decanoate did not induce significant alterations in blood
pressure and heart morphology and function. Short-term administration of AAS for periods up
to 16 weeks did not lead to detectable echocardiographic alterations of heart morphology
and systolic and diastolic function in experienced strength athletes. The administration
regimen used nor the length of AAS abuse did influence the results. Moreover, it is
concluded that echocardiographic evaluation may provide incomplete assessment of the
actual cardiac condition in AAS users since it is not sensitive enough to detect alterations at
the cellular level. Nevertheless, from the present study no conclusions can be drawn of the
cardiotoxic effects of long term AAS abuse [03048].
1057
Chronic abuse of anabolic steroids is widespread. Hypertrophy of skeletal and heart muscle
is a well-known effect of chronic anabolic steroid abuse. Structural alterations of blood
vessels are new side effects. It was reported a case of a 32-year-old bodybuilder after long-
term use of anabolic steroids who died of cardiac arrest. Coronary angiography and autopsy
findings showed especially a hypertrophic heart, structural changes of coronary arteries,
intracoronary thrombosis and myocardial infarction, ventricular thrombosis and systemic
embolism [03049]
Previous investigations reported alterations in myocardial fibres and systolic function

associated with anabolic-androgenic steroid consumption by athletes. Advances in bio-
medical technology have allowed further investigation in assessing the possible effects of
anabolic-androgenic steroids on gross left ventricular kinetics. Twenty-three male strength
and power athletes with a past and current history of anabolic-androgenic steroid
consumption (x 46 days, range 28 days to 70 days), were compared to 23 controls. Testing
consisted of resting and immediate post-exercise transthoracic left ventricular wall
cardiokymograms. Statistical results identified no difference over time between groups or
condition. Cardiokymographic waveform analysis found 33 percent of all (n=184) waveforms
to be abnormal (Type II, n=56 or Type III, n=4). There were 14 treatment subjects (61 %)
who demonstrated an abnormal waveform as compared to 9 controls (39 %). A significant
difference in the overall proportions of waveform types was identified where the treatment
group exhibited 41 percent abnormal waveforms, compared to 24 percent by controls.
Additionally, two athletes (1 treatment, 1 control) demonstrated abnormal left ventricular wall
motions (Type III) analogous to impaired left ventricular performance. The results indicated:
highly strength trained athletes with no history of anabolic-androgenic steroid usage
exhibited an unexpected high incidence of Type II waveforms (28 % pre/24 % post); and a
comparable group of strength trained athletes using anabolic-androgenic steroids exhibited a
significantly higher percentage of abnormal waveforms as compared to controls (35 % pre/37
% post). Based on these results, high intensity strength training with and without anabolic-
androgenic steroid supplementation induced alterations in the left ventricular wall motion
[03050].
Explanatory models
The cardiovascular system may be affected via at least four different pathways. Although
hypothetical, they provide interesting models to explain AAS-induced adverse effects on this
system [04002]:
- The atherogenesis model. The atherogenesis model is based on the association
between AAS and HTGL, an enzyme that regulates serum lipids and lipoproteins.
AAS administration enhances HTGL activity that decreases regression of
atherosclerotic plaques by suppression of serum HDL-cholesterol and elevation of
LDL-cholesterol
- The thrombosis model. The thrombosis model is characterised by influence on the

haemostatic system, with the strongest effects of AAS on platelet aggregation that
results in enhanced blood-clot formation, including an increased cardiovascular risk
- The coronary artery vasospasm model. Since no evidence of atherosclerosis or

thrombosis of the coronary arteries was involved in several reports of sudden cardiac
death, nitric oxide has been suggested to play a role in the third model, the coronary
artery vasospasm model. Nitric oxide acts as an endothelial-derived relaxing factor in
smooth muscles of arteries. AAS may inhibit nitric oxide properties and may induce
1058
vasospasm, although the authors suggested that other models may be involved in
conjunction with the vasospasm theory. The latter is supported by recent findings in
animal studies. It was demonstrated that AAS may impair capillary supply of the heart
as a result of an increase in myocardial muscle mass and a relative decrease in
capillary density, which may provoke compression of coronary vessels that could
trigger myocardial infarction
- The direct injury model. The fourth hypothesis is the direct injury model. AAS is
hypothesised to induce direct myocardial cell injury, leading to myocardial cell death
and replacement of dead cells by scar tissue within the myocardium. Development of
fibrotic areas predisposes to arrhythmias, which exposes the individual to an
increased risk of fatal events. Postmortem pathological findings in previous AAS
users included focal, regional, interstitial and disseminated fibrosis of the
myocardium, although the impact of myocardial fibrosis is still unclear
- Other models. Several other hypotheses have been postulated, especially those
affecting red blood cells and volume. It has been suggested that cardiac arrest may
be mediated catecholamine myotoxicity associated with ventricular fibrillation due to
myocardial necrosis and degenerative changes within the intramyocardiac
sympathetic neurons. All mechanisms proposed for explaining cardiovascular disease
due to AAS use have interesting points; however, future research is needed to clarify
the relevance of each theory.
Experimental
The aim of one study was the investigation of effects of the metenolone enanthate (ME) that
is used among athletes as doping and muscle amplifier, on hearts of male and female rats
that are in puberty using morphometrical methods. A total of 36 rats which were divided into
three separate groups (Experiment, ME; vehicle, PO; control, C) each consisting of 6 male
and 6 female rats were used. 0.5mg/kg metenolone enanthate was applied intraperitoneally
into experiment subjects 5 times a week over a period of 4 weeks. At the end of experiment,
rats were euthanized and their hearts were cut at the level of musculus papillaris after the
fixation in formalin. Hearts were taken out and embedded in paraffin wax. Photos were taken
at cut surfaces, and thickness, diameters and surface area levels were measured. Left
ventriculus mass (LVM) and left ventriculus mass index (LVMI) were calculated. In the study
LVM and LVMI were found to be significantly higher in the ME group in females whereas left
ventricular lumen diameter (LVLD) were found to be significantly lower. Thus left ventricular
hypertrophy development was observed. LVM and LVMI were found to be similar in ME and
C groups among male rats and the highest level of these data were found in the group. LVM
and LVMI were higher among females. In conclusion, it has been shown that the adverse
effects of ME on heart were developing starting from puberty and resulting with the
enlargement of the heart and left ventricular hypertrophy and especially among females this
condition was more evident. It has also been discussed that the continuous use of drugs may
further enhance this condition [12127].
Cardiac aldosterone might be involved in nandrolone decanoate (ND) deleterious effects on

the heart. Therefore, we investigated the involvement of cardiac aldosterone, by the
pharmacological block of AT1 or mineralocorticoid receptors, on cardiac hypertrophy and
fibrosis. Male Wistar rats were randomized into 8 groups (n=14/group). Nandrolone (10
mg/kg/week), was administered during 10 weeks of swimming training (5 times/wk). Losartan
(20 mg/kg/day) and spironolactone (10mg/kg/day) were administered in drinking water.
Cardiac hypertrophy was increased 10 percent by using nandrolone and 17 percent by
nandrolone plus training. In both groups, there was a significant increase in the collagen
1059
volumetric fraction (CVF) and cardiac collagen type III expression. The nandrolone treatment
increased: LV-ACE activity, AT1 receptor expression, aldosterone synthase (CYP11B2) and
11-beta hydroxysteroid dehydrogenase 2 (11betaHSD2) gene expression and inflammatory
markers, TGFbeta and osteopontin. Both losartan and spironolactone inhibited the increase
of CVF and collagen type III. In addition, both treatments inhibited the increase in LV-ACE
activity, CYP11B2, 11betaHSD2, TGFbeta and osteopontin induce by the nandrolone
treatment. The results suggest that these effects may be associated to TGFbeta and
osteopontin. Thus, it was conclude that the cardiac aldosterone has an important role on the
deleterious effects on the heart induced by nandrolone [11065].
To investigate the effects of exercise training and anabolic androgenic steroids (AAS) on
hemodynamics, glycogen content, angiogenesis, apoptosis and histology of cardiac muscle.
Forty rats were divided into 4 groups; control, steroid, exercise-trained and exercise-trained
plus steroid groups. The exercise-trained and trained plus steroid groups, after one week of
water adaptation, were exercised by jumping into water for 5 weeks. The steroid and trained
plus steroid groups received nandrolone decanoate, for 5 weeks. Systolic blood pressure
and heart rate (HR) were monitored weekly. Heart weight/body weight ratio (HW/BW ratio)
was determined. Serum testosterone, vascular endothelial growth factor (VEGF), cardiac
caspase-3 activity and glycogen content were measured. Compared with control, the steroid
group had significantly higher blood pressure, HR, sympathetic nerve activity, testosterone
level, HW/BW and cardiac caspase-3 activity. Histological examination revealed apoptotic
changes and hypertrophy of cardiomyocytes. In exercise-trained group, cardiac glycogen,
VEGF and testosterone levels were significantly higher while HR was significantly lower than
control. HW/BW was more than control confirmed by hypertrophy of cardiomyocytes with
angiogenesis on histological examination. Trained plus steroid group, had no change in HR,
with higher blood pressure and HW/BW than control, cardiac glycogen and serum VEGF
were higher than control but lower than exercise-trained group. Histological examination
showed hypertrophy of cardiomyoctes with mild angiogenesis rather than apoptosis. Thus,
when exercise is augmented with AAS, exercise-associated cardiac benefits may not be fully
gained with potential cardiac risk from AAS if used alone or combined with exercise [13129].
One study focused on the short term effects of repeated low level administration of turinabol
and methanabol on cardiac function in young rabbits (4 months). The experimental scheme
consisted of two oral administration periods, lasting 1month each, interrupted by 1 month
wash-out period. Serial echocardiographic evaluation at the end of all three experimental
periods was performed in all animals. Oxidative stress markers have also been monitored at
the end of each administration period. Treated animals originally showed significantly
increased myocardial mass and systolic cardiac output, which normalized at the end of the
wash out period. Re-administration led to increased cardiac output, at the cost though of a
progressive myocardial mass reduction. A dose-dependent trend towards impaired
longitudinal systolic, diastolic and global myocardial function was also observed. The
adverse effects were more pronounced in the methanabol group. For both anabolic steroids
studied, the low dose had no significant effects on oxidative stress markers monitored, while
the high dose created a hostile oxidative environment. In conclusion, anabolic administration
has been found to create a possible deleterious long term effect on the growth of the
immature heart and should be strongly discouraged especially in young human subjects
[13130].
High doses of anabolic androgenic steroids (AAS) impair the cardioprotective effects of
exercise against ischemia/reperfusion (I/R) insult, possibly through cellular redox imbalance.
Here, the effect of nandrolone decanoate (DECA) treatment on heart redox metabolism was
investigated during I/R in sedentary and exercised rats. DECA treatment significantly
reduced superoxide dismutase and glutathione reductase activities in exercised rats after
1060
heart reperfusion. Catalase and glutathione peroxidase activities were not affected by DECA
in both sedentary and trained rats, regardless the I/R period. DECA also induced myocardial
oxidative stress, as evidenced by the reduced levels of total reduced thiols after heart
reperfusion in exercised rats treated with the anabolic steroid. These results indicate that
cardiotoxic effects of supraphysiological doses of AAS involve reduced heart antioxidant
capacity [13131].
One study focuses on the short term effects of repeated low level administration of turinabol
and methanabol on cardiac function in young rabbits (4 months-old). The experimental
scheme consisted of two oral administration periods, lasting 1 month each, interrupted by 1-
month wash-out period. Serial echocardiographic evaluation at the end of all three
experimental periods was performed in all animals. Oxidative stress markers have also been
monitored at the end of each administration period. Treated animals originally showed
significantly increased myocardial mass and systolic cardiac output, which normalized at the
end of the wash out period. Re-administration led to increased cardiac output, at the cost
though of a progressive myocardial mass reduction. A dose-dependent trend towards
impaired longitudinal systolic, diastolic and global myocardial function was also observed.
The adverse effects were more pronounced in the methanabol group. For both anabolic
steroids studied, the low dose had no significant effects on oxidative stress markers
monitored, while the high dose created a hostile oxidative environment. In conclusion,
anabolic administration has been found to create a possible deleterious long term effect on
the growth of the immature heart and should be strongly discouraged especially in young
human subjects [13133].
For decades, individual case reports or small case series have described a variety of
cardiovascular effects, including cardiomyopathy, myocardial infarction (MI), cerebrovascular
accidents, conduction abnormalities, and coagulation abnormalities, in known or suspected
AAS users. Several recent reviews have summarized these reports. More recently, larger
controlled studies, using a variety of methodologies, have supported these findings. In a
recent postmortem pathological study, comparing 87 deceased men testing positive for AAS
with 173 control men, AAS users exhibited significantly greater cardiac mass even after
adjusting for body mass, age, and history of trauma. Another pathological study found
ventricular hypertrophy, associated with fibrosis and myocytolysis, after cardiac death in four
AAS users. Recent conduction studies have demonstrated decreased cardiac electrical
stability, abnormal tonic cardiac autonomic regulation, and ventricular repolarization
abnormalities in AAS users; the last finding has also been demonstrated in rats that received
AAS. Perhaps most importantly, numerous recent controlled studies (using echocardio-
graphy or cardiac magnetic resonance imaging (MRI) to compare AAS-users with non-AAS-
using athletes and/or nonathletes) have demonstrated cardiomyopathy in AAS users,
characterized by decreased ventricular ejection fractions and reduced diastolic tissue
velocities. One study also found decreased aortic elasticity in AAS users. These changes
may be profound, but may be at least partially reversible following AAS abstinence. However,
loss of tissue elasticity appears likely due at least in part to increased fibrotic content
resulting from direct AAS-induced cellular injury, and hence may be irreversible. In addition
to their direct effects on cardiac tissue, AAS cause dyslipidemia, characterized by decreased
high-density lipoprotein cholesterol (HDL-C) and increased low-density lipoprotein
cholesterol (LDL-C) – an established risk profile for atherosclerotic disease. This effect is
particularly associated with orally administered 17-alkylated AAS. One imaging study of 14
professional weightlifters with long-term AAS exposure found coronary-artery calcium scores
much higher than expected for men of comparable age. Atherosclerotic coronary disease
may contribute to many of the cases of myocardial or cerebral infarction reported in young
men with known or suspected AAS use [14045].
1061
Effects of methyltestosterone on myocardial function in vitro
Testosterone analogs have been used as performance enhancers by athletes for more than
40 years. It was asked whether the anabolic steroid 17 alpha-methyl-4-androstene-17-ol-3-
one (17 alpha-MT) would affect intrinsic contractile function of the heart. Male Sprague-
Dawley rats, 125-150 g, were treated with 17 alpha-MT either parenterally or orally for up to
8 wk. Intrinsic contractile function of the hearts was assessed utilizing both the isolated
working heart and isovolumic perfused heart preparations. Isolated working hearts from 17
alpha-MT-treated rats had a 45 percent decrease in heart work attributable largely to a
similarly decreased stroke volume. Isovolumic perfused hearts from treated animals had
elevated left ventricular systolic and diastolic pressures at similar interventricular volumes
compared to controls. Rates of ventricular pressure development (+dP/dT) or relaxation (-
dP/dT) were unchanged as a result of the treatment. However, static elastance was reduced
in potassium-arrested hearts from the 17 alpha-MT treatment (63 % increase in
interventricular pressure), consistent with a limitation being imposed on stroke volume by a
decreased myocardial compliance. Hydroxyproline content of the hearts was not altered by
17 alpha-MT treatment suggesting that increased stiffness was not a consequence of
collagen proliferation. Treatment of the steroid rats with beta-aminopropionitrile, a compound
that inhibits lysyl oxidase, restored the left ventricular volume-pressure relationship
(elastance curve) to that of control hearts. Thus, chronic treatment with anabolic steroids
appears to reduce left ventricular compliance, possibly related to an enhanced activity of
lysyl oxidase, and results in increased crosslink formation between collagen strands in the
extracellular matrix [00059].
Combined cardiac effects of cocaine and nandrolone in the rat

Despite reports of an increase in the incidence of simultaneous cocaine and anabolic steroid
abuse, potential adverse interactions between these two drugs on the cardiovascular system
are largely unquantified. Cocaine has been reported to induce coronary vasoconstriction,
cardiac arrhythmias and conduction delays. Anabolic steroids have been associated with
cardiac hypertrophy and hypertension. Utilising both in vivo (radiotelemetry) and in vitro
(isolated Langendorff-perfused heart) techniques, our aim was to determine whether
anabolic steroids cause cardiac hypertrophy and alter cardiac function, and consequently
alter the response of the heart to cocaine. It was found that 15 days of treatment of rats with
nandrolone decanoate (20 mg/kg, s.c.) was not sufficient to cause hypertrophy, alter cardiac
function or the spread of electrical activity through the heart. However, nandrolone
pretreatment was found to significantly potentiate the heart rate response to cocaine (45
mg/kg, i.p.) in vivo. The study indicates that nandrolone significantly elevates the heart rate
response to high dose cocaine without changing heart morphology. The mechanism of this
interaction remains uncertain [00060].
A national population-based cohort study
Non-therapeutic use of anabolic androgenic steroids (AAS) has been associated with various
adverse effects; one of the most serious being direct cardiovascular effects with unknown
long-term consequences. Therefore, large studies of the association between AAS and
cardiovascular outcomes are warranted. It was investigated cardiovascular morbidity and
mortality in individuals who tested positive for AAS. Between 2002 and 2009, a total of 2013
men were enrolled in a cohort on the date of their first AAS test. Mortality and morbidity after
cohort entry was retrieved from national registries. Of the 2013 individuals, 409 (20 %) tested
positive for AAS. These men had twice the cardiovascular morbidity and mortality rate as
those with negative tests (adjusted hazard ratio (aHR) 2.0; 95 % confidence interval (CI) 1.2
to3.3). Compared to the Swedish population, all tested men had an increased risk of
premature death from all causes (standardized mortality ratio for AAS-positive: 19.3, 95 % CI
1062
12.4 to 30.0; for AAS-negative: 8.3, 95 % CI 6.1 to 11.0). It was concluded that non-
therapeutic exposure to AAS appears to be an independent risk factor for cardiovascular
morbidity and premature death [150176].
Metabolic syndrome
Obesity is one of the constellations of factors that make up the definition of the metabolic
syndrome. Metabolic syndrome is also associated with insulin resistance, dyslipidemia,
hypertriglyceridemia, and type 2 diabetes mellitus. The presence of obesity and metabolic
syndrome in men and women is also associated with increased risk of cardiovascular
disease and hypertension. In men, obesity and metabolic syndrome are associated with
reductions in testosterone levels. In women, obesity and metabolic syndrome are associated
with increases in androgen levels. In men, reductions in androgen levels are associated with
inflammation, and androgen supplements reduce inflammation. In women, increases in
androgens are associated with increases in inflammatory cytokines, and reducing androgens
reduces inflammation [11093].
Coronary artery calcifications
The authors measured coronary artery calcification as a means of examining the impact of
anabolic steroids on the development of atherosclerotic disease in body builders using
anabolic steroids over an extended period of time. Fourteen male professional body builders
with no history of cardiovascular disease were evaluated for coronary artery calcium, serum
lipids, left ventricular function, and exercise-induced myocardial ischemia. Seven subjects
had coronary artery calcium, with a much higher than expected mean score of 98. Six of the
7 calcium scores were >90th percentile. Mean total cholesterol was 192 mg/dL, while mean
high-density lipoprotein was 23 mg/dL and the mean ratio of total cholesterol to high-density
lipoprotein was 8.3. Left ventricular ejection fraction ranged between 49 percent and 68
percent, with a mean of 59 percent. No subject had evidence of myocardial ischemia. This
small group of professional body builders with a long history of steroid abuse had high levels
of coronary artery calcium for age. The authors conclude that in this small pilot study there is
an association between early coronary artery calcium and long-term steroid abuse. Large-
scale studies are warranted to further explore this association [06069].
Myocardial infarction
The potential cardiotoxicity of anabolic steroids is not well known. The authors report the
case of a young man who was a top class body builder and who developed severe ischaemic
cardiomyopathy presenting with an inferior wall myocardial infarction. The clinical history
revealed prolonged and intensive usage of two types of anabolic steroids to be the only risk
factor. This cardiotoxicity may be related to several physiopathological mechanisms:
accelerated atherogenesis by lipid changes, increased platelet aggregation, coronary spasm
or a direct toxic effect on the myocytes. The apparent scarcity of the reported clinical details
in the literature is probably an underestimation of the consequences of this usage [00057].
Anabolic-androgenic steroids are associated with numerous side effects, including acute
myocardial infarction. It was reported a case of myocardial infarction in a young 31-year-old
bodybuilder. Because of the serious cardiovascular complications of anabolic steroids,
physicians should be aware of their abuse and consequences [08142].
A few case reports suggest that the use of androgenic anabolic steroids may be associated
with myocardial infarction. It was reported a case of a 27-year-old male bodybuilder with
1063
acute myocardial infarction due to occlusion of the proximal left anterior descending coronary
artery. He was treated with primary angioplasty with stent implantation and intra-aortic
balloon support, but still developed a large myocardial infarction as determined by both
echocardiography and myocardial perfusion tomography. The patient had been using
androgenic anabolic steroids regularly for ten years. There was no family history of heart
disease or lipid disorder. The actual frequency of myocardial infarction and even sudden
death among users of anabolic steroids is presumably underreported in the medical
literature. A causal relationship is not established, but a pathogenic role is plausible. Use of
androgenic anabolic steroids has been associated with platelet hyperactivity, effects on
vasoreactivity and changes in lipid levels. It is important for clinicians to be aware of this
association and to counsel patients carefully about this and other side effects that may occur
with these agents [04058].
Sudden cardiac deaths secondary to myocardial infarction and related to AAS use in
previously healthy athletes have been reported; however, it must be pointed out that these
effects are reported in individual case reports and no large, randomized study has been
conducted to verify these results. Exposure to AAS alters endothelial cell growth with a
strong antiproliferative effect, induces apoptosis, and modifies intracellular levels of calcium.
These observed endothelial alterations may be considered events that predispose to serious
damage at the cell vasculature level. Androgens impair arterial vasomotor response and
consequently increase collagen and other fibrous proteins in arterial vascular tissue and they
impair flow-mediated, endothelium-dependent vasodilation. This effect may improve after the
discontinuation of agents. Moreover, experimental studies have shown that androgens
potentiate platelet aggregation in vitro and in vivo. Androgens may exert their effect on
platelets through an effect on the prostaglandin system and lead to increasing platelet
production of thromboxane A2 (a potent platelet aggregator), decreasing the production of
prostacycline (prostaglandin I2, an inhibitor of platelet aggregation) and increasing fibrinogen
levels. They also increase human platelet thromboxane A2 receptor density and their
aggregation responses. The above-mentioned physiological changes may predispose
individuals to be at higher risk for myocardial infarction. It also has been shown that aortic
distensibility decreases in athletes who use AAS. This went against previously reported
increases in aortic distensibility in athletes in comparison with age-matched sedentary
volunteers. Aortic stiffness by increasing ventricular load predisposes to the development of
LVH, progressing to left ventricular dysfunction and cardiac failure, and creating an
unfavorable oxygen supply/demand ratio. It also causes a reduction in aortic pressure during
diastole, which decreases the coronary perfusion pressure and contributes to myocardial
ischemia even in the absence of coronary artery atherosclerotic narrowing [12119].
Infarctions and strokes
Changes in the electrocardiogram QT interval are associated with high doses of AAS use
and an increasing predisposition to arrhythmias and acute myocardial infarction. Increased
vascular tone, blood pressure, platelet aggregation, and producing atherothrombotic
phenomena are other causes attributed to AAS use. The use of AAS has been associated
with sudden death among young bodybuilders with no history of cardiovascular diseases. In
an interesting study, the mortality of 62 male power lifters between 1977 and 1982 was
compared with the mortality of a control population. The data showed 13 percent mortality
among power lifters and only 3 percentin the control population. In this same study, the
authors report 62 deaths in power lifters and 34 controls in a population of 1,094 people. The
misuse of AAS resulted in greater mortality 4.6. The causes of death were mainly acute
myocardial infarction but also included suicide, liver disease, and lymphoma. In autopsies
performed with 34 Swedish AAS users, chronic cardiac abnormalities such as cardiac
hypertrophy, fibrosis, and atheromatous changes were found, although AAS were believed to
1064
contribute to the death of only two users. In another study, based on necropsies of AAS
users, it was found that cardiac hypertrophy exceeded the physiological adaptations that
reasonably explained the sudden death. Several cardiac structural alterations, such as left
ventricular hypertrophy, ischemia, and autonomic dysfunction have been found in individuals
using AAS who died suddenly [14247].
Anabolic steroid use has been shown to have detrimental cardiac effects including left
ventricular hypertrophy, increased thickening of the interventricular septum, dyslipidemia,
cardiac arrhythmias, increased blood pressure, and most notably acute myocardial
infarctions (MIs). Previous cases have reported multiple fatalities from the abuse of anabolic
steroids as well as numerous incidents of acute MI. Furthermore, anabolic steroids have
been linked to changes in homocysteine with increases so severe some patients develop
hyperhomocysteinemia. Advanced research suggests that increased levels of homocysteine,
including mild hyperhomocysteinemia, can lead to atherosclerosis of the coronary vessels as
a result of damage to the endothelium and can be considered a risk factor for arterial
vascular disease. A previous study showed that bodybuilders chronically taking anabolic
steroids developed acute hyperhomocysteinemia. More research still needs to be conducted
on the mechanism in which increased levels of homocysteine affect coronary vessels. We
report the first case of a patient presenting to the emergency department (ED) with an acute
MI due to anabolic steroids and associated elevated homocysteine levels. A 27-year-old
male anabolic steroid user presented to the emergency department with chest pain radiating
down his left arm. After initial assessment and electrocardiogram, the patient was diagnosed
with an ST-elevation myocardial infarction (STEMI), and STEMI protocol was initiated.
Cardiac catheterization revealed a 70 percent stenosis of the left anterior descending artery.
Further in-patient testing revealed remarkably elevated homocysteine levels, which led to an
additional diagnosis of hyperhomocysteinemia. Special attention should be paid to patients
who abuse anabolic steroids due to their association with elevated homocysteine levels and
subsequent stenosis of the coronary vessels and MI. The patient was found to have
markedly high homocysteine levels (45.2 micromol/L) after anabolic steroid use. There has
never been a case reported of a patient with a STEMI and hyperhomocysteinemia while
taking anabolic steroids. This case is noteworthy because homocysteine levels should be
considered when patients admit to anabolic steroids [14648].
Changes in the electrocardiogram QT interval are associated with high doses of AAS use
and an increasing predisposition to arrhythmias and acute myocardial infarction. Increased
vascular tone, blood pressure, platelet aggregation, and producing atherothrombotic
phenomena are other causes attributed to AAS use. The use of AAS has been associated
with sudden death among young bodybuilders with no history of cardiovascular diseases. In
an interesting study, the mortality of 62 male power lifters between 1977 and 1982 was
compared with the mortality of a control population. The data showed 13 percent mortality
among power lifters and only 3.1 in the control population. In this same study, the authors
report 62 deaths in power lifters and 34 controls in a population of 1,094 people. The misuse
of AAS resulted in greater mortality 4.6. The causes of death were mainly acute myocardial
infarction but also included suicide, liver disease, and lymphoma. In autopsies performed
with 34 Swedish AAS users, chronic cardiac abnormalities such as cardiac hypertrophy,
fibrosis, and atheromatous changes were found, although AAS were believed to contribute to
the death of only two users. In another study, based on necropsies of AAS users, it was
found that cardiac hypertrophy exceeded the physiological adaptations that reasonably
explained the sudden death. Several cardiac structural alterations, such as left ventricular
hypertrophy, ischemia, and autonomic dysfunction have been found in individuals using AAS
who died suddenly [14645].
1065
Cerebral venous sinus thrombosis
Cerebral venous sinus thrombosis is an infrequent disease with a variety of causes.
Pregnancy, puerperium, contraceptive pills and intracranial infections are the most common
causes. The patient may present with headache, focal neurological deficits and seizures.The
clinical outcome is highly variable and treatment with heparin is advised. The patient is a 22
year old male who presented with headache, repeated vomiting and papilledema.He was a
bodybuilder doing exercise since 5 years ago, who had used nandrolone decaonoate 25
milligrams intramuscularly during the previous 5 months. Brain MRI and MRV showed
superior sagital and transverse sinus thrombosis and extensive investigations did not reveal
any known cause. It was suggested that androgen was the predisposing factor in our patient.
Androgens may increase coagulation factors or platelet activity and cause arterial or venous
thrombosis. As athletes may hide using androgens it should be considered as a predisposing
factor for thrombotic events in such patients [04059].
Cardiac thrombosis
Increased thrombogenicity and acute embolism are well-recognized complications of chronic
anabolic steroid abuse. The following cases highlight such dangers in steroid-enhanced
bodybuilders who developed intracardiac thrombosis that subsequently embolized. Systemic
anticoagulation and surgical thrombectomy constituted the mainstay treatment. This
represents the first report of such devastating cardiovascular complications after anabolic
steroid abuse and their management [00058].
Chronic abuse of anabolic steroids is widespread. Hypertrophy of skeletal and heart muscle
is a well-known effect of chronic anabolic steroid abuse. Structural alterations of blood
vessels are new side effects. It was reported a case of a 32-year-old bodybuilder after long-
term use of anabolic steroids who died of cardiac arrest. Coronary angiography and autopsy
findings showed especially a hypertrophic heart, structural changes of coronary arteries,
intracoronary thrombosis and myocardial infarction, ventricular thrombosis and systemic
embolism [03051].
Heart failure due to anabolic-androgenic steroids
The objective of one study was to analyse the outcome of patients with advanced heart
failure due to abuse of anabolic-androgenic steroids. A retrospective chart review of
patients admitted or referred for advanced heart failure, due to anabolic-androgenic steroid
abuse, in the period 2009-2013 was performed. In 6 of 9 patients (median age: 31, all males)
referred in the study period, some potential for recovery of left ventricular (LV) function was
seen, with a maximal improvement in LV ejection fraction reached within 6 months of
treatment with angiotensin-converting enzyme inhibitors and beta-blockers. The remaining 3
patients required implantation of a LV assist device (LVAD) and were listed for heart
transplantation. No recovery of LV function in the patients treated with assist device was
seen. It was concluded that anabolic-androgenic steroid-induced advanced heart failure is
generally not a reversible condition. If diagnosed in the early stages some recovery of
ventricular function is possible, but the long-term prognosis is uncertain. Likely, a substantial
proportion of patients will eventually require LVADs or cardiac transplantation [14749].
Cardiac arrhythmias and abnormal electrocardiography
Cardiac arrhythmias are among the most important causes of non-eligibility to sports
activities, and may be due to different causes (cardiomyopathies, myocarditis, coronary
abnormalities, valvular diseases, primary electrical disorders, abuse of illicit drugs). The list
1066
of illicit drugs banned by the International Olympic Committee and yearly updated by the
World Anti-Doping Agency includes the following classes: stimulants, narcotics, anabolic
agents (androgenic steroids and others such as beta-2 stimulants), peptide hormones,
mimetics and analogues, diuretics, agents with an antiestrogenic activity, masking agents.
Almost all illicit drugs may cause, through a direct or indirect arrhythmogenic effect, in the
short, medium or long term, a wide range of cardiac arrhythmias (focal or reentry type,
supraventricular and/or ventricular), lethal or not, even in healthy subjects with no previous
history of cardiac diseases. Therefore, given the widespread abuse of illicit drugs among
athletes, in the management of arrhythmic athletes the cardiologist should always take into
consideration the possibility that the arrhythmias be due to the assumption of illicit drugs
(sometimes more than one type), especially if no signs of cardiac diseases are present. On
the other hand, in the presence of latent underlying arrhythmogenic heart disease including
some inherited cardiomyopathies at risk of sudden cardiac death, illicit drugs could induce
severe cardiac arrhythmic effects [03052].
Sudden cardiac arrhythmia resulting from inflammatory process and myocardial fibrosis has
been suggested to be the cause of death in athletes using AAS. AAS cause the deepest and
most prolonged depression of stimulation threshold in the heart muscles. Long-term use may
be responsible for an alteration in the electrophysiology of the myocardium that may
predispose to reentry mechanism. It also has been shown that QTc interval and dispersion
are increased in individuals who abuse androgens, suggesting the presence of ventricular
repolarization abnormalities that could increase the risk of cardiac arrhythmias and sudden
cardiac death. Atrial fibrillation secondary to high-dose steroids was reported in two cases of
athletes who were using AAS and had no other known cause of atrial fibrillation [12119].
In an electrophysiological study power athletes taking AAS were found to have increased QT
dispersion and short QT intervals. The authors associated these changes with the
manifestation of arrhythmias. In addition, in an experimental study it was found that the
administration of nandrolone decanoate to rats led to a disturbance of cardiac autonomic
nervous system function. It was also found analogous results in a study of athletes who used
anabolic steroids, who presented a reduction in baroreflex sensitivity and maintained that
anabolic steroids lead to degenerative changes in endomyocardial sympathetic neurons,
resulting in the appearance of malignant arrhythmias. This degenerative mechanism is
referred to as catecholamine myotoxicity [12126].
Cardiac arrhythmias are associated with AAS abuse. Most commonly, atrial fibrillation but
also ventricular tachycardia and ventricular fibrillation has been described secondary to AAS
abuse in human case reports. In a study on rats treated with high-dose nandrolone for 10
weeks, heart rate variability measurements revealed a reduction in parasympathetic activity
compared with the vehicle-treated group. Sympathetic indices were also higher in the AAS-
treated group. It was also shown that AAS-treated animals show prolonged action potentials
as a result of reduced density of transient potassium outward current in the left ventricle
[12125].
The current management of athletes with cardiac arrhythmias has become complicated by
the widespread use of illicit drugs, which can be arrhythmogenic. The World Anti-Doping
Agency annually updates a list of prohibited substances and methods banned by the
International Olympic Committee that includes different classes of substances namely,
anabolic androgenic steroids, hormones and related substances, beta2-agonists, diuretics,
stimulants, narcotics, cannabinoids, glucocorticosteroids, alcohol, beta-blockers and others.
Almost all illicit drugs may cause, through a direct or indirect arrhythmogenic effect, a wide
range of cardiac arrhythmias (focal or reentry type, supraventricular and/or ventricular) that
can even be lethal and which are frequently sport activity related. A large use of illicit drugs
1067
has been documented in competitive athletes, but the arrhythmogenic effect of specific
substances is not precisely known. Precipitation of cardiac arrhythmias, particularly in the
presence of a latent electrophysiologic substrate including some inherited cardiomyopathies,
at risk of sudden death or due to long-term consumption of the substances, should raise the
suspicion that illicit drugs may be a possible cause and lead cardiologists to investigate
carefully this relationship and appropriately prevent the clinical consequences [07063].
Effect on Tp-E Interval, Tp-E/Qt Ratio, and Tp-E/Qtc ratio in male bodybuilders
The chronic consumption of androgenic anabolic steroids has shown to cause atrial
arrhythmias. Several studies have suggested that the interval from the peak to the end of the
electrocardiographic T wave (Tp-e) may correspond to the transmural dispersion of
repolarization and that increased Tp-e interval and Tp-e/QT ratio are associated with
malignant ventricular arrhythmias. The aim of on study was to evaluate repolarization
dispersion measured from the 12-lead surface electrocardiogram (including Tp-e interval, Tp-
e/QT ratio, and Tp-e/cQT ratio) in bodybuilders who are using anabolic androgenic steroids
(AAS). It was selected a population of 33 competitive bodybuilders, including 15 actively
using AAS for ≥ 2 years (users) and 18 who had never used AAS (nonusers), all men. QT,
cQT, QTd, cQTd, JT, and cJT were significantly increased in AAS users bodybuilders
compared to the nonusers. Tp-e interval, Tp-e/QT ratio, and Tp-e/cQT ratio were also
significantly higher in AAS user group compared to the nonuser group. QRS duration was not
different between the groups. There were negative correlation between Em and Tp-e, Tp-
e/QT ratio, Tp-e/cQT ration.There were also negative correlation between Sm and Tp-e, Tp-
e/QT ratio, Tp-e/cQT ration. In conclusion, it was presented a strong evidence suggesting
that Tp-e interval, Tp-e/QT ratio, and Tp-e/QTc ratio were increased in AAS users, which
suggest that there might be a link between AAS use and ventricular arrthymias and sudden
death [150178].
QT-interval
The association between physiologic levels of sex hormones and QT-interval duration in
humans was evaluated using data from 727 men enrolled in the Third National Health and
Nutrition Examination Survey and 2,942 men and 1,885 postmenopausal women enrolled in
the Multi-Ethnic Study of Atherosclerosis. Testosterone, estradiol, and sex hormone-binding
globulin levels were measured in serum and free testosterone was calculated from those
values. QT interval was measured using a standard 12-lead electrocardiogram. In men from
the Third National Health and Nutrition Survey, the multivariate adjusted differences in
average QT-interval duration comparing the highest quartiles with the lowest quartiles of total
testosterone and free testosterone were -8.5 ms (95 % confidence interval: -15.5 to -1.4) and
-8.0 ms (95 % confidence interval -13.2, -2.8), respectively. The corresponding differences
were -1.8 ms and -4.7 ms, respectively. Estradiol levels were not associated with QT-interval
duration in men, but there was a marginally significant positive association in
postmenopausal women. The findings suggest that testosterone levels may explain
differences in QT-interval duration between men and women and could be a contributor to
population variability in QT-interval duration among men [11330].
Androgenic anabolic steroid (AAS) abuse is associated with changes in cardiac

electrophysiology. Recently heart rate corrected QT interval (QTc) has been suggested as a
method of screening for AAS use in athletes despite conflicting reports. This study aimed to
further investigate the effect of AAS on QTc in a cohort of long-term AAS users in whom the
affects may be more pronounced. Using a cross-sectional cohort design with AAS using
resistance trained athletes (AS n = 15) and a group of non-AAS using resistance trained, age
matched controls (C n = 15). AS had a long history of AAS use (18 ± 2 years) and AS and C
both had >19 years of resistance training. Participants underwent a resting electrocardiogram
(ECG), from which, the QTc interval was calculated using the Bazett formula. The main
1068
outcome measure was significant differences in mean corrected QTc between groups. A
secondary outcome was to calculate a QTc that best differentiated between C and AS.
Results indicated that QTc was shorter in AS than in C (382.0 ± 21.01 ms versus
409 ± 18.77 ms for AS and C respectively). Chi squared analyses revealed a greater
incidence of QTc < 380 ms in AS versus C, specificity 93 % sensitivity 60 %). In conclusion
these results supports previous findings that AAS use causes a reduction in QTc, however,
the specificity and sensitivity in our sample is lower than reported previously and precludes
use as a screening tool [150179].
Echocardiography
The introduction of echocardiography was important for investigating the physiological
responses of the heart to exercise and training. Echocardiography has also been applied for
evaluation of AAS on heart structure and function. Eight cross-sectional studies observed
differences in one or more echocardiographic variables between AAS users and non-using
strength athletes, whereas five studies did not register any difference. Compared with non-
users, steroid users have been demonstrated to show larger left ventricular mass and/or left
ventricular index, and larger posterior wall and interventricular septum thicknesses. The
majority of studies seem to show that the left ventricular cavity during diastole and systole is
not subject to alterations under the use of steroids. Six prospective echocardiographic
studies have been published and only one study reported steroid-induced changes in
echocardiographic variables. It was observed significant changes of left ventricular mass,
interventricular septum thickness and left ventricular end-diastolic diameter, but the left
ventricular posterior wall thickness remained unaffected by AAS. However, the researchers
did not pay attention to an increase in work load of the AAS users during the study period.
Therefore, these results must be interpreted with caution, especially since all other studies
unanimously reported no changes in any echocardiographic variable measured during AAS
administration. Although a nonblinded design was applied in all studies except one, these
results indicate that changes in heart structure and function are not to be expected when an
athlete takes AAS for periods of up to 4 months.The effects of prolonged AAS abuse and/or
the use of many successive AAS courses remains unknown. Nevertheless, animal studies
clearly have shown that short-term use of androgens and anabolic agents may exert strong
hazardous effects on cardiac structure and function and, therefore, it has been proposed that
echocardiography might be not sensitive enough to detect early and small changes due to
AAS administration [04002].
Increased risk of ventricular arrhythmias in the rat

It was examined the influence of chronic administration of nandrolone decanoate with low-
intensity endurance swimming exercise on susceptibility to lethal ventricular arrhythmias in
rat. The animal groups included the control group, exercise group (EX), nandrolone group
(Nan), vehicle group (Arach), trained vehicle group (Arach + Ex) and trained nandrolone
group (Nan + Ex) that treated for 8 weeks. Then, arrhythmia induction was performed by
intravenous infusion of aconitine and electrocardiogram recorded. Then, malondialdehyde
(MDA), hydroxyproline (HYP) and glutathione peroxidase of heart tissue were measured.
Chronic administration of nandrolone with low-intensity endurance swimming exercise had
no significant effect on blood pressure, heart rate and basal ECG parameters except RR
interval that showed increase. Low-intensity exercise could prevent the incremental effect of
nandrolone on MDA and HYP significantly. It also increased the heart hypertrophy index and
reduced the abating effect of nandrolone on animal weighting. Nandrolone along with
exercise significantly increased the duration of VF and reduced the VF latency. The findings
suggest that chronic co-administration of nandrolone with low-intensity endurance swimming
exercise to some extent facilitates the occurrence of ventricular fibrillation in rat.
Complementary studies are needed to elucidate the involved mechanisms of this abnormality
[150181].
1069
Increased atrial electromechanical delay
It was investigated the effect of long-term supraphysiologic doses of anabolic androgenic
steroids (AAS) on atrial electromechanical delay (AEMD) in male bodybuilders. It was clearly
demonstrated that long-term consumption of supraphysiologic doses of AAS is associated
with higher values of inter- and intra-AEMD in healthy young bodybuilders. Self-
administration of high doses of anabolic androgenic steroids (AAS) is a widespread practice
among athletes to increase lean body mass and muscular strength. Long-term illicit use of
supraphysiologic doses of AAS may cause several adverse cardiovascular effects. Recent
studies have found pathological left ventricular (LV) hypertrophy, diastolic dysfunction, and
subclinical LV systolic impairment in long-term AAS users. In addition, ventricular and atrial
arrhythmic events were described secondary to the intake of AAS. Atrial fibrillation (AF) is the
most frequently observed arrhythmia in bodybuilders who are using AAS. Moreover, various
case reports of AF among AAS users suggest a causal link between AAS use and AF in
power athletes. However, the mechanisms underlying such predispositions to AF are poorly
understood and also it is not clear that AAS using athletes are more prone to atrial rhythm
disturbances than non-AAS users. The prolongation of intra-atrial and interatrial conduction
times and the inhomogeneous propagation of sinus impulses are typical electrophysiological
features of the atrium which is prone to fibrillate. Moreover, atrial electromechanical delay
(AEMD) as measured by tissue Doppler imaging (TDI) has been shown to detect atrial
impairment in paroxysmal AF. Another important point is that AEMD may also predict the
development of new-onset AF. Since AF is a reentrant arrhythmia, it is logical that the
triggering factor generally is a critically timed atrial activation that may give rise to reentry in a
vulnerable structure. Atrial and ventricular structural alteration, increased atrial stretch,
autonomic imbalance, atrial interstitial fibrosis, inflammation, and ischemia may act in this
respect as an internal or external factor by modulating atrial refractoriness through both atria
and modifying intra-atrial conduction. Because these factors are effected by long-term use of
supraphysiologic doses of AAS, there might be an association between AAS use and AEMD
[14249].
Previous studies reported that long-term illicit use of supraphysiologic doses of AAS was
associated with reduced LV diastolic functions (impaired relaxation and reduced compliance
of LV), increased LV mass, LV/atrial hypertrophy, subclinical systolic impairment, increased
myocardial stiffness and myocardial fibrosis, and altered cardiac autonomic system
regulation. Furthermore, it has been reported that myocardial infarction, cardiomyopathy,
sudden death, cardiovascular morbidity, and mortality have significantly increased in long-
term AAS using bodybuilders more than nonusers. In addition, arrhythmic events were
described secondary to the long-term intake of AAS. Although AF is the most frequently
observed arrhythmia, ventricular arrhythmias were also described. However, it is not clear
that AAS using bodybuilders are more prone to rhythm disturbances compared with
nonusers. The prolongation of intra- and inter-AEMD and the inhomogeneous propagation of
sinus impulses are well-known electrophysiologic characteristics of the atria which is prone to
fibrillation. The evaluation of AEMD by using TDI has been studied in patients with rheumatic
mitral stenosis, paroxysmal AF, acute sleep deprivation, and type I diabetes mellitus. Also, it
has been found that atrial electromechanical interval is a predictor of AF emerging after
coronary artery bypass grafting and demonstrated that the preoperative administration of
amiodarone to patients having longer atrial electromechanical interval has decreased the
postoperative atrial fibrillation incidence. Furthermore, it was shown that prolonged PA-TDI
interval (indicator of AEMD) predicted the development of new-onset AF in one study, which
included 249 patients. In addition, prolonged AEMD in patients with paroxysmal AF was
reported with TDI and pulsed-wave Doppler echocardiographic studies. In the present study,
we found that inter- and intra-atrial AEMD were prolonged in AAS users compared with both
nonusers [14249].
1070
There may be several mechanisms involved in increasing inter-AEMDs in chronic
consumption of supraphysiologic doses of AAS. There are several studies that indicate
impairment of LV diastolic function, which is known to play a role in the pathogenesis of AF,
which was also found to be impaired in AAS using athletes. When left ventricular diastolic
dysfunction occurs, emptying of the left atrium is impaired as well. Following impaired left
ventricular diastolic relaxation, there is increased atrial contribution to the mitral flow in the
left ventricular diastolic flow, thus leading to atrial overstretching and enlargement. The left
atrium diameter is known to be correlated with cardiovascular events and is a risk factor for
AF. In one study, the left atrial diameters of the AAS user and nonuser groups were similar.
However, the presence of left ventricular diastolic dysfunction in AAS user athletes is a
controversial issue. It was investigated the diastolic functions by using the tissue TDI method
as well because the conventional Doppler method is load dependent and TDI constitutes a
good index of LV relaxation properties. In previous studies, the E/Em and Em/Am were
demonstrated to be significantly correlated with the left ventricle end-diastolic pressure and
diastolic dysfunctions. It was now found that E/Em ratio was significantly higher in AAS users
than in nonusers. In addition, the Em/Am ratio was significantly lower in AAS users than in
nonusers. Also it was found that IVRT prolonged in AAS using group, indicating the
impartment of diastolic function. The other possible mechanism for increasing inter-AEMD
and intra-AEMD in AAS using athletes is LV pathological hypertrophy. LV pathological
hypertrophy induced by AAS appears to be generated by a direct action on cardiac androgen
receptors, whose effects are directly proportional to the doses, time, and duration of drug
administration. LV wall thickness and LV mass index were enlarged in AAS using athletes
compared to nonusers. The presence of LV hypertrophy is an indicator of increased
myocardial demand for oxygen and hence decreases coronary reserve. When coronary
blood flow is fixed or reduced, there is a supply-demand mismatch, resulting in increased risk
for ischemia. In such a scenario, a decrease in blood flow can be catastrophic to the already
increased demand of the myocardial cells. Patients with LV pathological hypertrophy are at
increased risk for ischemia, probably causing prolongation of inter- and intra-AEMD [14249].
Probably adverse effects of AAS on the cardiovascular system are also due to direct toxicity
on myocardial structure with increased collagen deposition, fibrosis, and altered
microcirculation with intimal hyperplasia of the intramural coronary arteries resulting in
chronic ischemic damage. Vascular endothelial cells may be directly affected by AAS, which
may result in vasospasm. As the cause of these alterations, AAS may directly affect the
atrium, causing heterogeneity in the atrial conduction. It was speculated that long-term illicit
use of supraphysiologic doses of AAS might directly affect atrial conduction time (inter-
AMED). The last possible mechanism to increase AEMD may be sympathetic activation. It
has been shown that chronic consumption of supraphysiologic doses of AAS induces cardiac
autonomic imbalance by reduction in parasympathetic cardiac modulation and increase in
sympathetic cardiac modulation. Experimental studies showed that greater sympathetic
activation leads to myocardial injury. Increased sympathetic activity may also trigger atrial
arrhythmias. Therefore, altered autonomic system regulation occurring secondary to the
chronic consumption of supraphysiologic doses of AAS may be the other reason underlying
the delayed interatrial electromechanical coupling intervals [14249].
Long-term intensive training is associated with distinctive cardiac adaptations which are
known as athlete's heart. The aim of one study was to determine whether the use of anabolic
androgenic steroids (AAS) could affect echocardiographic parameters of left ventricular (LV)
morphology and function in elite strength and endurance athletes. A total of 20 elite strength
athletes (10 AAS users and 10 non-users) were compared to 12 steroid-free endurance
athletes. All the subjects underwent comprehensive standard echocardiography and tissue
Doppler imaging. After being indexed for body surface area, both left atrium (LA) and LV
1071
end-diastolic diameter (LVEDD) were significantly higher in the endurance than strength
athletes, regardless of AAS use. A significant correlation was found between LA diameter
and LVEDD in the steroid-free endurance athletes, showing that 75 percent of LA size
variability depends on variability of LVEDD. No significant differences in ejection fraction and
cardiac output were observed among the groups, although mildly reduced LV ejection
fraction was seen only in the AAS users. The AAS-using strength athletes had higher A-peak
velocity when compared to steroid-free athletes, regardless of training type. Both AAS-using
and AAS-free strength athletes had lower e' peak velocity and higher E/e' ratio than
endurance athletes. It was concluded that there is no evidence that LV ejection fraction in
elite athletes is altered by either type of training or AAS misuse. Long-term endurance
training is associated with preferable effects on LV diastolic function compared to strength
training, particularly when the latter is combined with AAS abuse [14250].
Maximal heart rate
Previous study showed that muscle sympathetic nerve activity (MSNA) was augmented in
anabolic steroids users (AASU). In one study, it was tested the hypothesis that the heart rate
(HR) responses after maximal exercise testing would be reduced in AASU. Ten male AASU
and 10 AAS nonusers (AASNU) were studied. Cardiopulmonary exercise was performed to
assess the functional capacity and heart rate recovery. MSNA was recorded directly from the
peroneal nerve by microneurography technique. Peak oxygen consumption (VO₂) was lower
in AASU compared to AASNU (44 ± 2 vs 53 ± 2 mL/kg/min). HR recovery (HRR) at first and
second minute was lower in AASU than AASNU (21 vs 27 bpm, and 37 vs 45 bpm,
respectively). MSNA was higher in AASU than AASNU (29 vs 20 bursts/min). Further
analysis showed a correlation between HRR and MSNA, HRR at first minute and peak VO₂
and HRR at second minute and peak VO₂. The exacerbated sympathetic outflow associated
with a lower parasympathetic activation after maximal exercise, which impairs heart rate
recovery, strengthens the idea of autonomic imbalance in AASU [13132].
Heart rate recovery
Previous study showed that muscle sympathetic nerve activity (MSNA) was augmented in
anabolic steroids users (AASU). In the present study, we tested the hypothesis that the heart
rate (HR) responses after maximal exercise testing would be reduced in AASU. Ten male
AASU and 10 AAS nonusers (AASNU) were studied. Cardiopulmonary exercise was
performed to assess the functional capacity and heart rate recovery. MSNA was recorded
directly from the peroneal nerve by microneurography technique. Peak oxygen consumption
(VO2) was significantly lower in AASU compared to AASNU. HR recovery (HRR) at first and
second minute was significantly lower in AASU than AASNU. MSNA was higher in AASU
than AASNU. Further analysis showed a correlation between HRR and MSNA, HRR at first
minute and peak VO2 and HRR at second minute and peak VO2. The exacerbated
sympathetic outflow associated with a lower parasympathetic activation after maximal
exercise, which impairs heart rate recovery, strengthens the idea of autonomic imbalance in
AASU [13136].
Reflex control in heart rate

The aim of one study was to analyze the cardiovascular effects of chronic stanozolol
administration in male rats. The rats were randomly assigned to one of three groups: control
(n=12), chronic treatment with low dose of stanozolol (LD, n=18, 5 mg/kgweek) and;
treatment with high dose of stanozolol (HD, n=28, 20 mg/kgweek). Mean arterial pressure
(MAP) was higher in both HD and LD than control. The LD group showed an increase in
cardiac output, whereas in the HD group total peripheral resistance increased. Acute
1072
sympathetic blockade caused a similar decrease in MAP in all groups. In conscious rats, the
baroreflex index for bradycardia and tachycardia responses changed only in the LD group.
Cardiac hypertrophy was observed in both treated groups. In conclusion, hypertension with
differential hemodynamic changes and alterations in the reflex control in heart rate is seen at
different stanozolol doses, which may be important variables in the cardiovascular effects of
anabolic steroids [05049].
The harmful cardiovascular effects of indiscriminate use of AAS to the cardiac structure and
functioning can include increased cardiac tissue collagen, imbalance of vasomotor tone,
reduction in the number of capillaries, and pathological cardiac hypertrophy in animal and
human models. In addition, supraphysiological doses of AAS have already demonstrated that
they may inhibit angiogenesis induced by physical training. In a study involving 152 men
(between 18 and 40 years of age), non-athletes (n=52), strength-endurance athletes (n=52),
and athletes (n=52) who admitted using AAS, showed ventricular volumes and ventricular
wall thickness statistically higher than in non-users, and lower ejection fraction in both
ventricles. An increase in cardiac collagen can induce electrophysiological changes in the
myocardium with abnormal propagation of the excitation wave favoring tachycardia, which
may explain the repeated occurrence of sudden death in users of AAS. But this possibility is
raised based on a case report of a user 31 years of age. On the other hand, another study
reports other factors occurring after massive doses of AAS for several years, namely, a case
of ventricular fibrillation during exercise, one heart failure, and arterial thrombus in the lower
left leg. Interestingly, all these patients had increased cardiac hypertrophy and fibrosis in the
myocardium. It is certain that the character of case reports makes these inconsistent data.
The development of experimental models is useful in helping us better understand the
deleterious effects of SEA on cardiovascular tissue, but must consider the obvious limitations
of extrapolating data to humans. So that our knowledge of this adverse effect is dependent
on cases recorded and documented in the scientific literature. Owing to this limitation, it was
also investigated the effect of administration of nandrolone decanoate in rats for 10 weeks,
associated with swim training. They found a 10 percent increase in cardiac hypertrophy in
rats treated with nandrolone decanoate and 17 percent who had been administered this drug
associated with physical training, which were significantly higher than that found in mice not
treated with the drug [14645].
Despite the limitations of data with an animal model, it was demonstrated by

echocardiography that 17 bodybuilders and power-lifters and AAS users manifested cardiac
hypertrophy. The most interesting aspect of this study was that 15 of the subjects who did
not use AAS for at least 12 months still showed a slight concentric ventricular hypertrophy
compared with those who remained on the drug. Several studies have demonstrated
changes in the cardiac functioning aspects of AAS users, especially on diastolic functioning.
Diastolic dysfunction has been strongly associated with collagen deposition. Fibrillar
collagens, types I and III, are the major structural proteins of the myocardial collagen matrix
exerting an important influence on ventricular compliance. Exposure to supraphysiological
doses of AAS can lead to tissue necrosis and diastolic dysfunction, resulting in structural
changes similar to those seen in the earlier stages of heart failure. There are several
mechanisms that appear to be related to cardiac hypertrophy and collagen accumulation.
AAS can induce such hypertrophy through nuclear receptors, acting directly on RNA and
increasing the protein synthesis and acting as well on specific enzymes, on ions flow and the
structural matrix in the myocardium. Increased circulating pro-inflammatory cytokines such
as TNF-alpha and increased cAMP concentration have been documented, which contributes
to the positive inotropic response through the calcium in the cytosol of the myocardial cell.
However, the exact mediators of such effects are diverse and not fully elucidated [14645].
1073
Impaired exercise-induced growth of the cardiac capillary bed
Concomitant application of anabolic-androgenic steroids and physical exercise can induce
cardiac hypertrophy. These experiments investigate the still unknown response of the
cardiac myocytes and capillaries to the combined influence of various anabolic steroids and
muscular exercise. Female SPF-NMRI mice were divided into the following groups: a)
sedentary control, b) exercise (treadmill running); c) sedentary receiving Dianabol; d)
exercise + Dianabol; e) exercise + Oral-Turinabol. After 3 and 6 weeks the left ventricular
papillary muscles were studied morphometrically. Evaluated variables: minimal myocyte
diameter, number of capillaries around a single myocyte, capillary density and intercapillary
distance. Only the anabolic steroids + exercise groups showed a mild myocyte hypertrophy.
In contrast, only exercise alone caused a significant increase of the capillary density after
both experimental periods; e.g. capillary density after 6 weeks. Moreover, unlike all other
regimens, only exercise alone shortened the intercapillary distance. Finally, exercise without
drugs induced the greatest increase in the number of capillaries around a single myocyte. It
was concluded that anabolic steroids combined with exercise: 1) induce mild hypertrophy of
the cardiac myocytes, 2) impair the cardiac microvascular adaptation to physical
conditioning. The microvascular impairment may cause a detrimental alteration of the
myocardial oxygen supply, especially during muscular exercise [00054].
Experimental application of anabolic-androgenic steroids and exercise training induce

cardiac hypertrophy. One study quantified for the first time, on microscopical level, the
adaptation of the cardiac capillaries and myocytes to the concomitant application of
testosterone-propionate and exercise training. Female SPF-NMRI mice were studied over 3
and 6 wk. Experimental groups: (i) sedentary control (C); (ii) exercise (treadmill running, E);
(iii) testosterone-propionate (TP); and (iv) testosterone-propionate+exercise (TPE).
Morphometric parameters: 1) papillary muscles: capillary density, intercapillary distance,
number of capillaries around a myocyte, and minimal myocyte diameter; and 2) left
ventricular wall: capillary density and intercapillary distance. Papillary muscle: A striking
suppression of the exercise-induced improvement in capillary supply occurs in the
testosterone-propionate+exercise groups over 3 and 6 wk. Exercise without drugs increases
significantly the capillary density, shortens significantly the intercapillary distance, whereas it
increases the number of capillaries around a myocyte. These alterations are not observed in
the testosterone-propionate treated sedentary animals; e.g. capillary density after 6 wk C:
4272 + 287, E: 5411 + 758, TP: 4221 + 364, and TPE: 3997 + 397. Moreover, only in the
testosterone-propionate+exercise groups occurs a mild myocyte hypertrophy after both time
periods: there is a trend toward hypertrophy in comparison with the C groups and a
significant hypertrophy in comparison with the E groups. It was concluded that testosterone-
propionate profoundly inhibits the exercise-induced augmented capillarization, whereas
(under training conditions) it leads to a mild myocyte hypertrophy. The microvascular
impairment could trigger an imbalance between the myocardial oxygen supply and demand,
especially during physical exercise [00055].
Decreased ventricular compliance

Testosterone analogs have been used as performance enhancers by athletes for more than
40 yr. We asked whether the anabolic steroid 17 alpha-methyl-4-androstene-17-ol-3-one (17
alpha-MT) would affect intrinsic contractile function of the heart. Male Sprague-Dawley rats,
125-150 g, were treated with 17 alpha-MT either parenterally or orally for up to 8 wk. Intrinsic
contractile function of the hearts was assessed utilizing both the isolated working heart and
isovolumic perfused heart preparations. Isolated working hearts from 17 alpha-MT-treated
rats had a 45 percent decrease in heart work attributable largely to a similarly decreased
stroke volume. Isovolumic perfused hearts from treated animals had elevated left ventricular
systolic and diastolic pressures at similar interventricular volumes compared to controls.
Rates of ventricular pressure development (+dP/dT) or relaxation (-dP/dT) were unchanged
1074
as a result of the treatment. However, static elastance was reduced in potassium-arrested
hearts from the 17 alpha-MT treatment (63 % increase in interventricular pressure),
consistent with a limitation being imposed on stroke volume by a decreased myocardial
compliance. Hydroxyproline content of the hearts was not altered by 17 alpha-MT treatment
suggesting that increased stiffness was not a consequence of collagen proliferation.
Treatment of the steroid rats with beta-aminopropionitrile, a compound that inhibits lysyl
oxidase, restored the left ventricular volume-pressure relationship (elastance curve) to that of
control hearts. Thus, chronic treatment with anabolic steroids appears to reduce left
ventricular compliance, possibly related to an enhanced activity of lysyl oxidase, and results
in increased crosslink formation between collagen strands in the extracellular matrix [00055].
Attenuated beta-adrenoceptor-mediated cardiac contractile responses

Androgenic steroids administered in doses at pharmacological levels to sedentary animals
have been shown to result in a reduced beta-adrenoceptor-mediated increase in systolic
cardiac performance when assessed in vivo. Whether the attenuated adrenergic response
occurs as a consequence of alterations in either cardiac loads, heart rate, modifications in
left ventricular (LV) geometry, or a decrease in myocardial contractile performance has not
been determined. In this study the effect of chronic administration (over 3 months) of an
androgenic steroid (nandrolone decanoate, 5 mg./kg biweekly) on the response of load-
insensitive indices of myocardial contractile function [the slope of the LV systolic stress-strain
relationship (LV-E(n)(max), where E(n)(max) is systolic myocardial elastance)] to an
adrenergic-inotropic stimulus was examined ex vivo in paced rat hearts. Systolic cardiac
performance was assessed at 300 beats/min in isolated constant flow perfused heart
preparations both before and during 10-8.5 mol/L isoproterenol (ISO) infusion (approximate
concentration of ISO eliciting 50 % maximal inotropic response to ISO). Steroid
administration resulted in left-shifted LV systolic and diastolic pressure-volume (P-V)
relationships. The leftshifted P-V relationships were attributed, in part, to increased slopes of
these relationships. However, the steroid-mediated increment in the slope of the systolic P-V
relationship (systolic chamber elastance, E(max)) was not associated with a similar change
in LV E(n)(max) as determined in the absence of ISO. Isoproterenol infusion resulted in an
increase in both E(max) and E(n)(max) in the control rats, without altering systolic
performance in the steroid treated rats. Consequently, in the presence of ISO, the steroid
treated rats exhibited a similar E(max), but a reduction in E(n)(max) compared to the control
rats. In conclusion, these results would suggest that chronic high dose androgenic steroid
administration produces a decrease in myocardial contractile reserve to beta-adrenoceptor
stimulation [00056].
Myocardial fibrosis caused of anabolic steroids
Experimental studies have demonstrated that AAS abuse leads to skeletal muscle
hypertrophy and increased collagen accumulation; changes that are similarly detected in the
myocardium. Studies in rats and mice have shown that AAS abuse leads to both myocardial
hypertrophy and fibrosis, destruction of the mitochondria and other elements of the
cardiomyocytes, disturbances of the microcirculation, and to a deterioration in systolic
function and to diastolic dysfunction. Post-mortem studies of athletes who used AAS have
found infiltration of eosinophils into myocardial cells, as well as destruction of myofibrils.
Endothelial dysfunction was also observed [12126].
A 2005 study reported two cases of sudden cardiac death in young male athletes related to
AAS abuse. Both cases involved healthy individuals without any history of coronary artery
disease (CAD) and with no evidence of significant abnormality in arterial microscopic
examination. Autopsy of both hearts showed focal myocardial fibrosis suggestive of prior
myocardial injury. In a study of a sudden unexpected death in a female fitness athlete using
1075
steroids and ephedrine, the only pathological finding was a few small foci of granulation
tissue, which was interpreted as evidence of earlier myocardial necrosis. Sudden cardiac
arrhythmia resulting from inflammatory process and myocardial fibrosis was suggested to be
the cause of death in these cases. Other researchers have reported sudden cardiac deaths
related to steroids that also showed myocardial fibrosis in the absence of CAD [12119].
A study recently published describes the potentially beneficial cellular effects of testosterone
against cardiac fibrosis. In a rat model, it was observed that testosterone inhibited cardiac
fibroblast migration and proliferation in addition to myofibroblast differentiation induced by
transforming growth factor-beta1. Moreover, the authors suggested that modulated cell
signaling and response of cardiac fibroblasts through a decreased production of collagen
after stimulation by transforming growth factor-beta1 and angiotensin II is the mechanism by
which testosterone may attenuate cardiac fibrosis and progression of heart failure [150180].
The effects of testosterone on the heart and the cardiovascular system are, however, still a
matter of debate. Some influences of testosterone on cardiac function and morphology have
been established. This is primarily in regard to a nonphysiologic situation of testosterone
misuse when the side-effects include left ventricular hypertrophy with systolic and diastolic
dysfunction. However, in patients with heart failure and reduced left ventricular ejection
fraction, testosterone supplementation significantly improves exercise capacity in both men
and women, without affecting left ventricular systolic function. In addition, in male patients
with heart failure, neither total, free nor bioavailable testosterone levels correlate with the left
ventricular ejection fraction. Although some disagreement exists, clinical evidence suggests
that men with coronary disease have lower levels of endogenous testosterone. The most
likely explanation is that low testosterone is associated with diabetic and metabolic
derangements such as hypertension, insulin resistance dyslipidemia and obesity. However,
one of the most intriguing questions is whether low testosterone is one of the
ethiopathogenic mechanisms of coronary disease; is it just comorbidity or perhaps a
consequence of generalized atherosclerotic vascular disease? Moreover, an inverse
association between the degree of hypogonadism and the severity of coronary disease
exists. Lower testosterone levels are associated with increased all-cause and cardiovascular
mortality as well as vascular mortality defined as death from cardiac arrest, heart failure and
atherosclerosis [150180].
Regarding the vascular mechanisms of testosterone and its effects on blood vessels, a body
of evidence suggests that one of testosterone's beneficial cardiovascular effects is the
vasodilation of systemic, coronary and pulmonary vessels.Mechanisms available from
experimental studies have been proposed to explain testosterone induced vasodilation.
Testosterone may influence the tonus of vascular smooth muscle cells by modulating the
activity of several ion channels such as the voltage-sensitive potassium ion channels, non-
ATP-sensitive potassium ion channels, calcium-activated potassium ion channels and L-type
calcium ion channels. Whether the effect on only one type of channel is dominant or a
concomitant effect on several types of channels produces vasodilation remains to be
elucidated. In heart failure, the chief beneficial cardiovascular mechanism of testosterone
seems to be primarily vascular and peripheral since peripheral vasodilation produces a
reduced cardiac afterload and increased cardiac output. In addition, coronary vasodilation
improvesmyocardial oxygenation which is important in heart failure patients with ischemic
ethiopathogenesis. However, the association of serum testosterone levels with clinical
severity of heart failure seems to be present only innon-obese heart failure patients. In obese
patients with heart failure, a lack of such association and comparably lower testosterone
levels may suggest altered hormonal and hemodynamic mechanisms which could contribute
to the obesity paradox and a better prognosis of such patients [150180].
1076
In addition to vasodilation, testosterone effects such as increased skeletalmuscle strength
and peak oxygen consumption, increased baroreflex sensitivity and higher hemoglobin levels
all help to improve quality of life and functional capacity of patients with heart failure. A direct
influence of testosterone onmuscle strength may be explained by a stimulation of change of
skeletal muscle composition toward type I muscle fibers which are, compared to type II
fibers, associated with enhanced physical capability and strength. Vice versa, exercise
induces an increase in endogenous testosterone levels, which in heart failure patients seems
to be particularly associated with interval training exercise. Altogether, available research
suggests that the beneficial effects of either endogenous testosterone levels or its
supplementation on exercise capacity and quality of life are completely independent of the
level of heart dysfunction and its impact on the clinical severity of heart failure [150180].
Apart from important peripheral mechanisms, additional cardiac effects of testosterone most
likely include the influence on electrophysiology and arhythmogenesis. Higher levels of
endogenous testosterone are associated with shorter QT and QTc interval and consequently
a reduced arhythmogenesis. Physiologic testosterone supplementation has been shown to
reduce QT dispersion in patients with congestive heart failure. In contrast, supraphysiologic
doses of testosterone and other anabolic androgen steroids may facilitate ventricular
arrhythmias by increasing QTc interval and dispersion and by predisposing to reentry
mechanism. Androgen misuse has been also associated with episodes of atrial fibrillation.
Nevertheless, the possibility of a protective effect of testosterone against atrial fibrillation has
been contradicted by both a meta-analysis which included middle-aged and older men and
by animal research associating testosterone supplementation with higher occurrence of atrial
fibrillation [150180].
One study explores another possible cardiac mechanism of testosterone – a modulatory role
in cardiac fibrosis. The authors reported no change in baseline cardiac fibroblast proliferative
and migration potential, but described an androgen receptor-mediated antiproliferative, anti-
collagen and anti-fibrotic effect of physiological testosterone levels in the myocardium
unaffected by a pathological process. Although they recognized that the cardiac fibroblasts
were from normal hearts, the authors' conclusion was that testosterone decreased the
production of collagen after transforming growth factor-beta1 and angiotensin II stimulation
which can attenuate the genesis of cardiac fibrosis under pathological conditions. However,
this sounds rather speculative because in pathological conditions, testosterone effects could
be quite the opposite. Cardiac remodeling is characterized by removal of destroyed necrotic
tissue, formation of scar tissue, compensatory myocyte hypertrophy and enlargement of the
affected cardiac chamber. An important study has investigated the impact of sex hormones
on cardiac remodeling after myocardial infarction in a mouse model and showed that
testosterone worsened cardiac dysfunction and remodeling in both sexes. Testosterone was
linked to decreased ejection fraction, increased left ventricular dimension and increased
myocyte size. A detrimental chronic effect on postinfarction healing and aggravation of
cardiac dysfunction was observed through the association of testosterone with occurrence of
cardiac rupture and lethal outcome. In male mice, both castration and estrogen
supplementation reduced the occurrence of most of those adverse testosterone effects
[150180].
The mechanisms of a profound impact of sex hormones on cardiac fibrosis and remodeling
in both healthy and diseased hearts must be further explored. Such mechanisms are only a
part of cardiac effects which act in concert with peripheral mechanisms and are exceptionally
important for a growing population of patients with heart failure. There is a possibility that
normal testosterone levels within a physiological range have beneficial biological effects only
in relatively healthy individuals from the cardiovascular point of view. Conversely, in aged or
diseased hearts, like those after myocardial infarction or in obesity, when the pathological
1077
cascade of myocardial remodeling has already started, higher testosterone may have a
detrimental effect. Certainly we should more clearly delineate the conditions inwhich higher
testosterone is useful from the conditions where it may have adverse effects [150180].
Androgenic anabolic steroids and arterial structure and function
The physiologic and pharmacologic effects of androgens on arterial structure and function
are poorly characterized. Several lines of evidence implicate a pro-atherogenic effect.
Epidemiologic studies demonstrate that cardiovascular disease is more prevalent and severe
in adult men than in women at all ages. It has previously been observed that androgens may
promote monocyte adhesion to endothelial cells and macrophage lipid loading. Regarding
vascular function, androgens are associated with impaired arterial reactivity in genetic
females taking high-dose androgenic steroids, and endothelial function is enhanced in
androgen-deprived older men. In contrast, certain observations are consistent with an anti-
ischemic effect of androgens. Testosterone is an acute coronary vasodilator. Furthermore,
although men have greater cardiovascular risk than women, men with low androgen levels
have a higher risk of cardiovascular events. The vascular effect of androgens is important in
assessing the potential influence of illicit androgenic anabolic steroid (AAS) use on arterial
structure and function in healthy young athletes. One study examined arterial and cardiac
structure and function in bodybuilders using androgenic anabolic steroids (AAS), compared
to non-steroid-using bodybuilder controls. Adverse cardiovascular events have been reported
in bodybuilders taking anabolic steroids. The cardiovascular effects of AAS, however, have
not been investigated in detail. It was recruited 20 male bodybuilders (aged 35 + 3 years), 10
actively using AAS and 10 who denied ever using steroids. Serum lipid and hormone levels,
carotid intima-media thickness (IMT), arterial reactivity, and left ventricular (LV) dimensions
were measured. Vessel diameter was measured by ultrasound at rest, during reactive
hyperemia (an endothelium-dependent response, leading to flow-mediated dilation, FMD),
and after sublingual nitroglycerin (GTN, an endothelium-independent dilator). Arterial
reactivity was also measured in 10 age-matched non-bodybuilding sedentary controls. Use of
AAS was associated with significant decreases in high density lipoprotein cholesterol, sex
hormone binding globulin, testosterone and gonadotrophin levels, and significant increases
in LV mass and self-reported physical strength. Carotid IMT arterial FMD and GTN
responses were similar in both bodybuilding groups. The GTN responses were significantly
lower and carotid IMT significantly higher in both bodybuilding groups, however, compared
with the non-bodybuilding sedentary controls. It was concluded that although high-level
bodybuilding is associated with impaired vascular reactivity and increased arterial thickening,
the use of AAS per se is not associated with significant abnormalities of arterial structure or
function [01056].
Left ventricular myocardial dysfunction and cardiac hypertrophy
Chronic anabolic steroid use suppresses left ventricular functions. However, there is no
information regarding the chronic effects of anabolic steroids on right ventricular function
which also plays a key role in global cardiac function. The main objective of one study was to
investigate the effects of androgenic anabolic steroids usage among athletes on remodeling
the right part of the heart. Androgenic-anabolic steroids-using bodybuilders had smaller
diastolic velocities of both ventricles than drug-free bodybuilders and sedentary counterparts.
This study shows that androgenic anabolic steroids-using bodybuilders exhibited depressed
diastolic functions of both ventricles [08141].
Anabolic steroids cause a variety of side effects, among them a slight concentric left
ventricular hypertrophy. The objective of the present study was to clarify if they also induce
1078
alterations in left ventricular function. Fourteen male body builders with substantial intake of
anabolic steroids (users) were examined by standard echocardiography and cardiac tissue
Doppler imaging. They were compared to 11 steroid-free strength athletes (non-users) and
15 sedentary control subjects. Users showed an increased left ventricular muscle mass
index. The ratio of peak transmitral blood flow velocities during early diastolic filling and atrial
contraction did not differ between groups. In contrast an analogous tissue Doppler
parameter, the ratio of myocardial velocities during early and late ventricular filling in the
basal septum, was significantly lower in users when compared to non-users or controls. The
velocity gradient during myocardial E-wave in the posterior wall showed significantly lower
values in users as compared to controls. There were no differences in systolic function.
Summarizing strength athletes abusing anabolic steroids show negative alterations in
diastolic function [07064].
Recent echocardiographic studies of AAS users demonstrate an increase in septal and left
ventricular posterior wall thickness. This hypertrophy is greater in weight-trained individuals
using AASs than in weight-trained individuals provided placebo or not using AASs and
persists for years among former AAS users. Cardiac wall hypertrophy may not occur after
short-term ASA use. AAS use impairs measures of diastolic function (e.g. isovolumetric
relaxation time and altered tissue Doppler imaging of the left ventricle) that reflect impaired
relaxation and altered filling during diastole. Possible etiologies for impaired diastolic function
include increased collagen content or areas of focal necrosis, seen at autopsy of AAS users.
Cardiovascular performance also can be assessed by way of formal exercise testing;
although AASs may increase bulk and strength, they do not improve endurance. Despite
having similar aerobic and weight-training schedules as control subjects, AAS users had a
significantly decreased maximum oxygen consumption (VO2max; an index of metabolic and
cardiovascular endurance ability). Impaired diastolic function could contribute to decreased
VO2max [07058].
Bioptical data have shown that in athletes under the pharmacological effects of AAS, a focal
increase in myocardial collagen content might occur as a repair mechanism against
myocardial damage. To investigate the potential underlying left ventricular myocardial
dysfunction after chronic misuse of AAS in athletes by use of Doppler myocardial imaging
(DMI) and strain rate imaging (SRI). Standard Doppler echocardiography, DMI, SRI and ECG
treadmill test were undertaken by 45 bodybuilders, including 20 athletes misusing AAS for at
least 5 years (users), by 25 anabolic-free bodybuilders (non-users) and by 25 age-matched
healthy sedentary controls, all men. The mean number of weeks of AAS use per year was 31
in users, compared with 9 years in non-users, and the mean weekly dosage of AAS was 525
mg. The groups were matched for age. Systolic blood pressure was higher in athletes (145
vs 130 mm Hg) than in controls. Left ventricular mass index did not significantly differ
between the two groups of athletes. In particular, both users and non-users showed
increased wall thickness and relative wall thickness compared with controls, whereas left
ventricular ejection fraction, left ventricular end-diastolic diameter and transmitral Doppler
indexes were comparable for the three groups. Colour DMI analysis showed significantly
lower myocardial early: myocardial atrial diastolic wave ratios in users at the level of the
basal interventricular septum (IVS) and left ventricular lateral wall, in comparison with both
non-users and controls. In addition, in users, peak systolic left ventricular strain rate and
strain were both reduced in the middle IVS and in the left ventricular lateral free wall. By
stepwise forward multivariate analyses, the sum of the left ventricular wall thickness, the
number of weeks of AAS use per year and the weekly dosage of AAS were the only
independent determinants of middle IVS strain rate. In addition, impaired left ventricular
strain in users was associated with a reduced performance during physical effort. Several
years after chronic misuse of AAS, power athletes show a subclinical impairment of both
systolic and diastolic myocardial function, strongly associated with mean dosage and
1079
duration of AAS use. The combined use of DMI and SRI may therefore be useful for the early
identification of patients with more diffused cardiac involvement, and eventually for
investigation of the reversibility of such myocardial effects after discontinuation of the drug
[07065].
Anabolic androgenic steroids (AAS) are used by some athletes to enhance performance
despite the health risk they may pose in some persons. This work was carried out to evaluate
the possible structural and functional alterations in the heart using two-dimensional, M-mode,
tissue Doppler imaging (TDI) and strain rate imaging (SRI) in athletes using
supraphysiological doses of AAS. Additionally, the histological and ultrastructural changes in
cardiac muscles of adult albino rats after injection of sustanon, as an example of AAS, were
studied. Fifteen male bodybuilders using anabolic steroids constituted group 1, five male
bodybuilders who are not using anabolic steroids constituted group 2, and five nonathletic
males constituted negative control group (group 3). They were investigated by two-
dimensional, M-mode, TDI and SRI. Moreover, a study was performed on 30 adult albino
rats. They were divided into two groups. Group I (Control group) (n=10) was subdivided into
negative control, subgroup 1a (n=5), and subgroup 1b (n=5), which received 0.8 ml olive oil
intramuscular once a week for 8 weeks. Group II (Experimental group) (n=20) received
sustanon 10 mg/kg intramuscularly once a week for 8 weeks. The heart specimens were
prepared for light microscopy and transmission electron microscopy. Echocardiographic
results showed that bodybuilders who use steroids have smaller left ventricular dimension
with thicker walls, impaired diastolic function, as well as higher peak systolic strain rate in
steroid-using bodybuilders as compared to the other two groups. Light microscopy
examination of cardiac muscle fibers showed focal areas of degeneration with loss of
striations and vacuolation in the experimental group. Ultrastructural examination showed
disturbance of the banding pattern of the cardiac muscle fiber with disintegration, loss of
striations, dehiscent intercalated disc, and interrupted Z-bands. Administration of
supraphysiological doses of AAS caused severe deleterious effects in the myocardium both
in athletes and in experimental animals. The SRI shows promise in the early detection of
systolic dysfunction in those athletes who use steroids [09056].
Anabolic androgenic steroids (AAS) are sometimes used by power athletes to improve
performance by increasing muscle mass and strength. Recent bioptical data have shown that
in athletes under the pharmacological effects of AAS, a focal increase in myocardial collagen
content might occur as a repair mechanism against myocardial damage. To investigate the
potential underlying left ventricular myocardial dysfunction after chronic misuse of AAS in
athletes by use of Doppler myocardial imaging (DMI) and strain rate imaging (SRI). Standard
Doppler echocardiography, DMI, SRI and ECG treadmill test were undertaken by 45
bodybuilders, including 20 athletes misusing AAS for at least 5 years (users), by 25 anabolic-
free bodybuilders (non-users) and by 25 age-matched healthy sedentary controls, all men.
The mean number of weeks of AAS use per year was 31 in users, compared with 9 years in
non-users, and the mean weekly dosage of AAS was 525 mg. The groups were matched for
age. Systolic blood pressure was higher in athletes than in controls. Left ventricular mass
index did not significantly differ between the two groups of athletes. In particular, both users
and non-users showed increased wall thickness and relative wall thickness compared with
controls, whereas left ventricular ejection fraction, left ventricular end-diastolic diameter and
transmitral Doppler indexes were comparable for the three groups. Colour DMI analysis
showed significantly lower myocardial early: myocardial atrial diastolic wave ratios in users at
the level of the basal interventricular septum (IVS) and left ventricular lateral wall (p<0.01), in
comparison with both non-users and controls. In addition, in users, peak systolic left
ventricular strain rate and strain were both reduced in the middle IVS and in the left
ventricular lateral free wall. By stepwise forward multivariate analyses, the sum of the left
ventricular wall thickness the number of weeks of AAS use per year and the weekly dosage
1080
of AAS were the only independent determinants of middle IVS strain rate. In addition,
impaired left ventricular strain in users was associated with a reduced performance during
physical effort. Several years after chronic misuse of AAS, power athletes show a subclinical
impairment of both systolic and diastolic myocardial function, strongly associated with mean
dosage and duration of AAS use. The combined use of DMI and SRI may therefore be useful
for the early identification of patients with more diffused cardiac involvement, and eventually
for investigation of the reversibility of such myocardial effects after discontinuation of the drug
[06066].
The effects of anabolic androgenic steroids (AASs) on left ventricular (LV) diastolic function
in strength-trained athletes are controversial. The main objective of this study was to
evaluate the effects of regular AAS administration in bodybuilders using pulsed tissue
Doppler imaging (TDI) to evaluate LV relaxation properties. Fifteen male bodybuilders with a
history of intensive, long-term strength training and 16 age-matched sedentary controls were
recruited. Six of the bodybuilders reported regular use of AASs, and 9 were drug free. To
assess LV diastolic function, each subject underwent standard Doppler echocardiography
and pulsed TDI. Drug-using bodybuilders exhibited altered LV diastolic filling characterized
by a smaller contribution of passive filling to LV filling compared with their drug-free
counterparts. TDI measurements indicated that drug-using bodybuilders had smaller peak
E(m) than drug-free bodybuilders and sedentary controls, except at the level of the anterior
wall, at which peak E(m) was significantly smaller than in drug-free bodybuilders only. The
E/E(m) ratio, an index of LV filling pressures, was not affected by strength training or by AAS
use. Drug-using bodybuilders exhibited larger LV end-diastolic diameters, volumes, and
masses than their drug-free counterparts. However, no difference was found in LV wall
thickness between the groups. In conclusion, drug-using bodybuilders showed a decrease in
the contribution in LV passive filling to LV filling associated with a decrease in LV relaxation
properties. Because no wall thickening was obtained in drug-using bodybuilders, the
decrease in LV relaxation properties might have been be due to an alteration in the active
properties of the myocardium, but that has yet to be confirmed [06067].
It was compared cardiac parameters in weightlifters reporting long-term AAS use to those in
otherwise similar weightlifters without prior AAS exposure. It was performed 2D tissue-
Doppler and speckle-tracking echocardiography to assess left ventricular (LV) ejection
fraction, LV systolic strain, and conventional indices of diastolic function in long-term AAS
users (n=12) and otherwise similar AAS nonusers (n=7). AAS users (median [quartile 1,
quartile 3] cumulative lifetime AAS exposure, 468 [169, 520] weeks) closely resembled
nonusers in age, prior duration of weightlifting, and current intensity of weight training. LV
structural parameters were similar between the two groups; however, AAS users had
significantly lower LV ejection fraction (51 % [48, 54] versus 59 % [58 %, 62 %]), longitudinal
strain (17 % [14 %, 19 %] vs 21 % [20 %, 23%]), and radial strain (38 % [29 %, 44 %] vs 50
% [44 %, 62 %]). Ten of the 12 AAS users showed LV ejection fractions below the accepted
limit of normal (>55 %). AAS users also demonstrated decreased diastolic function compared
to nonusers as evidenced by a markedly lower early peak tissue velocity (7.4 [6.8, 7.9] cm/s
vs 9.9 [8.3, 10.5] cm/s) and early-to-late diastolic filling ratio (0.93 [0.88, 1.39] vs 1.80 [1.48,
2.00]). It was concluded that cardiac dysfunction in long-term AAS users appears to be more
severe than previously reported and may be sufficient to increase the risk of heart failure
[10327].
An increase in LV mass is an independent risk factor for CV disease. AS use has been
associated with an increase in LV mass, but there is conflicting data. There are some data in
AS users that suggest a reduction in systolic cardiac function although this is not a consistent
finding between studies. A reduction in diastolic function has been observed more frequently
and it has been suggested that a reduction in myocardial relaxation/elastance is associated
1081
with AS use [12114].
Left ventricular hypertrophy (LVH) has been reported in androgen abusers. Several groups
have shown that athletes using AAS have reduced end diastolic dimension, a thicker
posterior wall and interventricular septum, and a larger left ventricular mass than athletes not
using AAS. Cardiac muscle cells have receptors for androgens, and both testosterone and
dihydrotestosterone produce a hypertrophic response by acting directly on cardiac muscle
cells, increasing amino acid incorporation into protein. The problem is that LVH may persist
after discontinuation of AAS [12119].
In athletes who are mainly involved in bodybuilding, echocardiographic studies have derived
conflicting results regarding the effects of AAS on left ventricular mass and function. Most
studies compared the echocardiographic results between AAS users and nonusers or
healthy controls. It has been found significant left ventricular wall thickening in elite power
athletes using AAS compared to non AAS-users. Indeed, in one case the wall thickness was
16 mm. However, none of them demonstrated diastolic dysfunction. In contrast, a number of
studies found no significant difference in left ventricular hypertrophy between AAS users and
non-users. In most cases, the hypertrophy observed was concentric, as would be expected
after long-term static exercise training, while only a few showed eccentric hypertrophy due to
dilatation of the cardiac cavities. It is noteworthy that studies until early 2000 found no
particular evidence for systolic and diastolic dysfunction in athletes using AAS. However, with
the use of the latest echocardiographic techniques, such as tissue Doppler, some
researchers detected left ventricular diastolic dysfunction in athletes who are AAS users. In a
study of ours the use of pulsed tissue Doppler was helpful in the early detection of diastolic
dysfunction caused by AAS abuse, which was not detectable using the classical estimation
of transmitral flow. Moreover, the diastolic dysfunction was found to be correlated with the
dosage and the duration of use. Apart from left ventricular diastolic dysfunction, it was found
using Doppler myocardial imaging and strain rate imaging, also recorded early findings of
deteriorated systolic function in drug users. It is likely that studies using the latest non-
invasive diagnostic techniques will confirm the possibility that AAS lead to cardiomyopathy in
athletes, mainly due to a direct toxic effect on the myocardium. There are reports of athletes
with dilated cardiomyopathy and heart failure after AAS abuse [12126].
The use of anabolic androgenic steroids (AASs) has been associated with hypertrophy of the
left cardiac ventricle (LVH) as diagnosed by echocardiography. Case reports suggest that
AAS-related LVH may lead to sudden death. It was performed an investigation of the gross
cardiac pathological findings in deceased male AAS users in order to further elucidate the
proposed role of AAS in cardiac hypertrophy. Eighty-seven deceased males who tested
positive for AAS at autopsy and 173 age-adjusted control deceased males without suspected
AAS use were studied for cardiac hypertrophy. The AAS-positive subjects had been
examined at any of the six departments of forensic medicine in Sweden during the period
from 1989 to 2009. Data were assessed employing multivariate analyses controlling for body
weight, height, age, bleeding after trauma, and the impact of weight training. The analysis of
the logarithm of heart mass by multivariate statistics implied that strong correlations existed
between body mass and heart mass, height and heart mass, age and heart mass, and
trauma (bleeding) and heart mass. After controlling for these factors, a significantly higher
heart mass was found among the AAS-positive males. The findings suggest that use of AAS
may lead to cardiac hypertrophy with a direct cardiotropic effect [12128].
The role of AAS abuse in myocardial hypertrophy has been shown in animal and human
studies. In a recent investigation on rats treated with high-dose nandrolone for 8 weeks,
electrical remodelling and increasing myocytes nuclei diameter in the AAS group suggested
early stages of myocardial hypertrophy. Significant increase in left ventricular mass index,
1082
ranging from 7 to 24 percent, has been shown in studies on rats treated with low-dose and
high-dose AAS for 8-10 weeks. Another study on the AAS treatment for 3 weeks in mice
subjected to aerobic training and sedentary mice showed that high-dose AAS treatment in
sedentary mice results in increased ventricular mass index by 25 percent. Also, many case
reports of sudden cardiac death in athletes who abused AAS have shown clinically important
left ventricular hypertrophy. Association between AAS abuse and echocardiographical
detected myocardial hypertrophy has been shown in a study on athletes who chronically
abused AAS (median 24 months). In this study, hypertrophic index (interventricular septum
plus posterior wall thickness divided by the internal diameter) was significantly higher in AAS
(ex-)abusers compared with nonuser athletes. Moreover, the extent of AAS abuse was
linearly correlated with mean left ventricular wall thickness.It has been shown that long-term
AAS abuse increases peripheral vascular resistance, blood pressure, and myocardial
sympathetic nerve activity, which can explain mechanical stress-induced myocardial
hypertrophy in AAS abusers. Moreover, androgen receptors which are responsible for AAS-
induced hypertrophic effects on skeletal muscles are also present in myocytes and result in
increased protein anabolism within myocardial cells and interstitium [12125].
Anabolic androgenic steroids (AAS) abuse for improving physical appearance and
performance in body builders is common and has been considered responsible for serious
cardiovascular effects. Due to disagreement about cardiovascular side effects of these drugs
in published articles, this case control study was designed to evaluate the echocardiographic
findings in body builder athletes who are current and chronic abusers of these drugs. Body
builder athletes with continuous practice for the preceding two years and were training at
least twice weekly were selected and divided into AAS abuser and non user and compared
with age and BMI matched non athletic healthy volunteers (15 cases in each group). There
was no significant difference in left ventricular size or function either systolic or diastolic in
comparison to cases and control groups. The only difference was in diastolic size of septum
and free wall but observed differences were only significant between first (athletic with AAS
abuser) and third group (non athletic and nonuser). The difference between the above-
mentioned indexes was not significant between two groups of athletes. Observed differences
in diastolic size of septum and free wall is in favor of that long term abuse of anabolic steroid
results in accentuation of physiologic hypertrophy due to long term sport most probably due
to higher rate pressure product. Furthermore long term abuse and supra pharmacologic
doses do not have significant effect in size and left ventricular function [13798].
Concentric left ventricular myocardial hypertrophy is a common pathologic finding following

the chronic use of AAS. A 21-year-old, previously healthy weight lifter collapsed during a
benchpress workout, and paramedics found him in ventricular fibrillation. For the preceding
several months, he used parenteral AASs (nandrolone, 19-nortestosterone). Postmortem
findings included marked cardiac hypertrophy, regional myocardial fibrosis,a nd focal
myocardial necrosis along with hepatosplenomegaly and renal hypertrophy. There was no
evidence of recent myocardial inflammation. Other autopsies of AAS abusers have not
demonstrated cardiac hypertrophy, but histologic examination of cardiac tissue also detected
focal myocardial necrosis. The postmortem examination of 2 cases of sudden death in 2
previously healthy chronic AAS abusers (i.e. bodybuilders) demonstrated also focal
myocardial necrosis (contraction band necrosis) without evidence of significant
coronaryartery disease or myocardial hypertrophy. Other pathologic changes associated with
cardiac arrest in previously healthy athletes following AAS use (e.g. oxymesterone) include
hypertrophic cardiomyopathy, acute cellular ecrosis, interstitial fibrosis, and myocarditis.
Typically, there is no evidence ofc oronaryt hrombosis in these cases of sudde ndeath.
However, evidence of recent (i.e. 2 weeks) myocardial infarction may occur in these cases
without evidenc eof coronary artery disease [13003].
1083
Resistance training (RT) is a popular method of conditioning to enhance sport performance
as well as an effective form of exercise to attenuate the age-mediated decline in muscle
strength and mass. Although the benefits of RT on skeletal muscle morphology and function
are well established, its effect on left ventricular (LV) morphology remains equivocal. Some
investigations have found that RT is associated with an obligatory increase in LV wall
thickness and mass with minimal alteration in LV internal cavity dimension, an effect called
concentric hypertrophy. However, others report that short- (<5 years) to long-term (>18
years) RT does not alter LV morphology, arguing that concentric hypertrophy is not an
obligatory adaptation secondary to this form of exertion. This disparity between studies on
whether RT consistently results in cardiac hypertrophy could be caused by: (i) acute
cardiopulmonary mechanisms that minimise the increase in transmural pressure (i.e.
ventricular pressure minus intrathoracic pressure) and LV wall stress during exercise; (ii) the
underlying use of anabolic steroids by the athletes; or (iii) the specific type of RT performed.
It was proposed that when LV geometry is altered after RT, the pattern is usually concentric
hypertrophy in Olympic weightlifters. However, the pattern of eccentric hypertrophy
(increased LV mass secondary to an increase in diastolic internal cavity dimension and wall
thickness) is not uncommon in bodybuilders. Of particular interest, nearly 40 percent of all
RT athletes have normal LV geometry, and these athletes are typically powerlifters. RT
athletes who use anabolic steroids have been shown to have significantly higher LV mass
compared with drug-free sport-matched athletes. This brief review will sort out some of the
factors that may affect the acute and chronic outcome of RT on LV morphology. In addition, a
conceptual framework is offered to help explain why cardiac hypertrophy is not always found
in RT athletes [02031].
Cardiomyopathy
Anabolic-androgenic steroids are synthetic derivatives of testosterone that some athletes
have used to enhance muscle mass and improve their athletic performance. Ephedrine is a
potent sympathomimetic agent that can lead to cardiomyopathy similar to that seen with
catecholamine excess. Adverse cardiovascular events attributed to anabolic steroid and
ephedra use, such as arrhythmias, myocardial infarction, cardiomyopathy, and sudden
death, are rarely reported. Bodybuilders have used gamma-hydroxybutyrate, a potent
secretagogue of growth hormone, to promote muscle development. Although dilated
cardiomyopathy is a known complication of excess growth hormone levels, it has not been
associated with use of gamma-hydroxybutyrate. A healthy 40-year-old man was admitted to
our hospital for new-onset congestive heart failure and severe acute hepatitis that developed
several months after he began using anabolic-androgenic steroids, ephedra, and gamma-
hydroxybutyrate supplements. Analysis with an objective causality assessment scale
revealed a probable adverse drug reaction between the patient's use of anabolic steroids,
ephedra, and gamma-hydroxybutyrate and the development of his cardiomyopathy and
acute liver injury [05048].
Though doping has become increasingly ostracized in the context of professional sports, an
enormous number of unrecorded cases must be assumed in semi-professional competitive
sports as well as in popular sports. This holds especially true for those forms of sports which
are done in order to obtain a well-proportioned, athletic, healthy looking body. This case
report describes a formerly healthy young man who had to be urgently admitted to an
intensive care unit due to severe myocardial pump failure. As anamnestic information was
insufficient and inadequate, the taking of anabolic steroids in high doses was proven, as their
metabolites could be detected by urine analysis. Until now, myocardial contractile
dysfunction has persisted for more than twelve months after the initial admission. Though
other diagnoses which might have led to this impaired myocardial contractile performance
have been excluded, cardiomyopathy associated with the taking of anabolic steroids must be
assumed. Even in non-professional and public sports, a widespread abuse of doping
1084
substances exists. Hence, cardiomyopathy associated with the misuse of anabolic steroids
has to be considered especially in young, formerly healthy patients [02032].
TNF-alpha. The exact mediators of myocardial hypertrophy are diverse and vary from
mechanical stimuli to circulating humoral factors released by the heart and peripheral organs.
Exercise-induced cardiac hypertrophy is thought to be due to increases in the pre-load
(diastolic filling) on the heart, while the exact mechanisms for anabolic steroid-induced
myocardial hypertrophy are at present unknown. Recent studies have shown that circulating
cytokines such as TNF-alpha may play a role in cardiac remodeling and that anabolic
steroids strongly stimulate leukocyte TNF-alpha production. The question of whether a link
exists between anabolic steroid use, serum and myocardial TNF-alpha concentrations and
myocardial hypertrophy remains to be established. Exercise training in rats has been shown
to improve myocardial resistance to ischaemia-reperfusion injury. In addition, exercise-
induced physiological cardiac hypertrophy alters the heart’s susceptibility to ischaemia and
reperfusion and renders it more resistant to ischaemia-reperfusion injury in in vivo rat hearts.
These changes were associated with an increased energy charge and decreased lipid
peroxidation during ischaemia and reperfusion in the hearts from these animals. What,
however, remains unclear is whether anabolic steroid-induced hypertrophy alters the
susceptibility of the heart to ischaemia-reperfusion injury. In addition, should these hearts be
more susceptible to ischaemia-reperfusion injury, the mechanisms that contribute to this
phenomenon need to be elucidated. TNF-alpha has been implicated in
ischaemia/reperfusion injury. Mice devoid of myocardial TNF-alpha (knockouts) have been
shown to be more resistant to ischaemia-reperfusion injury than their wild-type counterparts
and treatment of rat hearts with anti-murine TNF-alpha antibody before ischaemia also
improved reperfusion function of these hearts. Besides cytokines, elevations in cytosolic
cAMP concentrations during ischaemia would be expected to increase cytosolic calcium
concentrations and exacerbate ischaemia-reperfusion injury in this model of ischaemia and
reperfusion. Interestingly, the basal myocardial cAMP concentrations are elevated by
anabolic androgenic steroids in isolated rat atrial muscle and contribute to the positive
inotropic response observed with steroid treatment. These recent findings suggest that
anabolic steroids could also potentially promote calcium overload during ischaemia in the
anabolic steroid-treated heart. The final question remains whether chronic anabolic steroid
treatment alters ventricular myocardial cAMP concentrations, and if so, what the effects of
this alteration are on susceptibility to ischaemia-reperfusion injury. Several studies have also
suggested that there is a correlation between myocardial cAMP and cGMP concentrations
and that the ratio between these cyclic nucleotides during ischaemia may be important in
determining the severity of ischaemia/reperfusion injury. The effects of anabolic steroids on
NO-cGMP pathway activity, as assessed by measuring myocardial cGMP concentrations,
also remain unknown [05047].
Cardiac (autonomic) dysfunction
To date no published data exist regarding the effects of chronic high-dose anabolic-
androgenic steroid administration on tonic cardiac autonomic control. The aim of this study
was to evaluate, by power spectral analysis of heart rate variability (HRV), the effects of
chronic treatment with supraphysiological doses of nandrolone decanoate (DECA) on tonic
cardiac autonomic regulation in sedentary rats. Male Wistar rats were treated weekly with 10
mg/kg of DECA (n=7) or vehicle (CONTROL, n=7) for 10 weeks. At the 8th week of
treatment, electrocardiogram was recorded in the conscious state, for time- and frequency-
domain HRV analysis. Parasympathetic indexes were reduced in DECA group: high-
frequency power, RMSSD, and pNN5. The sympathetic index LF/HF tended to be higher in
DECA group. In conclusion, chronic treatment with DECA, in rats, impairs tonic cardiac
autonomic regulation, which may provide a key mechanism for anabolic steroid-induced
1085
arrhythmia and sudden cardiac death [06068].
Anabolic-androgenic steroids abuse has been shown to affect the cardiomyocyte survival
and heart function in cell cultures, animal models and humans. A recent study reported that
both diastolic and systolic functional parameters are impaired in AAS abuser athletes
comparing with nonabuser athletes. In this study, echocardiography in AAS abusers showed
a significantly lower ejection fraction (50 % vs 59 %) and longitudinal strain compared to AAS
nonabusers. A similar trend was observed in diastolic functional parameters. The
mechanisms of high-dose AAS-associated heart dysfunction are still not thoroughly
investigated. However, some studies showed deleterious molecular and cellular effects of
high-dose AAS administration on myocardium which overlap early injury pathways of heart
failure. It is known that in hypertrophic myocardium, hypertrophy can be linked with any of
the heart failure signalling pathways, resulting in heart failure. It has also been shown that
AAS indirectly mediates the processes that precede mitochondrial damage, apoptosis and
sarcomere disruption. It has also been reported that high-dose AAS treatment in small
animal models is associated with interstitial collagen deposition and fibrosis. Fibrosis is
assumed to occur initially as an adaptation in myocardial hypertrophy to preserve the
function of the ventricles and, thereafter, as a repair mechanism to compensate apoptotic
myocardial cell loss [12125].
.
An echocardiographic study of 47 strength-training individuals (46 male subjects), 28 of
whom were regular AAS users, revealed a lower systolic function in AAS users versus
nonusers, ejection fractions 58 versus 63 percent, respectively. In addition, there was
evidence of reduced diastolic function by tissue Doppler measurement in the AAS users (i.e.
their hearts were weaker and stiffer). Another study of 12 long-term AAS users noted that
compared with controls, they were noted to have significant systolic cardiac dysfunction as
measured by lower left ventricular ejection fraction (50 % vs 59 %). An Italian Doppler
imaging study also showed reduced systolic function but in a regional distribution [12119].
One study aimed to evaluate if androgenic-anabolic steroids (AAS) abuse may induce
cardiac autonomic dysfunction in recreational trained subjects. Twenty-two men were
volunteered for the study. The AAS group (n=11) utilized AAS at mean dosage of 410 ± 78.6
mg/week. All of them were submitted to submaximal exercise testing using an Astrand-
Rhyming protocol. Electrocardiogram (ECG) and respired gas analysis were monitored at
rest, during, and post-effort. Mean values of VO2 , VCO2 , and VE were higher in AAS group
only at rest. The heart rate variability variables were calculated from ECG using MATLAB-
based algorithms. At rest, AAS group showed lower values of the standard deviation of R-R
intervals, the proportion of adjacent R-R intervals differing by more than 50 ms (pNN50), the
root mean square of successive differences (RMSSD), and the total, the low-frequency (LF)
and the high-frequency (HF) spectral power, as compared to Control group. After
submaximal exercise testing, pNN50, RMSSD, and HF were lower, and the LF/HF ratio was
higher in AAS group when compared to control group. Thus, the use of supraphysiological
doses of AAS seems to induce dysfunction in tonic cardiac autonomic regulation in
recreational trained subjects [13134].
The harmful cardiovascular effects of indiscriminate use of AAS to the cardiac structure and
functioning can include increased cardiac tissue collagen, imbalance of vasomotor tone,
reduction in the number of capillaries, and pathological cardiac hypertrophy in animal and
human models. In addition, supraphysiological doses of AAS have already demonstrated that
they may inhibit angiogenesis induced by physical training. In a study involving 152 men
1086
(between 18 and 40 years of age), non-athletes (n=52), strength-endurance athletes (n=52),
and athletes (n=52) who admitted using AAS, showed ventricular volumes and ventricular
wall thickness statistically higher than in non-users, and lower ejection fraction in both
ventricles. An increase in cardiac collagen can induce electrophysiological changes in the
myocardium with abnormal propagation of the excitation wave favoring tachycardia, which
may explain the repeated occurrence of sudden death in users of AAS. But this possibility is
raised based on a case report of a user 31 years of age. On the other hand, another study
reports other factors occurring after massive doses of AAS for several years, namely, a case
of ventricular fibrillation during exercise, one heart failure, and arterial thrombus in the lower
left leg. Interestingly, all these patients had increased cardiac hypertrophy and fibrosis in the
myocardium. It is certain that the character of case reports makes these inconsistent data.
The development of experimental models is useful in helping us better understand the
deleterious effects of SEA on cardiovascular tissue, but must consider the obvious limitations
of extrapolating data to humans. So that our knowledge of this adverse effect is dependent
on cases recorded and documented in the scientific literature. Owing to this limitation, it was
investigated the effect of administration of nandrolone decanoate in rats for 10 weeks,
associated with swim training. They found a 10 percent increase in cardiac hypertrophy in
rats treated with nandrolone decanoate and 17 percent who had been administered this drug
associated with physical training, which were significantly higher than that found in mice not
treated with the drug. Despite the limitations of data with an animal model, it was
demonstrated by echocardiography that 17 bodybuilders and power-lifters and AAS users
manifested cardiac hypertrophy. The most interesting aspect of the study was that 15 of the
subjects who did not use AAS for at least 12 months still showed a slight concentric
ventricular hypertrophy compared with those who remained on the drug [14247].
Several studies have demonstrated changes in the cardiac functioning aspects of AAS users,
especially on diastolic functioning. Diastolic dysfunction has been strongly associated with
collagen deposition. Fibrillar collagens, types I and III, are the major structural proteins of the
myocardial collagen matrix, exerting an important influence on ventricular compliance.
Exposure to supraphysiological doses of AAS can lead to tissue necrosis and diastolic
dysfunction, resulting in structural changes similar to those seen in the earlier stages of heart
failure. There are several mechanisms that appear to be related to cardiac hypertrophy and
collagen accumulation. AAS can induce such hypertrophy through nuclear receptors, acting
directly on RNA and increasing the protein synthesis and acting as well on specific enzymes,
on ions flow and the structural matrix in the myocardium. Increased circulating pro-
inflammatory cytokines such as TNF-alpha and increased cAMP concentration have been
documented, which contributes to the positive inotropic response through the calcium in the
cytosol of the myocardial cell. However, the exact mediators of such effects are diverse and
not fully elucidated [14247].
Vascular reactivity
The use of supraphysiological doses of anabolic androgenic steroids can have serious side
effects. One article reported the case of a young man who suffered potentially life-
threatening arterial thromboses following the use of these drugs [14046].
Anabolic androgenic steroids are used by some bodybuilders to enhance performance. While
the cardiovascular implications of supraphysiological androgen levels requires further
clarification, use is associated with sudden death, left ventricular hypertrophy, thrombo-
embolism and cerebro-vascular events. To further understand the effect of androgenic
anabolic steroid abuse on vascular function, this study assessed vascular stiffness (pulse-
wave analysis) and cardiovascular risk factors in 28 male, bodybuilding subjects, of whom
ten were actively receiving anabolic agents (group A; 26 years) and eight had undergone a
1087
3-month "wash-out" period (group B; 32 years). The remaining ten bodybuilding subjects
(group C; 24 years) denied any past use of anabolic steroids or other performance
enhancing drugs. Comparisons were made with ten sedentary male controls (group D, 29
years). Endothelial independent dilatation in response to glycerol trinitrate was significantly
impaired in the group currently using anabolic steroids (group A) compared with the other
three groups, whereas no significant differences in endothelial-dependent dilatation were
detected between the groups. Previous studies described a decline in vascular reactivity
occurring in bodybuilding subjects which is independent of anabolic steroid use and may
result from smooth muscle hypertrophy with increased vascular stiffness. This study revealed
impaired vascular reactivity associated with anabolic agents and that improvement in
vascular function may occur following their discontinuation [06070].

Increased systemic inflammation and oxidative stress participate in the mechanism of
various cardiovascular diseases. These two phenomena are associated with venous
thrombosis and endothelial dysfunction. Curiously, prolonged use of stanozolol decreased
the mitochondrial oxidative stress of skeletal muscle in rats. However, hepatic oxidative
stress has increased in rats at high doses (2 mg/kg body weight). Hepatic oxidative stress is
associated with the production of C-reactive protein, a potent pro-inflammatory agent
involved in vascular dysfunction, arterial hypertension, and ischemic heart disease. However,
it is worth noting that the increase in hepatic oxidative stress was seen in rats, so it cannot be
said that the same would occur in human AAS users. In human experiments, levels of
homocysteine were significantly higher in users of AAS for 21 ± 2 years compared with a
group that also used AAS for 21 ± 3 years, but discontinued the use for 3 months, and
control groups of non-users of AAS. Homocysteine can be involved in the etiology of various
cardiovascular diseases. Androgens can exert vasorelaxant effects, but chronic exposure of
replacement doses decreases the vascular reactivity to vasodilators. In fact, analysis of pulse
waves in response to glyceryltrinitrate shows that endothelium independent vasodilator
function was impaired in young adult bodybuilders. The vasodilatation was reduced by half
compared to previous users who discontinued the use and was only 30 percent of the value
obtained in a control group of AAS non-users. An impaired vasodilator functioning was also
found in young adults manifesting hypogonadism (35 ± 4 years old) even at physiological
doses used in testosterone replacement therapy. Finally, a lower vasodilator functioning was
accompanied by an increased sympathetic nerve activity to the vasculature among AAS
users based on microneurography measures. This adverse effect of AAS calls attention, but
the data are inconsistent [14247].
Dyslipidemia
Somatic adverse reactions of AAS abuse include disturbances in the lipid profile,
cardiovascular effects, dermal manifestations, and endocrine adverse reactions. Decreased
secretion of the pituitary luteinizing hormone (LH) and follicle-stimulating hormone (FSH) is
commonly reported. These effects result from the negative feedback of androgens on the
hypothalamic–pituitary–gonadal axis, and possibly from the local suppressive effects of
exogenous androgens on the testes. Long persistence of low levels of gonadotropins has
been described in nandrolone abusers, with a significant correlation between 19-
norandrosterone and LH and FSH. Abuse of AASs leads to increased levels of low-density
lipoprotein (LDL) and apolipoprotein B (ApoB; the major component of the LDL particle), but
also a decreased level of high-density lipoprotein (HDL) and apolipoprotein A1 (ApoA1; the
major component of the HDL particle). A perturbation in the lipoprotein profile has been
observed even after one single dose of testosterone in healthy volunteers through increased
total cholesterol levels and induced expression of 3-hydroxy-3-methyl-glutaryl-CoA
1088
reductase, 2 days after administration. Stanozolol has been studied with respect to its effect
on HDL and found to give a 71 percent decrease in HDL levels after 7 days’ treatment in
association with changes in hepatic triglyceride lipase. These effects on the lipid profile
during long-term abuse are associated with an increased risk of coronary artery disease. In
contrast to their unfavorable effects on lipids, AASs may favorably lower lipoprotein(a) (Lp[a])
concentrations. “Lp(a)” is an LDL-like particle and contains, in addition to LDL, a specific
protein component, apolipoprotein(a) (apo[a]). High levels of Lp(a) have been reported as a
risk factor for ischemic heart disease and peripheral vascular disease. To study the effect
and time profile of different doses of testosterone enanthate on the blood lipid profile and
gonadotropins 25 healthy male volunteers aged 27-43 years were given 500 mg, 250 mg,
and 125 mg of testosterone enanthate as single intramuscular doses of Testoviron(®) Depot.
Luteinizing hormone (LH), follicle-stimulating hormone (FSH), blood lipid profile (total
cholesterol, plasma [p-] low-density lipoprotein, p-high-density lipoprotein [HDL], p-
apolipoprotein A1 [ApoA1], p-apolipoprotein B, p-triglycerides, p-lipoprotein(a), serum [s-]
testosterone, and 25-hydroxyvitamin D3) were analyzed prior to, and 4 and 14 days after
dosing. Testosterone and epitestosterone in urine (testosterone/epitestosterone ratio) were
analyzed prior to each dose after a washout period of 6-8 weeks. All doses investigated
suppressed the LH and FSH concentrations in serum. LH remained suppressed 6 weeks
after the 500 mg dose. These results indicate that testosterone has a more profound
endocrine effect on the hypothalamic-pituitary-gonadal axis than was previously thought.
There was no alteration in 25-hydroxyvitamin D3 levels after testosterone administration
compared to baseline levels. The 250 and 500 mg doses induced decreased concentrations
of ApoA1 and HDL, whereas the lowest dose (125 mg) did not have any effect on the lipid
profile. It was concluded that a single doses of testosterone produced a dose-dependent
increase in serum testosterone concentrations together with suppression of s-LH and s-FSH.
Alterations in ApoA1 and HDL were observed after the two highest single doses. It is
possible that long-time abuse of anabolic androgenic steroids will lead to alteration in vitamin
D status. Knowledge and understanding of the side effects of anabolic androgenic steroids
are important to the treatment and care of abusers of testosterone [150191].
Although several reports have been reported of adverse cardiovascular effects associated
with the use of supraphysiological doses and/or prolonged use of such drugs, changes in
lipid profile have also been observed both at therapeutic doses and in the short term. A
meta-analysis of 19 studies, totaling 272 men manifesting hypogonadism (44 ± 4 years old)
who used physiological doses of intramuscular testosterone (179 ± 13 mg every 16 days for
6 months), was conducted in 2001. There was a reduction of 14 mg/dL (17-11 mg/dL) in total
cholesterol, 5 mg/dL (8-1 mg/dl) in LDL cholesterol, 4 mg/dL (5-2 mg/dL) in HDL cholesterol,
and 5 mg/dL (−6 to 4 mg/dL) in triglycerides. However, effect size was not significant. Only
HDL cholesterol reduction tended to result in a significant outcome Another meta-analysis of
studies involving 1,083 middle-aged and older men (mean age 65 years) designed to study
the prevention of loss of muscle mass and bone found a significant reduction in HDL
cholesterol, at therapeutic doses between controls and experimental participants. Therefore,
the evidence points to possibilities of damage only in HDL cholesterol, and these numbers
are not for athletes. The reason for the absence of data from athletes is the fact that when
athletes do use AAS, they use them in supraphysiological doses [14613].
While the use of physiological doses promotes a mild reduction in serum concentration of
HDL cholesterol, the self-administration of several AAS simultaneously (at least three drugs
and up to nine drugs for those who used the most, without prescription, and in
supraphysiological doses in all cases) during 14 weeks decreased the concentration of HDL
cholesterol to less than half in 19 bodybuilders (31 ± 7 years old) who had undergone an
average of seven cycles (range: 1–30) over an average period of time of 4.8 years (range: 1–
14). This phenomenon was accompanied by a reduction, also by half, of apolipoprotein A1,
1089
which mediates the HDL cholesterol formation. Six weeks following cessation of the use of
AAS, the worsening of the lipid profile still persisted. Halving in HDL cholesterol was also
observed by Lane and colleagues in 10 youths who admitted using AAS compared to
nonusers. Although the administration of nandrolone decanoate for 8 weeks at physiological
doses (200 mg/week) did not affect the HDL cholesterol or other serum lipoproteins, a single
dose of 500 mg of testosterone increased the serum concentration of total cholesterol by 15
percent. Interestingly, molecular biology tests revealed that this increase was accompanied
by increased mRNA and protein expression of HMG-CoA reductase, a key enzyme in the
formation of cholesterol by the liver. The trend found in reduction of HDL cholesterol at
physiological doses is confirmed by the very significant reduction following treatment with
high doses of AAS. Although there are few studies, the data presented indicate an adverse
effect of AAS use on HDL cholesterol [14613].
An association between premature cardiovascular events and the misuse of AAS in athletes
has been observed. This is believed to be primarily mediated through changes in the lipid
profile. Although endurance exercise favorably affects the lipid profile primarily through an
antiatherogenic effect of raising high-density lipoprotein (HDL) cholesterol and lowering
triglycerides, heavy resistance training alone fails to show a similar effect. This implies that
the effect of AAS on lipid profile may confounded by the type of training the athlete pursues.
Multiple prospective studies of AAS effects on cholesterol have yielded varied results. The
majority of results (studies ranging from 3 to 26 weeks) show no overall change in levels of
total serum cholesterol; however, some individual studies show increases and others show a
decrease in total cholesterol. Despite the varied results on total cholesterol, the effects on
HDL seem more consistent. Reductions in HDL range from 39 to 70 percent depending on
the type of AAS and also appear to be dose dependent. Several studies show reductions of
HDL down into the teens, which, based on Framingham data, places these patients at a
three times greater risk for coronary artery disease compared with men with HDL above 50
mg/dL There even appears to be some variation in the dose effect based on gender. In one
study of hemodialysis patients, weekly administration of nandrolone resulted in a reduction of
HDL-2 and apolipoprotein A-1 levels, complemented by a corresponding increase in
apolipoprotein B and triglycerides. The oral 17-alpha alkylated steroids, as opposed to
parenteral nandrolone, seem to exert the greatest effects on lipids and lipoproteins, which
can be seen as early as the first few days of administration. This reduction often reaches a
plateau effect after 8 weeks of use. Although the direct mechanisms of action and impact on
cardiovascular disease remain unproven this negative effect on HDL suggests a higher risk
of atherogenesis. The alteration in lipid profile seems completely reversible upon
discontinuation, but may take at least 4 to 12 weeks, often depending on dose and duration
of steroid use [06031].
In addition to HDL effects, an increase in low-density lipoprotein (LDL) appears to parallel the
HDL reduction. Significant LDL increases were appreciated in just 8 weeks of anabolic
steroid use in one study, and often had not returned to baseline 6 weeks after cessation of
AAS use. Because HDL acts as a primary scavenger of LDL particles, LDL changes possibly
reflect a secondary rather than primary effect. These alterations in HDL and LDL cholesterol
are more profound in athletes engaged in heavy resistance sports taking AAS as compared
with endurance athletes, possibly reflecting the influence of the athlete’s training regimen. In
one arm (self-administered, prospective, nonblinded portion) of the study from the
Netherlands, reductions in lipoprotein (a), which is an independent risk factor for vascular
disease, seem to provide a slightly beneficial effect on the lipid profile. Reductions of as
much as 50 percent of lipoprotein (a) were observed in as little as 8 weeks of AAS use, and
remained decreased at 6 weeks postcessation. Longer duration of AAS did not correlate
directly with further serum reductions, but did demonstrate a more prolonged return to
baseline of lipoproteins. In the second phase (randomized controlled trial, double-blinded
1090
portion) of the Dutch study, both placebo groups and nandrolone decanoate both
demonstrated reductions (19 % and 40 %, respectively) in lipoprotein (a) that was
nonsignificant. One explanation for the difference is that the oral 17-alpha alkylated steroids,
taken in the first portion, seem to exert the greatest effects on lipids and lipoproteins as
opposed to the parenterally administered nandrolone used in the second arm. This is
mediated by the first-pass metabolism of the orally administered drugs through the liver.
These effects can be seen as early as the first few days of administration, and seem to be
more dependant on the type of steroid as opposed to the duration, although no long-term
studies exist currently. Concentrations of lipoprotein (a) have been shown to have a close
correlation with deposition in vascular walls, are often genetically determined, and seem
resistant to current lipid [06031].
Although several reports have been reported of adverse cardiovascular effects associated
with the use of supraphysiological doses and/or prolonged use of such drugs, changes in
lipid profile have also been observed both at therapeutic doses and in the short term. A
meta-analysis of 19 studies, totaling 272 men manifesting hypogonadism (44 ± 4 years old)
who used physiological doses of intramuscular testosterone (179 ± 13 mg every 16 days for
6 months), was conducted in 2001. There was a reduction of 14 mg/dL (17-11 mg/dL) in
total cholesterol, 5 mg/dL (8-1 mg/dL) in LDL cholesterol, 4 mg/dL (5-2 mg/dL) in HDL
cholesterol, and 5 mg/dL (−6 to 4 mg/dL) in triglycerides. However, effect size was not
significant. Only HDL cholesterol reduction tended to result in a significant outcome. Another
meta-analysis of studies involving 1,083 middle-aged and older men (mean age 65 years,
range 50-78 years) designed to study the prevention of loss of muscle mass and bone found
a significant reduction in HDL cholesterol, at therapeutic doses between controls and
experimental participants. Therefore, the evidence points to possibilities of damage only in
HDL cholesterol, and these numbers are not for athletes. The reason for the absence of data
from athletes is the fact that when athletes do use AAS, they use them in supraphysiological
doses [14247].
While the use of physiological doses promotes a mild reduction in serum concentration of
HDL cholesterol, the self-administration of several AAS simultaneously (at least three drugs
and up to nine drugs for those who used the most, without prescription, and in
supraphysiological doses in all cases) during 14 weeks decreased the concentration of HDL
cholesterol to less than half in 19 bodybuilders (31 ± 7 years old) who had undergone an
average of seven cycles (range: 1-30) over an average period of time of 5 years (range: 1-
14). This phenomenon was accompanied by a reduction, also by half, of apolipoprotein A1,
which mediates the HDL cholesterol formation. Six weeks following cessation of the use of
AAS, the worsening of the lipid profile still persisted. Halving in HDL cholesterol was also
observed in 10 youths who admitted using AAS compared to nonusers. Although the
administration of nandrolone decanoate for 8 weeks at physiological doses (200 mg/week)
did not affect the HDL cholesterol or other serum lipoproteins, a single dose of 500 mg of
testosterone increased the serum concentration of total cholesterol by 15 percent.
Interestingly, molecular biology tests revealed that this increase was accompanied by
increased mRNA and protein expression of HMG-CoA reductase, a key enzyme in the
formation of cholesterol by the liver. The trend found in reduction of HDL cholesterol at
physiological doses is confirmed by the very significant reduction following treatment with
high doses of AAS. Although there are few studies, the data presented strongly indicate an
adverse effect of AAS use on HDL cholesterol [14247].
Anabolic-androgenic steroids (AAS) are used to enhance physical performance and/or

appearance. The aim of this study was to evaluate the influence of the concomitant use of
alcohol, tobacco, cocaine, and AAS on blood lipid profiles of 145 asymptomatic male
bodybuilders from the Northeast region of Brazil. Interviews, clinical exams, and serological
1091
evaluations were performed on all participants between 2007 and 2009. All subjects' self-
reported use of testosterone or its derivatives, 118 individuals reported alcohol intake, 27-
reported cigarette smoking, and 33 confirmed cocaine use. Four subjects were users of all
drugs at the same time. Higher levels of total cholesterol and LDL-cholesterol were observed
among concomitant users of alcohol, tobacco, cocaine, and AAS. The study's limitations are
noted [14251].
Altered lipid profiles in AAS users are reflected in increased low-density lipoprotein and
decreased high-density lipoprotein. The oral C-17 alkylated steroids seem to exert the
greatest effects on the lipid profile. Thrombus formation has been postulated by way of these
adverse lipid changes and is supported further by findings of AAS-induced increased platelet
aggregation, enhanced coagulation enzyme activity, and coronary vasospasm. Hypertension
in AAS users has been reported and is likely the result of blood volume increases and fluid
retention. This effect, as well as the finding of increased septal thickness and left ventricular
mass reported in AAS users, can lead to significant detrimental cardiac remodeling [07008].
The 17-alkylated compounds can provoke impairment of hepatic function and dyslipidemia.
There are no consistent changes reported for total cholesterol. The oral and the parenteral
forms of 17-a- alkylated steroids, though not the parenteral forms of androgen esters, have
been associated with a lowering effect on high density lipoprotein cholesterol (HDL-C),
thereby increasing the chance of the development of coronary heart disease (CHD).
However, HDL-C fractions start increasing after cessation of AS use and tend to normalize in
exercised men within 10 weeks [03002].
Postprandial triglyceridaemia, low-density lipoprotein particle size and lipoprotein

Although androgenic hormones decrease HDLC concentration, no direct evidence has linked
them to atherosclerosis. The present study was undertaken to extend our ability to assess
risk associated with androgen induced lipoprotein(Lp) changes by simultaneously gathering
information about postprandial triglyceridaemia (PPT), LDL particle size, HDL and Lp(a) in
men either taking exogenous androgens or with suppressed endogenous androgen
concentrations. The experimental groups comprised nine male bodybuilders who self-
administered anabolic-androgenic steroids (AAS) for a mean period of 6.5 weeks, and 10
healthy men whose testosterone concentration had been reversibly suppressed for 5 weeks
using the GnRH agonist triptorelin (Decapeptyl; D-Trp-6-LHRH). A separate group receiving
no hormonal treatment provided analytical control (n=7). Lipoprotein size was assessed by
gradient gel electrophoresis categorisation (GGE), lipoprotein concentrations by immuno and
enzymatic assays and PPT by a standardised oral fat tolerance test (65 g/m2). Testosterone
concentration was significantly reduced on triptorelin from 7.32 + 1.92 to 1.15 + 0.57 ng/mL.
High dose AAS use was confirmed by urinalysis. With AAS use, mean HDLC and Lp(a)
concentrations and PPT decreased from 0.9 + 0.3 to 0.7 + 0.3 mmol/L, 125 + 128 to 69 + 73
U/L and 11.6 + 10.0 mmol/L h to 7.5 + 5.4 mmol/L h respectively. Mean total cholesterol and
LDLC were unchanged. LDL size was unchanged in six AAS users, decreased in one but
remaining in the normal size range, and increased in two from small LDL to the normal
range. Size changes in the latter two subjects were associated with 42 and 58 percent
reductions in PPT respectively. In the triptorelin group, mean total cholesterol, HDLC and
Lp(a) were increased from 4.8 + 0.8 mmol/L to 5.2 + 1.0 mmol/L, 1.1 + 0.2 to 1.4 + -0.3
mmol/L and 278 + 149 to 377 + 222 U/L respectively. Mean LDLC concentration and PPT
were unchanged. LDL particle size increased in four, decreased in two, and was unchanged
in four subjects. LDL size decreased in two and showed no change in the other five control
subjects. Other lipid measures were unchanged in the control group. Thus, apart from
lowering HDLC concentrations, no other potentially atherogenic effects of endogenous
androgens or AAS were observed. A suppression of Lp(a) as well as a reduced PPT and
1092
increased LDL size in predisposed individuals may be antiatherogenic effects of AAS
[01055].
Lipid profiles in rats

Dietary protein amount and source, hypertrophy resistance training (RT) and anabolic
androgenic steroids (AAS) may affect body weight and plasma and hepatic lipid profile. 157
adult male Wistar rats were randomly distributed in 16 experimental groups resulting in:
normal-protein (NP) or high-protein (HP) diets, whey or soy-protein diets, with or without RT
and with or without AAS, for 3 months. Final body weight was lower in the RT and AAS
groups compared to sedentary and non- AAS groups, respectively. Plasma total cholesterol
(TC) was lower for the HP compared to the NP diets, for the whey compared to the soy-
protein diets and for the AAS compared to the non-AAS groups. Plasma HDL-cholesterol
was higher in the RT groups but lower for the AAS groups, the HP and the soy-protein diets.
Plasma triglycerides (TAG) were lower for the HP diet, for the RT and the non-AAS groups.
Liver TC was lower for the NP, for the soyprotein and for the AAS groups. Liver TAG were
lower for the whey-protein diet, RT and non-AAS groups. Some interactions were found,
such as the greater effect of AAS on reducing body weight of rats that performed RT or
ingested a HP diet. HDL-cholesterol was higher when RT was combined with HP diets or
non-AAS and when HP diets were combined with non-AAS. Groups that combined RT with
non-AAS administration obtained the lowest hepatic TAG. Among all the interventions tested,
AAS was the factor that most negatively affected plasma and hepatic lipid profile, whereas
HP diets and RT could benefit lipid profile, especially when combined [13143].
Impaired exercise-induced cardioprotection of antioxidant enzymes
High doses of anabolic androgenic steroids (AAS) impair the cardioprotective effects of
exercise against ischemia/reperfusion (I/R) insult, possibly through cellular redox imbalance.
Here, the effect of nandrolone decanoate (DECA) treatment on heart redox metabolism was
investigated during I/R in sedentary and exercised rats. DECA treatment significantly
reduced superoxide dismutase and glutathione reductase activities in exercised rats after
heart reperfusion. Catalase and glutathione peroxidase activities were not affected by DECA
in both sedentary and trained rats, regardless the I/R period. DECA also induced myocardial
oxidative stress, as evidenced by the reduced levels of total reduced thiols after heart
reperfusion in exercised rats treated with the anabolic steroid. These results indicate that
cardiotoxic effects of supraphysiological doses of AAS involve reduced heart antioxidant
capacity [13126].
Uncertainty remains about possible cardiac adaptation to resistance training. Androgenic

anabolic steroids (AAS) use plays a potential role and may have adverse cardiovascular
effects. To elucidate the effect of resistance training and of AAS-use on cardiac dimensions
and function cardiac magnetic resonance (CMR) were performed in 156 male subjects aged
18-40 years: 52 non-athletes (maximum of 3 exercise hours/week), 52 strength-endurance
(high dynamic-high static, HD-HS) athletes and 52 strength (low dynamic-high static, LD-HS)
trained athletes (athletes ≥ 6 exercise hours/week). Twenty-eight LD-HS athletes denied and
24 admitted to AAS use for an average duration of 5 years (range 3 months-20 years). No
significant differences were found between non-athletes and non-AAS-using LD-HS athletes.
AAS-using LD-HS athletes had significantly larger LV and RV volumes and LV wall mass
than non-AAS-using LD-HS athletes, but lower than HD-HS athletes. In comparison to all
other groups AAS-using LD-HS athletes showed lower ejection fractions of both ventricles
(LV/RV EF 51/48 % versus 55-57/51-52 %) and lower E/A ratios (LV/RV 1.5/1.2 versus 1.9-
2.0/1.4-1.5) as an indirect measure of diastolic function. Linear regression models
demonstrated a significant effect of AAS-use on LV EDV, LV EDM, systolic function and
mitral valve E/A ratio. It was concluded that strength athletes who use AAS show significantly
1093
different cardiac dimensions and biventricular systolic dysfunction and impaired ventricular
inflow as compared to non-athletes and non-AAS-using strength athletes. Increased
ventricular volume and mass did not exceed that of strength-endurance athletes. These
findings may help raise awareness of the consequences of AAS use [13127].
The beneficial effects of exercise in reducing the incidence of cardiovascular diseases are
well known and the abuse of anabolic androgenic steroids (AAS) has been associated to
cardiovascular disorders. Previous studies showed that heart protection to ischemic events
would be mediated by increasing the antioxidant enzyme activities. Here, we investigated the
impact of exercise and high doses of the AAS nandrolone decanoate (DECA), 10 mg/kg
body weight during 8 weeks, in cardiac tolerance to ischemic events as well as on the activity
of antioxidant enzymes in rats. After a global ischemic event, hearts of control trained (CT)
group recovered about 70 percent of left ventricular developed pressure, whereas DECA
trained (DT), control sedentary (CS) and DECA sedentary (DS) animals recovered only
about 20 percent. Similarly, heart infarct size was significantly lower in the CT group
compared to animals of the three other groups. The activities of the antioxidant enzymes
superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione reductase (GR)
were significantly higher in CT animals than in the other three groups, whereas catalase
activity was not affected in any group. Together, these results indicate that chronic treatment
with DECA cause an impairment of exercise induction of antioxidant enzyme activities,
leading to a reduced cardioprotection upon ischemic events [06071].
Total cholesterol
The effects of AAS on serum total cholesterol metabolism are not determined in detail. Most
prospective studies, investigating either low or high doses of single drug use or polydrug
administration for periods from 3 to 26 weeks, reported no alterations of serum total
cholesterol levels. Nevertheless, some studies found that AAS were able to induce an
increase of serum total cholesterol levels, whereas others observed a decrease. The nature
of this discrepancy of serum cholesterol response has yet to be established. However, the
response of serum total cholesterol levels to nandrolone decanoate administration seems to
be very consistent. The use of therapeutic and supratherapeutic doses of nandrolone
decanoate does not seem to affect serum cholesterol levels. In strength athletes,
testosterone enantate administered by intramuscular injection may have no effect on serum
total cholesterol levels after 3 weeks of administration, but after 6 weeks a reduction of total
cholesterol may occur. On the other hand, supratherapeutic doses of another testosterone
substance (i.e. testosterone cipionate) do not exert significant effects on serum total
cholesterol levels [04002].
High-density lipoprotein-cholesterol and its subfractions

High-density lipoprotein (HDL)-cholesterol and its subfractions have been recognised as
independent risk factors for the occurrence of cardiovascular disease. There is strong
evidence that AAS administration will induce remarkable reductions of the serum levels of
these lipoproteins. The suppressive effect varies between different androgenic-anabolic
compounds, with decrements of HDL-cholesterol ranging from 39-70 percent. The most
pronounced suppression has been observed in serum levels of HDL2-cholesterol rather than
HDL3-cholesterol, with suppression ranging from 55 to 89 percent for HDL2-cholesterol and
from 13 to 55 percent for HDL3-cholesterol. The reduction of serum HDL-cholesterol levels is
mediated by hepatic triglyceride lipase (HTGL), an enzyme that regulates serum lipids and
exposes the AAS-using athletes to an increased atherogenic risk. The orally taken 17-
[alpha]-alkylated substances (such as stanozolol, oxymetholone and metandienone) exert
much stronger effects than other AAS. The decline of HDL-cholesterol can be observed
within a few days of starting steroid administration. After an initial strong negative effect, the
suppression of the serum HDL-cholesterol and its subfractions continues at a more moderate
1094
level. After 8 weeks of AAS administration no further decline of HDL-, HDL2- and HDL3-
cholesterol can be observed. Short-term administration of androgens, such as testosterone
enantate and cipionate, also depress serum HDL-cholesterol levels significantly. Effects of
testosterone supplementation on lipoproteins, however, have been shown to be dose
dependent. It was thus demonstrated that intramuscular testosterone 600 mg/week reduced
HDL-cholesterol levels, whereas lower doses did not exert any effect on lipoprotein profiles.
HDL-cholesterol suppression by 19-nortestosterone esters seem to follow another pattern
since adverse effects in males have been demonstrated only after long-term administration,
whereas in women alterations have been observed after short-term use of low doses.
Parenteral administration of nandrolone decanoate for periods up to 2 months does not affect
HDL-cholesterol and subfractions in healthy athletes. However, in clinical studies this steroid
was found to affect HDL-cholesterol metabolism unfavourably in male haemodialysis patients
when administered for more than 6 months. In women with postmenopausal osteoporosis a
reduction of serum HDL-cholesterol levels was noticed even after 3 weeks. After steroid
withdrawal, the disturbed lipid and lipoprotein profiles recover completely, although at least 4-
12 weeks are needed for return to baseline values. It was demonstrated that recovery
depends strongly on the duration of an AAS course [04002].
Low-density lipoprotein-cholesterol
In general, the administration of multiple AAS is likely to increase serum low-density
lipoprotein (LDL)-cholesterol levels. The elevation parallels the decrease of HDL-cholesterol
and may be observed within a few days after initiation of steroid use. Single anabolic steroid
administration may exert different effects on serum LDL levels depending on the steroid and
route of administration. Oral administration of stanozolol increased LDL levels, whereas the
intramuscular injections of testosterone cipionate or testosterone enantate did not alter LDL
levels [04002].
Triglycerides
The effects on triglyceride metabolism appear to be more unequivocal. Most prospective
studies in athletes did not observe any alteration of serum triglyceride levels due to AAS
administration, although in one study an elevation of approximately 23 percenthas been
observed. The aberrant result of the latter study is hard to explain since in other studies of
this Finnish research group with the same strength athletes as volunteers no effect on
triglycerides were reported [04002].
Apolipoproteins and lipoprotein

The misuse of androgenic-anabolic steroids (AASs) in young, healthy strength athletes has
been associated with the occurrence of premature cardiovascular events. These events may
in part be mediated by the adverse effects on serum lipid variables that have been linked to
AAS administration. Previous studies have indicated that the use of AAS results in decreases
in high density lipoprotein cholesterol (HDL-C) and apolipoprotein A1 (Apo-A1; the major
component of the HDL particle), and increases in low density lipoprotein cholesterol (LDL-C).
A growing number of strength athletes misuse AASs to obtain a well shaped body or
increase muscular strength. Most athletes take AASs for periods of 8-12 weeks several times
a year. Self administration of AASs may result in much higher doses than recommended,
with possibly more severe side effects and more profound effects on serum lipids and
lipoproteins. In particular, the orally active 17-alpha-alkyl steroids have been shown to have
severe effects on LDL-C and HDL-C. Various studies have suggested that the concentration
of lipoprotein(a) (Lp(a)) is an independent risk indicator for the development of vascular
disease. The fat composition of Lp(a) is comparable to that of LDL-C, but the most important
difference is the presence of a specific apoprotein (a). This protein is attached to
apolipoprotein B (Apo-B) by a disulphide bridge. A close correlation has been reported
between the serum concentration of Lp(a) and the accumulation of this particle in the
1095
vascular wall. The serum concentration of Lp(a) seems to be genetically determined and,
when raised, cannot be lowered by alterations in food intake or taking cholesterol lowering
drugs. Previous reports have suggested that, in contrast with their detrimental effects on
lipids, AASs may favourably lower Lp(a) concentrations. To investigate the effects of two
different regimens of androgenic-anabolic steroid (AAS) administration on serum lipid and
lipoproteins, and recovery of these variables after drug cessation, as indicators of the risk for
cardiovascular disease in healthy male strength athletes in a non-blinded study (study 1)
serum lipoproteins and lipids were assessed in 19 subjects who self administered AASs for
eight or 14 weeks, and in 16 non-using volunteers. In a randomised double blind, placebo
controlled design, the effects of intramuscular administration of nandrolone decanoate (200
mg/week) for eight weeks on the same variables in 16 bodybuilders were studied (study 2).
Fasting serum concentrations of total cholesterol, triglycerides, HDL-cholesterol (HDL-C),
HDL2-cholesterol (HDL2-C), HDL3-cholesterol (HDL3-C), apolipoprotein A1 (Apo-A1),
apolipoprotein B (Apo-B), and lipoprotein (a) (Lp(a)) were determined. In study 1 AAS
administration led to decreases in serum concentrations of HDL-C, HDL2-C, HDL3-C, and
Apo-A1, whereas Apo-B increased. Serum Lp(a) declined. Total cholesterol and triglycerides
did not change significantly. Alterations after eight and 14 weeks of AAS administration were
comparable. No changes occurred in the controls. Six weeks after AAS cessation, serum
HDL-C, HDL2-C, Apo-A1, Apo-B, and Lp(a) had still not returned to baseline concentrations.
Administration of AAS for 14 weeks was associated with slower recovery to pretreatment
concentrations than administration for eight weeks. In study 2, nandrolone decanoate did not
influence serum triglycerides, total cholesterol, HDL-C, HDL2-C, HDL3-C, Apo-A1, and Apo-
B concentrations after four and eight weeks of intervention, nor six weeks after withdrawal.
However, Lp(a) concentrations decreased significantly from 103 (68) to 65 (44) U/l in the
nandrolone decanoate group, and in the placebo group a smaller reduction from 245 (245) to
201 (194) U/l was observed. Six weeks after the intervention period, Lp(a) concentrations
had returned to baseline values in both groups. It was concluded that self administration of
several AASs simultaneously for eight or 14 weeks produces comparable profound
unfavourable effects on lipids and lipoproteins, leading to an increased atherogenic lipid
profile, despite a beneficial effect on Lp(a) concentration. The changes persist after AAS
withdrawal, and normalisation depends on the duration of the drug abuse. Eight weeks of
administration of nandrolone decanoate does not affect lipid and lipoprotein concentrations,
although it may selectively reduce Lp(a) concentrations. The effect of this on atherogenesis
remains to be established [04060].
Only a few studies have investigated the effects of AAS on apolipoproteins in healthy young
athletes. These studies mainly focused on serum apolipoproteins A-1 and B levels. They
demonstrated that AAS diminish serum apolipoprotein A-1 levels and induce elevations of
apolipoprotein B levels. However, nandrolone decanoate does not seem to affect the
apolipoproteins at all. These findings are not surprising since apolipoproteins are very closely
related to HDL- and LDL-cholesterol and are in accordance with results in non exercising
humans. However, the magnitude of changes may depend on the drug and dose
administered. The 17-[alpha]-alkylated drugs (e.g. stanozolol) rather than testosterone esters
are responsible for inducing more profound effects. The same holds true for polydrug
regimens when compared with single-drug use. The time course until complete recovery of
serum apolipoproteins after drug withdrawal depends on the duration of the AAS course
used [04002].
Lipoprotein(a)
Lipoprotein(a) [Lp(a)] has been recognised as an independent risk factor for cardiovascular
disease. The fat composition of Lp(a) is comparable with that of LDL-cholesterol
accompanied by the presence of a specific apoprotein(a). The serum levels of Lp(a) seem to
be genetically determined and, when elevated, can be hardly influenced by nutrition and
1096
drugs. However, AAS have been demonstrated to improve serum Lp(a) levels in men and
women. Research in AAS-abusing athletes has been started very recently and, therefore,
only few data are available. In a cross-sectional study, it was observed that AAS-using
bodybuilders possessed beneficial serum Lp(a) levels, while non-using bodybuilders showed
atherogenic Lp(a) levels. In a series of prospective (blinded and unblinded) studies Hartgens
and coworkers demonstrated a strong Lp(a)-lowering effect of polydrug regimens of AAS in
strength athletes, while the effect of administration of intramuscular nandrolone decanoate
200 mg/week for 8 weeks was nonsignificant. More research is warranted to elucidate the
effects on Lp(a), and the impact of unfavourable changes of serum lipids and lipoprotein
levels in combination with a beneficially altered serum Lp(a) level [04002].
Summary on lipids and lipoproteins

In summary, many studies investigating therapeutic and supratherapeutic doses of AAS
administration have consistently demonstrated that serum lipids and lipoproteins are
unfavourably altered by these substances. However, the effects may vary considerably with
regimen and types of AAS used, and with route of administration. Serum total cholesterol
and triglyceride levels seem to remain unaffected by AAS abuse. Several AAS have been
demonstrated to suppress serum HDL-cholesterol levels, with a more distinct effect on
HDL2- rather than on HDL3-cholesterol. LDL levels will increase and parallels the pattern of
HDL-cholesterol suppression. The effects on apolipoprotein A and B-1 are in line with the
effects on HDL- and LDL-cholesterol, resulting in AAS-induced elevations of apolipoprotein A
and a decline of apolipoprotein B-1. Recent research indicated that Lp(a) levels may be
beneficially affected by the administration of a combination of several AAS in high doses.
The unfavourable effects of alkylated AAS exceed those of testosterone esters. The
influence of polydrug regimens on lipoprotein metabolism is more pronounced than the
administration of a single steroid. Moreover, short-term administration of nandrolone
decanoate, even at high doses, does not affect lipoprotein metabolism in young athletes. On
the other hand, long-term use of nandrolone decanoate in patients alters lipoprotein levels
considerably. The sometimes dramatic changes in serum lipids and lipoprotein levels
exposes the AAS user to an increased cardiovascular risk, although the impact of short-term
disturbances of the cardiovascular risk profile in otherwise healthy young athlete is unknown
yet. This ignorance is enhanced by the possible beneficial alteration of Lp(a) levels.
Disturbed serum lipids and lipoproteins may recover within a few months, although this is
strongly dependent on duration of the AAS course rather than on the dosages used [04002].
Hyperhomocysteinemia
Hyperhomocysteinemia has been accepted as an independent risk factor for atherosclerosis

and atherothrombosis. In recent years, several reports have appeared in the literature linking
the use of anabolic steroids with acute vascular events in bodybuilders. In this study, we
investigated whether hyperhomocysteinemia could contribute to the high vascular risk in
bodybuilders taking anabolic steroids. Twenty-three bodybuilders in different phases of their
training cycle and six control athletes participated in our study. Anthropomorphic measures
displayed a higher body mass index for bodybuilders in the competition phase than for
bodybuilders in the work-out and build-up phases, and for control athletes. Homocysteine
levels were 8.7 + 1.6 micromol/L in control athletes, 8.5 + 2.8 micromol/L in work-out phase
bodybuilders, and 8.3 + 1.5 micromol/L in competition phase bodybuilders, but 11.9 + 3.1
micromol/l in build-up phase bodybuilders. Vitamin B12 and folate levels did not differ
significantly between the four groups. The study shows that intake of anabolic steroids, as
used typically by bodybuilders in the build-up phase, induces acute hyperhomocysteinemia
and is likely to initiate an additional, potentially atherothrombotic mechanism in this group of
athletes [01053].
1097
Sudden death
Androgenic anabolic steroids (AAS) used for improving physical performance have been
considered responsible for acute myocardial infarction and sudden cardiac death. Two
young, healthy, male bodybuilders using AAS was investigated after “sudden death”
regarding pathologic cardiac findings associated with AAS ingestion. The autopsy revealed
normal coronary arteries. In one case, it was documented a typical infarct with a histologic
age of 2 weeks. A segmentation of myocardial cells at the intercalated disc level was
observed in the noninfarcted region. This segmentation was the only anomaly detected in the
second case. No other pathologic findings in the heart or other organs were found. Urine in
both subjects contained the metabolites of nortestosterone and stanozolol. It was concluded
that a myocardial infarct without vascular lesions is rare. Its association with AAS use,
bodybuilding, or both lacks any evidence of a cause-effect relationship. The histologic
findings in our 2 cases and in the few others reported in medical literature are nonspecific
and do not prove the cardiac toxicity of AAS. A better understanding of AAS action on the
neurogenic control of the cardiac function in relation to regional myocardial contraction and
vascular regulation is required [01052].
Several classes of recreational and prescription drugs have additional effects on the heart
and vasculature, which may significantly contribute to morbidity and mortality in chronic
users. The study presented herein focuses on pathological changes involving the heart
possibly due to anabolic androgenic steroid use. The role these hormones may play in their
occurrence of sudden cardiac death is also investigated. 98 medico-legal cases including 6
anabolic androgenic steroid users were retrospectively reviewed. Autopsies, histology,
immunohistochemistry, biochemistry and toxicology were performed in all cases.
Pathological changes consisted of various degrees of interstitial and perivascular fibrosis as
well as fibroadipous metaplasia and perineural fibrosis within the myocardium of the left
ventricle. Within the limits of the small number of investigated cases, the results appear to
confirm former observations on this topic and suggest anabolic androgenic steroid's potential
causative role in the pathogenesis of sudden cardiac deaths in chronic users [150182].
Anabolic androgenic steroids (AASs) represent a large group of synthetic derivatives of

testosterone, produced to maximize anabolic effects and minimize the androgenic ones. AAS
can be administered orally, parenterally by intramuscular injection and transdermally.
Androgens act by binding to the nuclear androgen receptor (AR) in the cytoplasm and then
translocate into the nucleus. This binding results in sequential conformational changes of the
receptor affecting the interaction between receptor and protein, and receptor and DNA.
Skeletal muscle can be considered as the main target tissue for the anabolic effects of AAS,
which are mediated by ARs which after exposure to AASs are up-regulated and their number
increases with body building. Therefore, AASs determine an increase in muscle size as a
consequence of a dose-dependent hypertrophy resulting in an increase of the cross-
sectional areas of both type I and type II muscle fibers and myonuclear domains. Moreover, it
has been reported that AASs can increase tolerance to exercise by making the muscles
more capable to overload therefore shielding them from muscle fiber damage and improving
the level of protein synthesis during recovery. Despite some therapeutic use of AASs, there
is also wide abuse among athletes especially bodybuilders in order to improve their
performances and to increase muscle growth and lean body mass, taking into account the
significant anabolic effects of these drugs. The prolonged misuse and abuse of AASs can
determine several adverse effects, some of which may be even fatal especially on the
cardiovascular system because they may increase the risk of sudden cardiac death (SCD),
myocardial infarction, altered serum lipoproteins, and cardiac hypertrophy. The aim of one
review is to focus on deaths related to AAS abuse, trying to evaluate the autoptic,
1098
histopathological and toxicological findings in order to investigate the pathophysiological
mechanism that underlines this type of death, which is still obscure in several aspects. The
review of the literature allowed us to identify 19 fatal cases between 1990 and 2012, in which
the autopsy excluded in all cases, extracardiac causes of death [150183].
Anabolic-androgenic steroids (AASs) are frequently misused. To determine causes of death,

characteristics, toxicology, and pathology of AAS positive cases, all cases (n=24) presenting
to the New South Wales Department of Forensic Medicine (1995-2012) were retrieved. All
were male, and the mean age was 32 years. Deaths were mainly due to accidental drug
toxicity (63 %), then suicide (17 %) and homicide (13 %). Abnormal testosterone/
epitestosterone ratios were reported in 63 percent, followed by metabolites of nandrolone (58
%), stanozolol (33 %), and methandienone (21 %). In 23 of 24 cases, substances other than
steroids were detected, most commonly psychostimulants (67 %). In nearly half, testicular
atrophy was noted, as was testicular fibrosis and arrested spermatogenesis. Left ventricular
hypertrophy was noted in 30 percent, and moderate to severe narrowing of the coronary
arteries in 26 percent. To summarize, the typical case was a male polydrug user aged in their
thirties, with death due to drug toxicity. Extensive cardiovascular disease was particularly
notable [14049].
It is estimated that 80 percent of weight lifters and body-builders take anabolic-androgenic

steroids. Their long-term use is associated with a variety of pathological conditions and
premature death. Anabolic-androgenic steroid abuse may lead to changes in the
presentation and progression of some conditions. It remains unclear whether anabolic
steroids should be given to patients with a history of abuse of these drugs who are to
undergo surgery. It was reported on a fatal outcome following surgery in a 48-year-old weight
lifter [05061].
It was reported two cases of sudden cardiac death (SCD) involving previously healthy
bodybuilders who were chronic androgenic-anabolic steroids users. In both instances,
autopsies, histology of the organs, and toxicologic screening were performed. The findings
support an emerging consensus that the effects of vigorous weight training, combined with
anabolic steroid use and increased androgen sensitivity, may predispose these young men
to myocardial injury and even SCD [05052].
Among 15,000 forensic post-mortem examinations performed on the coroner's order over a
24-year period (1981-2004) in the area of Lyon, France (population: 2,000,000), 2250 cases
of unexpected cardiac sudden death were identified retrospectively according to WHO
criteria. Of these, 108 occurred during recreational sport and 12 occurred in athletes. In the
latter category, a history of anabolic steroid abuse was found in 6 cases, whereas pre-
existing ordinary cardiac lesions were observed in the 6 remaining cases. To shed light on
the possible role of anabolic steroids in the induction of cardiac lesions, an experimental
study was conducted in rabbits that were treated orally with norethandrolone 8mg/kg/day for
60 days, and sacrificed at day 90. The histopathological examination of the heart from
treated animals showed coronary thrombosis associated with left ventricle hypertrophy in 3
cases, and lesions analogous to toxic or adrenergic myocarditis in all other treated animals.
These findings were very similar to those observed after cardiac sudden death in the 6
athletes with a history of anabolic steroid abuse. In addition, elevated caspase-3 activity in
the heart of treated rabbits as compared to controls suggests that apoptosis is involved in the
induction of norethandrolone-induced cardiac lesions. These results confirm the cardiotoxic
potential of anabolic steroid abuse [08140].
There have been several cases described of sudden death, SCD, in athletes using AAS.
However, the frequency and the pathophysiological mechanism of SCD remain unknown.
1099
Some researchers have concluded that AAS have an arrhythmogenic action, while others
believe that SCD is a secondary event, resulting from the cardiovascular side effects caused
by their abuse. Cases of atrial fibrillation and ventricular tachycardia have also been
described in athletes users. It has been suggested that the chronic administration of anabolic
agents prolongs and increases the inhomogeneity of repolarisation, thus creating an
arrhythmogenic substrate. These disturbances are more apparent in athletes with significant
cardiac hypertrophy as an adaptation to long term exercise training (“athlete’s heart”) or to
the use of anabolic substances [12126].
Anabolic androgenic steroids (AAS) are the main class of doping agents and their
consumption produces adverse effects involving several organs and systems. Three cases of
sudden cardiac death (SCD) and one of death due to congestive heart failure of previously
healthy athletes who were AAS users are herein reported. Concentric cardiac hypertrophy
with focal fibrosis (one case), dilated cardiomyopathy with patchy myocyte death (two cases)
and eosinophilic myocarditis (one case) were observed and most probably relate to the final
event. Molecular investigation for viral genomes was positive in one case (Ebstein virus). The
data confirm previous findings, showing that the most typical cardiac abnormality in AAS
abusers is left ventricular hypertrophy, associated with fibrosis and myocytolysis. An
exceptional cardiovascular substrate was represented by the case with drug induced
eosinophilic myocarditis. These features are at risk of ventricular arrhythmias as well as
congestive heart failure. The cause-effect relationship between AAS abuse and cardiac
death can be established only by a rigorous methodology with the use of standardized
protocols, including precise morphological studies of all target organs to search for chronic
toxic effects. Laboratory investigations should focus on AAS searching on a wide range of
biological matrices to demonstrate type, magnitude and time of exposure [12129].
Sudden cardiac death related to sports in young patients can have many causes.
Hypertrophic cardiomyopathy, congenital coronary abnormalities, and myocarditis make up
about half of the causes of sudden cardiac death after sports. Screening for all athletes is
important to prevent such episodes. This involves yearly examinations including clinical
examinations, stress echocardiograms, echocardiography, and laboratory investigations.
Also, behavioral follow up should be addressed, as cocaine administration and doping can
both lead to cardiac problems and sudden cardiac death after sports. It was presented a
case of a 17-year-old boy who collapsed after an ice hockey competition as a result of an
acute myocardial infarction, which was first represented by ventricular fibrillation. It was also
reviewed the main causes of sudden cardiac death in such young athletes and the main
investigations that have to be performed to reach the proper diagnosis and etiology of the
condition [06072].
Sudden death among athletes is very rare (1:50,000-1:100,000 annually) but it is still 2-4
times more frequent than in the age-matched control population and attracts significant
media attention. We propose a mechanism underlying sudden cardiac death in athletes that
does not relate to myocardial ischemia but is based on repolarization abnormalities due to
potassium channel downregulation and can also be best explained by the concurrent
presence of several factors such as cardiac hypertrophy (athlete's heart), and/or hypertrophic
cardiomyopathy, increased sympathetic tone, genetic defects, drugs, doping agents, food, or
dietary ingredients. These factors together can increase the repolarization inhomogeneity of
the heart ("substrate") and an otherwise harmless extrasystole ("trigger") occurring with a
very unfortunate timing may sometimes induce life-threatening arrhythmias. The effective
and possible prevention of sudden cardiac death requires the development of novel cost
effective cardiac electrophysiological screening methods. Athletes identified by these tests as
individuals at higher proarrhythmic risk should then be subjected to more costly genetic tests
in order to uncover possible underlying genetic causes for alterations in ionic channel
1100
structure and/or function [10066].
It was reporedt two cases of sudden cardiac death (SCD) involving previously healthy
bodybuilders who were chronic androgenic-anabolic steroids users. In both instances,
autopsies, histology of the organs, and toxicologic screening were performed. The findings
support an emerging consensus that the effects of vigorous weight training, combined with
anabolic steroid use and increased androgen sensitivity, may predispose these young men
to myocardial injury and even SCD [07066].
Several case reports associate the chronic use of AAS with serious cardiovascular
complications including acute myocardial infarction, cardiac arrest, and hypertrophic
cardiomyopathy without significant cardiac valvula ror coronary artery disease. Case reports
associate chronic AAS abuse with myocardial infarctions in young men with and without
evidence of coronary artery occlusion; the presence of coronary artery disease in these
athletes occurs despite the lack of known risk factors for coronary artery disease. The
development of an acute myocardial infarction was associated with high-dose AAS abuse
(e.g. 6 weeks daily, years intermittently) by a 44-year-old recreational bodybuilder with
diffuse coronary artery disease and multiple myocardial risk factors including polycythemia,
smoking, and family history of early coronary artery disease. Left ventricular hypertrophy is a
common structural abnormality in bodybuilders with AAS abuse. A study of 21 bodybuilders
with reported AAS abuse suggests that concentric hypertrophy of the left ventricular wall and
impaired diastolic function are common complications of steroid abuse. Echocardiographic
studies of these athletes demonstrated increased left ventricular posterior wall thickness and
end-diastolic volumes as well as decreased ratios of ventricular end-diastolic diameter to
body mass. A case report of 2 previously healthy bodybuilders associated sudden cardiac
death with chronic AAS abuse. There was evidence of focal myocardial necrosis without
clinically significant coronary artery disease, but the role of chronic AAS abuse in the cardiac
arrest remains unclear. Ventricular dysrhythmias are not commonly associated with chronic
AAS abuse. Several case reports associate persistent atrial fibrillation with chronic AAS use.
A 22-year-old man developed generalized weakness, diaphoresis, anxiety, and dyspnea.
The electrocardiogram revealed rapid atrial fibrillation and the echocardiogram indicated an
early cardiomyopathy. He had gynecomastia, and he admitted to the recent injection of
anabolic steroids. Although there is no direct evidence that AASs are thrombogenic in
humans, case reports suggest a possible causal relationship between AAS use and
thrombogenic events (e.g. massive pulmonary embolus, cerebral thrombosis, cardiomyop-
athy with congestive heart failure, biventricular thrombi, and hepatorenal dysfunction).
Studies on the association between chronic AAS use and hypertension or left ventricular
hypertrophy are inconsistent [13003].
Sudden death is the most frightening consequence of AAS use. The etiology of these events
likely is multifactorial, with AAS use contributing to the observed pathology. There are case
reports of myocardial infarctions, stroke, and peripheral vascular obstruction from thrombus
that likely are related to the changes in platelet function, inflammation, and cholesterol
metabolism discussed above. Autopsies of 34 users of AASs found chronic cardiac changes
consisting of cardiac hypertrophy, myocardial fibrosis, and coronary artery atheromatous
changes in 12 victims, although these were believed to contribute to the deaths of only 2
victims. Many sudden death events among AAS users have been due to ischemia secondary
to coronary artery disease; however, there is a report of ventricular tachycardia during
exercise testing of an AAS user who had myocardial fibrosis on biopsy. Other case reports of
sudden death demonstrate diffuse, patchy fibrotic changes in the myocardium of AAS users
without coronary artery atherosclerosis. The presence of scar or infiltrative processes is
commonly believed to be a cause for arrhythmia. The exact cause of sudden death in AAS
users is unclear but likely is due to ischemia or arrhythmia [07058].
1101
Anabolic-androgenic steroids (AASs) are frequently misused. To determine causes of death,
characteristics, toxicology, and pathology of AAS positive cases, all cases (n = 24)
presenting to the New South Wales Department of Forensic Medicine (1995-2012) were
retrieved. All were male, and the mean age was 32 years. Deaths were mainly due to
accidental drug toxicity (63 %), then suicide (17 %) and homicide (13 %). Abnormal
testosterone/epitestosterone ratios were reported in 63 percent, followed by metabolites of
nandrolone (58 %), stanozolol (33 %), and methandienone (21 %). In 23 of 24 cases,
substances other than steroids were detected, most commonly psychostimulants (67 %). In
nearly half, testicular atrophy was noted, as was testicular fibrosis and arrested
spermatogenesis. Left ventricular hypertrophy was noted in 30 percent, and moderate to
severe narrowing of the coronary arteries in 26 percent. To summarize, the typical case was
a male polydrug user aged in their thirties, with death due to drug toxicity. Extensive
cardiovascular disease was particularly notable [14647].
Doping – the abuse of anabolic-androgenic steroids in particular – is widespread in amateur

and recreational sports and does not solely represent a problem of professional sports.
Excessive overdose of anabolic steroids is well documented in bodybuilding or powerlifting
leading to significant side effects. Cardiovascular damages are most relevant next to adverse
endocrine effects. Clinical cases as well as forensic investigations of fatalities or steroid
consumption in connection with trafficking of doping agents provide only anecdotal evidence
of correlations between side effects and substance abuse. Analytical verification and self-
declarations of steroid users have repeatedly confirmed the presumption of weekly dosages
between 300 and 2000 mg, extra to the fact that co-administration of therapeutics to treat
side-effects represent a routine procedure. Beside the most frequent use of medications
used to treat erectile dysfunction or estrogenic side-effects, a substantial number of
antihypertensive drugs of various classes, i.e. beta-blockers, diuretics, angiotensin II receptor
antagonists, calcium channel blockers, as well as ACE inhibitors were recently confiscated in
relevant doping cases. The presumptive correlation between misuse of anabolic steroids and
self-treatment of cardiovascular side effects was explicitly confirmed by detailed user
statements. Two representative fatalities of bodybuilders were introduced to outline
characteristic, often lethal side effects of excessive steroid abuse. Moreover, illustrative
autopsy findings of steroid acne, thrombotic occlusion of Ramus interventricularis anterior
and signs of cardiac infarctions are presented.A potential steroid abuse should be carefully
considered in cases of medical consultations of patients exhibiting apparent constitutional
modifications and corresponding adverse effects. Moreover, common self-medications – as
frequently applied by steroid consumers – should be taken into therapeutic considerations
[150177].

It was a report of a 42 year old male amateur body builder using anabolic androgenic
steroids, who developed acute respiratory distress syndrome, acute kidney injury and
refractory supraventricular tachycardia. He required extracorporeal membrane oxygenation,
continuous veno-venous hemodialysis, and catheter ablation. It was thought that long-term
anabolic androgenic steroid abuse predisposed the patient to developing multiple organ
dysfunction syndromes from its immunomodulatory effects in an otherwise healthy patient.
Anabolic androgenic steroid use should be part of the history taking process since it may
complicate patient outcomes [13139].
Cardiovascular risk in older men on testosterone
1102
Ageing is accompanied by a reduction in circulating testosterone and progressive
accumulation of medical morbidities. There is an intense debate whether low testosterone
contributes to ill-health as opposed to being a biomarker for its presence. Prescriptions for
testosterone are rising on a background of concern over potential adverse effects. One
review examines evidence relating androgens to cardiovascular risk in older men.
Observational studies show lower risk of cardiovascular events in older men with higher
testosterone, and lower mortality from ischaemic heart disease in men with higher
concentrations of its more potent androgenic metabolite dihydrotestosterone. However,
randomized controlled trials of testosterone supplementation have been underpowered for
the outcome of cardiovascular events. Recent meta-analyses have reached contrasting
conclusions regarding cardiovascular adverse events associated with testosterone therapy.
Retrospective studies of prescription databases have produced controversial and conflicting
results. Thus, additional randomized controlled trials are required to clarify the role of
testosterone supplementation in older men in the absence of pituitary or gonadal disease.
Pending such studies, testosterone therapy should be considered in androgen-deficient men,
with evaluation of potential benefits and risks [150184].
Long-term risk
Non-therapeutic use of anabolic androgenic steroids (AAS) has been associated with various
adverse effects; one of the most serious being direct cardiovascular effects with unknown
long-term consequences. Therefore, large studies of the association between AAS and
cardiovascular outcomes are warranted. We investigated cardiovascular morbidity and
mortality in individuals who tested positive for AAS. Between 2002 and 2009, a total of 2013
men were enrolled in a cohort on the date of their first AAS test. Mortality and morbidity after
cohort entry was retrieved from national registries. Of the 2013 individuals, 409 (20 %) tested
positive for AAS. These men had twice the cardiovascular morbidity and mortality rate as
those with negative tests (adjusted hazard ratio (aHR) 2.0; 95 % confidence interval (CI) 1.2
to 3.3). Compared to the Swedish population, all tested men had an increased risk of
premature death from all causes (standardized mortality ratio for AAS-positive: 19.3, 95 % CI
12.4 to 30.0; for AAS-negative: 8.3, 95 % CI 6.1 to 11.0). Thus, non-therapeutic exposure to
AAS appears to be an independent risk factor for cardiovascular morbidity and premature
death [150185].
Abnormal plasma lipoprotein
Many studies have shown that AAS can cause dyslipidemia by increasing low-density
lipoprotein as high as 596 mg/dL and decreasing high-density lipoprotein as low as 5 mg/dL.
Alterations in high- and low-density lipoprotein levels occur in a dose-dependent manner
within 9 weeks of self-administration of steroids. These changes could accelerate coronary
artery atherosclerosis over the long term, resulting in an increased risk of coronary heart
disease three to six times that of normal. The effects of androgens on lipid profile have been
shown to be reversible after the discontinuation of administration [12119]
AS have been associated with negative alterations in lipid profiles. Changes reported include
a decrease in high-density lipoprotein (HDL), an elevation in low-density lipoprotein (LDL)
and reduced apolipoprotein levels, possibly through up-regulation of hepatic triglyceride
lipase. The changes in lipid profiles indicate an increase in atherosclerotic risk. Increases in
homocysteine, a naturally occurring amino-acid thought to have a role in vaso-control, and C-
reactive proteins (CRP), an acute-phase protein that rises in response to inflammation, have
been implicated as risk factors for CV disease. It has been demonstrated a significant
increase in CRP in AS users. It was noted a significant elevation in homocysteine in AS
1103
users as well as those who had abstained from AS use for 3 months, indicating a possible
effect of AS on vitamin B absorption. Previous studies have also suggested a possible link
between AS use and thrombotic risk through alterations in haemoglobin levels [12114].
To evaluate the effects of anabolic androgenic steroids (AAS) on chylomicron metabolism an
artificial lipid emulsion labeled with radioactive cholesteryl ester (CE) and triglycerides (TG)
mimicking chylomicrons was intravenously injected into individuals who regularly weight
trained and made regular use of AAS (WT+AAS group), normolipidemic sedentary
individuals (SDT group) and individuals who also regularly weight trained but did not use
AAS (WT group). Fractional clearance rates (FCR) were determined by compartmental
analysis for emulsion plasma decay curves. FCR-CE for the WT+AAS group was reduced
(0.0073 ± 0.0079 per min, 0.0155 ± 0.0100 per min, 0.0149 ± 0.0160 per min, respectively),
FCR-TG was similar for both the WT and SDT groups. HDL-C plasma concentrations were
lower in the WT+AAS group when compared to the WT and SDT groups (22 ± 13; 41 ± 7; 38
± 13 mg/dL, respectively). Hepatic triglyceride lipase activity was greater in the WT+AAS
group when compared to the WT and SDT groups (7243 ± 1822; 3898 ± 1232; 2058 ± 749,
respectively). However, no difference was observed for lipoprotein lipase activity. Data
strongly suggest that AAS may reduce the removal from the plasma of chylomicron
remnants, which are known atherogenic factors [12130].
Effect on metabolism
The effects of testosterone (T) supplementation on insulin sensitivity, inflammation-sensitive
markers, and apolipoproteins remain poorly understood. It is not known whether T's effects
on plasma lipids, apolipoproteins, and insulin sensitivity are dose dependent, or whether
significant anabolic effects can be achieved at T doses that do not adversely affect these
cardiovascular risk factors. To determine the effects of different doses of T, 61 eugonadal
men, 18-35 years of age, were randomly assigned to 1 of 5 groups to receive monthly
injections of long-acting GnRH agonist to suppress endogenous T secretion and weekly
injections of 25, 50, 125, 300, or 600 mg T enanthate for 20 wk. Dietary energy and protein
intakes were standardized. Combined administration of GnRH agonist and graded doses of T
enanthate resulted in nadir T concentrations of 253, 306, 542, 1345, and 2370 ng/dl at the
25-, 50-, 125-, 300-, and 600-mg doses, respectively. Plasma high density lipoprotein
cholesterol and apolipoprotein A-I concentrations were inversely correlated with total and
free T concentrations and were significantly decreased only in the 600 mg/week group.
Serum total cholesterol, low density lipoprotein cholesterol, very low density lipoprotein
cholesterol, triglycerides, apolipoprotein B, and apolipoprotein C-III were not significantly
correlated with T dose or concentration. There was no significant change in total cholesterol,
low density lipoprotein cholesterol, very low density lipoprotein cholesterol, triglycerides,
apolipoprotein B, or apolipoprotein C-III levels at any dose. The insulin sensitivity index,
glucose effectiveness, and acute insulin response to glucose, derived from the insulin-
modified, frequently sampled, iv glucose tolerance test using the Bergman minimal model,
did not change significantly at any dose. Circulating levels of C-reactive protein were not
correlated with T concentrations and did not change with treatment in any group. Significant
increments in fat-free mass, muscle size, and strength were observed at doses that did not
affect cardiovascular risk factors. Over a wide range of doses, including those associated
with significant gains in fat-free mass and muscle size, T had no adverse effect on insulin
sensitivity, plasma lipids, apolipoproteins, or C-reactive protein. Only the highest dose of T
(600 mg/week) was associated with a reduction in plasma high density lipoprotein cholesterol
and apolipoprotein A-I. Long-term studies are needed to determine whether T
supplementation of older men with low T levels affects atherosclerosis progression [02034].
1104
Chylomicron metabolism
An artificial lipid emulsion labeled with radioactive cholesteryl ester (CE) and triglycerides
(TG) mimicking chylomicrons was intravenously injected into individuals who regularly weight
trained and made regular use of AAS (WT+AAS group), normolipidemic sedentary
individuals (SDT group) and individuals who also regularly weight trained but did not use
AAS (WT group). Fractional clearance rates (FCR) were determined by compartmental
analysis for emulsion plasma decay curves. FCR-CE for the WT+AAS group was reduced,
FCR-TG was similar for both the WT and SDT groups. HDL-C plasma concentrations were
lower in the WT+AAS group when compared to the WT and SDT groups. Hepatic triglyceride
lipase activity was greater in the WT+AAS group when compared to the WT and SDT
groups. However, no difference was observed for lipoprotein lipase activity. Data thus
strongly suggest that AAS may reduce the removal from the plasma of chylomicron
remnants, which are known atherogenic factors [12146].
Trombocyte function
Several mechanisms have been implicated in atherothrombosis of the coronary and other
arteries in steroid users: atherogenesis, thrombosis, vasospasm, and direct myocardial
damage. Another cause of atherothrombosis may be the increase in erythrocytosis caused
by testosterone. A number of studies have demonstrated a significant disturbance of lipid
metabolism in athletes who have used anabolic steroids. The beneficial effect of AAS on
platelet aggregation and the mechanism of thrombosis, mainly through the activation of
prostaglandins and plasminogen, is well known from experimental studies. However, there
are conflicting results regarding the effects of anabolic steroids on the mechanism of
thrombosis in athletes. In addition, there are only a few reports on the effect of anabolic
steroids on vascular function in athletes [12126].
Adhesion molecules expression and platelets aggregation

Although therapeutic and physiological dosages of AAS seem to have beneficial effects on
platelet aggregation, deleterious effects of supraphysiological AAS dosages in promoting
expression of adhesion molecules in vessel walls and facilitating platelet-endothelium binding
have been reported as a mechanism that contributes to AAS-induced atherosclerosis.
Additionally, the role of AAS abuse in thrombogenicity has been reported in some studies.
Detection of adhesion molecules expression as an upstream process leading to binding of
platelets to the arterial wall can depict atherosclerotic plaque formation at early stages.
Vascular cell adhesion molecule-1 (VCAM-1) and integrins provide suitable targets for
molecular imaging of adhesion molecules expression. VCAM-1 is expressed by endothelial
cells, macrophages and smooth muscle cells. Integrins, that is, avb3 integrin, are adhesion
molecules that are expressed following endothelial cell injury, as well as at more progressed
stages of atherosclerotic plaque formation during neo-angiogenesis. avb3 integrin has high
binding affinity to arginine-glycine-aspartate amino acid sequence facilitating cell to
extracellular matrix interactions [12125].
Hypercoagulability
Onestudy evaluated the short-term effects of oxandrolone, an anabolic androgenic synthetic

steroid, on blood coagulation and the hemostatic/fibrinolytic system in healthy individuals.
Subjects (n=14) were administered oxandrolone (10 mg twice daily) for 14 days. Blood was
obtained on days 0, 1, 3, 7, 9, 14, and then at day 42 (28 days after discontinuation of the
drug). Samples were analyzed for the plasma plasminogen, plasminogen activator inhibitor
(PAI-1), fibrinogen, and coagulation factors (II, V, VII, VIII, and X). After 7 days of
1105
administration of oxandrolone, the plasma plasminogen level significantly increased
significantly. PAI-1 was significantly decreased at day 3. Coagulation factors II and V
significantly increased at day 14, respectively. Factor VII level decreased (not significantly)
by day 3, but after 14 days factor VII level returned to baseline. The increase of factor VIII
level was not significant. Factor X increased steadily over 14 days of drug treatment and
after discontinuation, decreased and returned to baseline by day 42. Fibrinogen decreased
by 22 + 12 percent. Administration of oxandrolone, to healthy young men was thus
associated with a significant increase in select blood coagulation factors and plasminogen.
These changes create a state of potential hypercoagulability that appears to be
counterbalanced by increased fibrinolytic activity to maintain homeostasis [06073].
Suppression of clotting factors II, V, VII, and X and bleeding in patients receiving concomitant
anticoagulant therapy have been reported with testosterone. Case reports demonstrated that
coadministration of oral anticoagulants and 17-alkylated androgens (fluoxymesterone,
oxandrolone, oxymetholone, methyltestosterone, methandrostenolone, stanozolol) resulted
in a prolonged prothrombin time and hemorrhages; AASs may reduce the need for
therapeutic anticoagulants by 25 percent [07058].
Anabolic androgenic steroids (AAS) are synthetic derivatives of testosterone with

thrombogenic potential in high doses and long-term administration. Taurine, a widely
distributed amino-sulfonic acid, is known for its beneficial effects in hypercoagulable states.
In order to assess the impact of chronic administration of high doses of AAS and taurine
upon haemostasis process in rats, 40 male Wistar rats were divided into four equal groups:
control group (group C) – no treatment; androgen group (group A) – received 10 mg/kg per
week of nandrolone decanoate (DECA); taurine (group T) – received oral supplementation of
2 percent taurine in drinking water; androgen and taurine group (group AT) – concomitant
administration of DECA and taurine. After 12 weeks, blood samples were collected and
haemostasis parameters were assessed with the thrombelastographic (TEG) analysis
system: reaction time, clot kinetics (K, alpha), final clot strength, coagulation index and the
clot lysis (Ly30). Nandrolone significantly decreased reaction time in group A compared with
control, whereas taurine significantly increase reaction time, the effect was maintained in
group AT compared with group A. Similar differences between groups have been recorded
for the clot kinetics parameters K, alpha. The final clot strength and coagulation index were
significantly increased in group A versus group C, but not in group AT versus group C. There
were no differences in clot lysis, as shown by Ly30. Nandrolone produces an accelerated clot
development and an increased clot firmness in Wistar rats. Taurine association ensures a
protective effect against this hypercoagulable state, partially restoring the altered parameters
of the coagulation profile [12131].
Altered coagulation profile

Anabolic androgenic steroids (AAS) are synthetic derivatives of testosterone with
thrombogenic potential in high doses and long-term administration. Taurine, a widely
distributed amino-sulfonic acid, is known for its beneficial effects in hypercoagulable states.
In order to assess the impact of chronic administration of high doses of AAS and taurine
upon haemostasis process in rats, 40 male Wistar rats were divided into four equal groups:
control group (group C) – no treatment; androgen group (group A) – received 10 mg/kg per
week of nandrolone decanoate (DECA); taurine (group T) – received oral supplementation of
2% taurine in drinking water; androgen and taurine group (group AT) – concomitant
administration of DECA and taurine. After 12 weeks, blood samples were collected and
haemostasis parameters were assessed with the thrombelastographic (TEG) analysis
system: reaction time, clot kinetics (K, alpha), final clot strength, coagulation index and the
clot lysis (Ly30). Nandrolone significantly decreased reaction time in group A compared with
control, whereas taurine significantly increase reaction time, and this effect was maintained
1106
in group AT compared with group A. Similar differences between groups have been recorded
for the clot kinetics parameters K, alpha. The final clot strength and coagulation index were
significantly increased in group A versus group C but not in group AT versus group C. There
were no differences in clot lysis, as shown by Ly30. Nandrolone produces an accelerated clot
development and an increased clot firmness in Wistar rats. Taurine association ensures a
protective effect against this hypercoagulable state, partially restoring the altered parameters
of the coagulation profile [13138].
Inherited antithrombin deficiency and anabolic steroids: a risky combination

A 20-year-old male with asymptomatic inherited type 1 antithrombin deficiency and a family
history of thrombosis started injecting himself with testosterone 250 mg intramuscularly twice
weekly for 5 weeks. He presented to the hospital with progressive dyspnea on exertion, chest
pain and hemoptysis. Workup revealed bilateral submassive pulmonary embolism and
proximal right lower extremity deep vein thrombosis. He was treated with intravenous (IV)
unfractionated heparin and underwent catheter-directed thrombolysis with alteplase to the
main pulmonary arteries. Postprocedure, he remained on IV alteplase infusion for 24 h and
unfractionated heparin in the intensive care unit. Concomitantly he received plasma-derived
antithrombin concentrate. He was transitioned to subcutaneous enoxaparin twice daily and
discharged from the hospital on oral rivaroxaban 15 mg twice a day. This case highlights the
heightened thrombogenic effect of anabolic steroids in the setting of underlying thrombophilia
especially in younger subjects [150190].
Arterial thrombosis
The use of supraphysiological doses of anabolic androgenic steroids can have serious side
effects. One article reports the case of a young man who suffered potentially life-threatening
arterial thromboses following the use of these drugs [13135].
Coronary thrombus
A 23 year old male body builder presented with a recent onset of central chest pain. He was
a smoker with cholesterol concentration of 7.l mmol/L on admission. He had been taking
anabolic steroids (methandrostenelone 20 mg daily) for three months. Anteroseptal T wave
inversion on a 12 lead ECG along with elevated troponin T prompted early coronary
angiography. This revealed multiple filling defects in the mid left anterior descending (LAD)
artery consistent with the presence of thrombus (below left). His LAD filled by collaterals from
the right coronary artery. The rest of his coronary arteries were smooth and unobstructed.
Repeat angiography was performed 48 hours later after treatment with abciximab, aspirin,
and low molecular weight heparin and revealed complete dissolution of all thrombus.
Intravascular ultrasound (IVUS) examination performed at this time revealed a segment of
eccentric atheroma at the site of the previous filling defects (below right); the echogenicity
was uniform with no focal echo lucent regions. The remainder of the LAD was normal at
IVUS examination. The patient made an uneventful recovery and was discharged well to
follow up. There have been several case reports of acute myocardial infarction in young male
athletes using anabolic steroids. The mechanism is unclear but may involve the adverse
effects on thrombosis and lipid profile. Some reports suggest thrombosis in “normal”
coronary arteries, but underlying atheroma cannot be excluded without IVUS. This case
supports the concept that both atheroma and the thrombogenic effects of anabolic steroids
may be necessary for vessel occlusion [02033].
Vascular effects in the brain

Lacunar infarction is traditionally ascribed to lipohyalinosis or microatheroma. It was reported
a case of 40-year-old man, without traditional risk factors for ischemic stroke, who presented
1107
to the Emergency Department with recurrent episodes of transient right-sided weakness and
paresthesia. Lacunar infarction was confirmed on diffusion-weighted MRI and blood tests
showed a marked polycythemia. Quantitative magnetic resonance perfusion imaging
demonstrated dramatically abnormal perfusion throughout both cerebral hemispheres, and
transcranial Doppler revealed reduced cerebral artery velocities, both consistent with the
proposed mechanism of hyperviscosity. His symptoms settled with treatment of the
polycythemia and workup did not find another cause of ischemic stroke. It was proposed that
hyperviscosity secondary to steroid-induced polycythemia caused ischemia in this case and
not lipohyalinosis or microatheroma, to which lacunar disease is commonly attributed
[14047].
It was reported a case of a 37-year-old man presented with acute stroke and hepatorenal
impairment which were associated with anabolic-androgenic steroids (AAS) abuse over 2
years. Despite the absence of apparent symptoms and signs of congestive heart failure at
presentation, an AAS-induced dilated cardiomyopathy with multiple thrombi in the left
ventricle was attributed to be the underlying cause of his condition. Awareness of the
complications of AAS led to the prompt treatment of the initially unrecognised dilated
cardiomyopathy, and improved the liver and kidney functions. However, the patient was
exposed to a second severe ischaemic event, which led to his death. This unique and
complex presentation of AAS complications opens for better recognition and treatment of
their potentially fatal effects [14048].
Thrombosis
It was presented a case of a 19-year-old male athlete with protein C deficiency who
developed proximal deep venous thrombosis and pulmonary embolism while abusing
anabolic-androgenic steroids. Anabolic-androgenic steroids have been reported to have
anticoagulatory and profibrinolytic effects in patients with protein C deficiency. Despite these
antithrombotic effects, the patient developed repeated venous thromboembolism during
treatment with low-molecular-weight heparin. The net effect of anabolic-androgenic steroids
on the haemostatic system may change from antithrombotic to prothrombotic in male
abusers of anabolic steroids with protein C deficiency [10065].
Cerebral venous thrombosis

There are only a few reports of patients developing cerebral venous sinus thrombosis
(CVST) after androgen therapy. It was presented a young man who developed cortical
venous thrombosis after using androgens to increase muscle mass. He was hospitalised for
parasthesia and dyspraxia in the left hand followed by a generalised tonic-clonic seizure. At
admission, he was drowsy, not fully orientated, had sensory inattention, pronation drift and a
positive extensor response, all on the left side. The patient had been using anabolic steroids
(dainabol 20 mg/day) for the last month for bodybuilding. CT angiography showed a right
cortical venous thrombosis. Anticoagulation therapy was started with intravenous heparin for
11 days and oral anticoagulation (warfarin) thereafter. A control CT angiography 4 months
later showed resolution of the thrombosis. He recovered fully [13148].
Pulmonary embolism
It was presented case of a 56-year-old man with deep vein thrombosis (DVT) and pulmonary
embolism (PE). He had been given intramuscular injections of testosterone and the anabolic-
androgenic steroid nandrolone, due to a muscle injury, a total of three times prior to
manifestation of the symptoms. An ultrasonographic examination of the right leg revealed a
DVT and computed tomography of the pulmonary arteries showed PE. The thromboembolic
1108
episodes in this previously healthy patient were in all probability associated with
intramuscular injections of testosterone and nandrolone, to which there is a clear correlation
in time [07070].
Endothelial cells
The aim of one study was to investigate the effects in vitro induced by androgenic anabolic
steroids (AAS) (testosterone, nandrolone, androstenedione, norandrostenedione, and
norandrostenediol) used illicitly in sport competitions, on the proliferation ability, apoptosis
and the intracellular calcium concentration ([Ca2+]i) in human umbilical vein endothelial cells
(HUVECs), selected as a prototype of a biological target system whose structure and
function can be affected by steroids. For this purpose, it was evaluated the proliferation
inhibition by cytotoxic assay expressed as the concentration of drug inducing a 50 percent
decrease in growth (IC50). The IC50 was reached for testosterone at 100 microM,
androstenedione at 375 microM, nandrolone at 9 microM, norandrostenedione at 500
microM. The IC50 value for norandrostenediol was not reached until a concentration of 6000
microM. The apoptotic effect was evaluated by flow cytometry at IC50 for each drug. It was
observed that testosterone induced 31 percent of apoptotic cells, norandrostenedione 25
percent, androstenedione 15 percent and nandrolone 18 percent. It was analyzed the effects
of these drugs on [Ca2+]i both in the immediate and long-term continuous presence of each
compound. The data show a statistically significant increase of [Ca2+]i in the acute condition
and in long-term treated cultures, suggesting that androgen steroids modulate intracellular
levels of calcium independent of incubation time or compound identity. As a whole, the study
demonstrates that AAS might alter endothelial homeostasis, predisposing to the early
endothelial cell activation that is responsible for vascular complications observed frequently
in AAS users [07067].
Atherothrombotic markers and endothelial dysfunction

The use of androgenic anabolic steroids (AAS) may be associated with changes in
atherothrombotic markers and endothelial function. The purpose of one study was to
compare atherothrombotic markers and endothelial function of AAS users and non-users.
Ten athletes who were users of AAS (confirmed by urine analysis) and 12 non-user athletes
were evaluated. Body weight, blood pressure, exercise load (hours/week), complete blood
count (CBC), platelets, fibrinogen, lipids, high-sensitivity C-reactive protein (hs-CRP), follicle-
stimulating hormone, testosterone and estradiol were measured. Endothelium-dependent
and independent functions were assessed by brachial artery ultrasound. AAS users had
significantly higher body mass and blood pressure. Platelet count was higher whereas HDL-
cholesterol was lower in AAS users compared with non-users. Levels of hs-CRP were higher
in AAS users. Follicle-stimulating hormone was suppressed in all users and not suppressed
in non-users. Compared with non-users, flow-mediated dilation was significantly reduced in
AAS users, whereas endothelium-independent function was similar in both groups.
Additionally, flow-mediated dilation was positively associated with levels of HDL- cholesterol.
AAS users present important changes in blood lipids as well as in inflammatory markers,
which are compatible with increased cardiovascular risk. Furthermore, this profile is
accompanied by a reduction in the endothelial function [13137].
Flow-mediated, endothelium-dependent vasodilatation

Self-administration of anabolic-androgenic steroids to increase muscular strength and lean
body mass has been used widely among athletes. Flow mediated dilatation (FMD)
determined by ultrasound of the brachial artery is accepted as both an in vivo index of
endothelial function and an indicator for future atherosclerosis. FMD was calculated in 20
male non-smoking body builders in different phases of their training cycle and in six male
1109
non-smoking control athletes. Ultrasound studies of the brachial artery were performed
according to the protocol of Celermajer et al. Of the entire training cycle, work-out phase was
training phase without actual intake of anabolic-androgenic steroids over 8 weeks; build-up
phase included actual intake of anabolic-androgenic steroids; and competition phase
consisted of 8 weeks post intake of anabolic-androgenic steroids. Baseline characteristics
did not differ between body builder groups except for a higher weight in competition phase
body builders. Hormonal analysis revealed suppressed luteinizing hormone and follicle
stimulating hormone levels in build-up phase body builders. The lipid profiles showed a
marked reduction of HDL-C in build-up phase body builders. FMD was reduced in body
builders of all phases when compared to control athletes. The glyceryl trinitrate-induced
vasodilatation was diminished, though not statistically significantly, in body builders when
compared with control athletes. The differences in FMD persisted after adjustment for vessel
size. The data indicate that intake of anabolic-androgenic steroids is associated with both an
atherogenic blood lipid profile and endothelial dysfunction and thus may pose an increased
risk of atherosclerosis [01057].
Increased iIntima-media thickness
AS use has also been associated with reduced endothelial function in conduit arteries. It was
noted a reduced flow-mediated dilation in AS users as well as a reduced vasodilator
response to glyceryl-trinitrate [12114].
It has been measured carotid intima-media thickness and radial and brachial artery reactivity
in bodybuilders using AAS. It was found a non-significant increase in the thickness and
diameter of the arteries in users compared to non-users, which was attributed mainly to fluid
retention. A small degree of endothelial dysfunction was also reported by other investigators.
It was also found changes of aortic wall elasticity in athletes who used steroids. Moreover,
using an electron beam tomography system, it was found increased calcium deposition in the
coronary arteries of bodybuilders using AAS. The authors hypothesised that this was due to
a direct toxic or inflammatory effect of steroids on the vascular endothelium [12126].
Aortic elasticity
The use of anabolic-androgenic steroids (AAS) has been linked to acute cardiovascular
events in athletes. The purpose of the present study was to investigate the aortic elastic
properties in athletes who had been self-administering AAS compared with a group of
athletes not using these drugs. Fourteen male bodybuilders using AAS and 27 male
wrestlers (non-users) volunteered to the study. All subjects were placed in a mild recumbent
position and the ascending aorta was recorded in the two-dimensional guided M-mode
tracings. The aortic distensibility was found to be reduced in user athletes. The results of this
study indicate that aortic stiffness is increasing in athletes using AAS [07068].
The use of anabolic-androgenic steroids (AAS) has been linked to acute cardiovascular
events in athletes. The purpose of one study was to investigate the aortic elastic properties in
athletes who had been self-administering AAS compared with a group of athletes not using
these drugs. Fourteen male bodybuilders using AAS and 27 male wrestlers (non-users)
volunteered to the study. All subjects were placed in a mild recumbent position and the
ascending aorta was recorded in the two-dimensional guided M-mode tracings. Although the
aortic distensibility was found to be reduced in user athletes (2.1 + 1.1 vs 3.8 + 1.4 cm2/dyn
10-6; 9.3 + 3.7 vs 5.9 + 2.5, respectively). The results of this study indicate that aortic
stiffness is increasing in athletes using AAS [05050].
1110
Arterial hypertension
The literature regarding the blood pressure response to AAS use is equivocal. In addition,
there is currently little data available on the rate pressure product (RPP) response to
anabolic androgenic steroids (AAS) use. The experimental aim of this study was to
investigate the effects of AAS administration in combination with resistance training on blood
pressure and rate pressure product in male amateur bodybuilders and compare the results
with a morphologically matched, resistance trained control group. Subjects were divided into
two groups (n=16 AAS users; n=16 controls). Systolic and Diastolic Blood Pressure, RPP.
Resting Heart Rate and Body Composition measurements were obtained before (Pre),
during (During) and 6-8 weeks following (Post) the AAS cycle in the AAS users with similar
time intervals for the control group. No significant cardiovascular or morphological changes in
the control group were found throughout the study. Significant increases in both diastolic and
mean arterial blood pressures were found from Pre to Post cycle in the AAS group. RPP also
increased significantly from pre to post AAS cycle. All cardiovascular parameters returned to
normal baseline measurements between 6 and 8 weeks post cycle. No blood pressure
measurements throughout the study were consistent with clinically defined hypertension. The
findings indicate that the AAS group exhibited significant increases in standard
cardiovascular measurements compared with the control bodybuilders, and provides a
contraindication to AAS use especially in borderline hypertensives [03053].
In a study conducted to evaluate the effects of the use and discontinuation of AAS on the
cardiovascular system, both AAS users (140 ± 10 mmHg) and former users (130 ± 5 mmHg)
had higher blood pressure than a control group of non-using weight lifters (125 ± 10 mmHg).
Five of the 16 users, 2 of the 15 former users, and 1 of the 15 non-users in this study
manifested blood pressure values at rest consistent with hypertension (values equal or
greater than 140/90 mmHg) and higher blood pressure response in response to a physical
exercise protocol. This phenomenon occurred despite the users having an average age of
only 31 ± 5 years. In another study, the blood pressure of 16 subjects who performed an
AAS cycle was significantly higher after 8 weeks than controls, and end values were
compatible with hypertension. The blood pressure values returned to normal 8 weeks later,
closing the cycle. The difference between these two studies related to blood pressure
returning to normal after discontinuation of the AAS treatment is the use for a short time in
this second study. The transitory aspect of the blood pressure increase was corroborated.
However, these data are insufficient to determine whether the extended use of AAS can
cause irreversible blood pressure increases. The return of blood pressure values to initial
conditions after treatment discontinuation still requires additional generalizable data,
particularly related to the sample size, time of use, and dose of AAS [14645].
Significant research attention has focused on the impact of AS use on cardiovascular (CV)
disease risk factors namely blood pressure, lipid profile, left ventricular (LV) mass, cardiac
function and arterial function. Elevated systemic arterial blood pressure is associated with an
increased CV disease risk. Compared to healthy controls, AS users have increased resting
and exercise systolic blood pressure. Conversely, other studies have not observed increased
blood pressure in AS user. Differences in the training level of the participants along with age
could be responsible for the differences seen in these studies [12114].
Although not shown in all studies, an association between elevated blood pressure and AAS
abuse has been reported. Enhanced reactivity of the vasculature to norepinephrine,
increased plasma renin activity, stimulation of aldosterone production by testosterone, and
sodium retention by the kidneys are suggested mechanisms for high blood pressure
following AAS use in athletes. Blood pressure response to androgen use typically shows a
1111
dose-response relation. The effects of AAS abuse on blood pressure may persist for long
periods; some studies have shown persistent elevations for 5 to 12 months after
discontinuing steroids [12119].
In experimental studies it has been found that the administration of AAS leads to
hypertension. An increase in the secretion of 11-deoxycorticosterone, norepinephrine, renin,
or aldosterone has been implicated as a possible mechanism, while others have noted an
increase in cardiac output and peripheral resistances. However, clinical studies in athletes
have led to conflicting results. Some observed a significant increase in both systolic and
diastolic blood pressure, whereas others noted only the latter. It has been attributed the
increase in blood pressure in steroid-using athletes to an increase in plasma volume. In
contrast, other authors found no significant increase in blood pressure at rest or during
exercise in athletes who used steroids compared to non-users [12126].
Systemic hypertension is a side effect of medical steroid administration and may require
antihypertensive therapy; therefore, high-dose ASA use also should result in systemic
hypertension. This is found in some reports, but not consistently. AAS-induced hypertension
may be related to vascular endothelial response, increased responsiveness to
catecholamines, and increased renin production. The magnitude and incidence of
hypertension likely are related to dosage and to the specific AAS [07058].
In a study conducted to evaluate the effects of the use and discontinuation of AAS on the
cardiovascular system, both AAS users (140 ± 10 mmHg) and former users (130 ± 5 mmHg)
had higher blood pressure than a control group of non-using weight lifters (125 ± 10 mmHg).
Five of the 16 users, 2 of the 15 former users, and 1 of the 15 non-users in this study
manifested blood pressure values at rest consistent with hypertension (values equal or
greater than 140/90 mmHg) and higher blood pressure response in response to a physical
exercise protocol. This phenomenon occurred despite the users having an average age of
only 31 ± 5 years. In another study, the blood pressure of 16 subjects who performed an
AAS cycle was significantly higher after 8 weeks than controls, and end values were
compatible with hypertension. The blood pressure values returned to normal 8 weeks later,
closing the cycle. The difference between these two studies related to blood pressure
returning to normal after discontinuation of the AAS treatment is the use for a short time in
this second study. However, these data are insufficient to determine whether the extended
use of AAS can cause irreversible blood pressure increases. The return of blood pressure
values to initial conditions after treatment discontinuation still requires additional
generalizable data – particularly related to the sample size, time of use, and dose of AAS
[14247].
Several studies investigating different AAS regimens showed no alteration inblood pressure
(BP) in healthy strength athletes. However, in other investigations, an elevation of systolic or
diastolic BP has been observed as a result of the administration of high doses of AAS. An
elevation of BP may be present within 4 weeks of taking steroids. The most pronounced
increase of diastolic pressure found an increase from 74 to 86 mmHg due to 10 weeks of
self-administration of high-dose AAS. Increments of systolic BP of about 10 and 12 mmHg in
normotensive strength athletes due to AAS have been reported. After drug cessation the BP
seems to return to pre-steroid levels within several weeks. However, a prospective study
could not confirm elevations of BP in athletes, even in those self-administering
supratherapeutic doses of AAS for periods of up to 16 weeks. The available literature is thus
not conclusive with respect to the effects of AAS on BP. It is suggested that elevations of
systolic and/or diastolic BP may occur in some individuals; however, the effect does not
seem to be consistent. Androgens seem to affect BP more than anabolic agents, although
1112
the exact mechanism remains to be established. However, if elevations of BP occur they
seem to be small and transient, indicating that the impact on health status of the athlete may
be limited [04002].
Increased systemic inflammation and oxidative stress participate in the mechanism of

various cardiovascular diseases. These two phenomena are associated with venous
thrombosis and endothelial dysfunction. Curiously, prolonged use of stanozolol decreased
the mitochondrial oxidative stress of skeletal muscle in rats. However, hepatic oxidative
stress has increased in rats at high doses (2 mg/kg body weight). Hepatic oxidative stress is
associated with the production of C-reactive protein, a potent pro-inflammatory agent
involved in vascular dysfunction, arterial hypertension, and ischemic heart disease. However,
it is worth noting that the increase in hepatic oxidative stress seen was in rats, so it cannot be
said that the same would occur in human AAS users. In human experiments, levels of
homocysteine were significantly higher in users of AAS for 21 ± 2. years compared with a
group that also used AAS for 21 ± 3 years, but discontinued the use for 3 months, and
control groups of non-users of AAS. Homocysteine can be involved in the etiology of various
cardiovascular diseases. Androgens can exert vasorelaxant effects, but chronic exposure of
replacement doses decreases the vascular reactivity to vasodilators. In fact, analysis of pulse
waves in response to glyceryltrinitrate shows that endothelium-independent vasodilator
function was impaired in young (26 ± 7 years old) adult bodybuilders. The vasodilatation was
reduced by half compared to previous users who discontinued the use and was only 30% of
the value obtained in a control group of AAS non-users. An impaired vasodilator functioning
was also found in young adults manifesting hypogonadism (35 ± 4 years old) even at
physiological doses used in testosterone replacement therapy. Finally, a lower vasodilator
functioning was accompanied by an increased sympathetic nerve activity to the vasculature
among AAS users based on microneurography measures. This adverse effect of AAS calls
attention, but the data are inconsistent. Besides being one of the few studies with an animal
model, we found only four reports between 2003 and 2013. Either way, the fact that all four
of these studies indicated some vasodilator dysfunction, there is the need for further studies
to confirm/discard the deleterious effects of AAS on inflammation, oxidative stress, and
vascular function [14645].
Other vascular effects
Reported severe adverse effects of anabolic-androgenic steroid use include cerebral venous
sinus thrombosis, ischemic cerebral stroke, and cardiovascular events in the absence of risk
factors. Two cases of limb-threatening arterial thrombosis were reported with the use of
danazol (Danocrine®), an antigonadotropin steroid-like compound with weak anabolic
properties [07031].
Lipid profile influence early atherogenesis. Therapeutic use of AAS has been shown in many
studies to affect the individuals’ lipid profile. A meta-analysis including 19 studies and
comprising 272 hypogonadal men showed that substitution therapy with intramuscularly
administered testosterone results in a decrease in plasma HDL cholesterol levels. The same
results were also demonstrated in a recent meta-analysis including 51 studies on men with
low or low-to-normal plasma testosterone levels who received testosterone in different doses
as therapy. Moreover, high-dose AAS abuse has been demonstrated to exert unfavourable
direct and indirect effects, through AAS-associated hyperhomocysteinaemia, on plasma lipid
levels. In a nonblinded investigation on 19 bodybuilders, short-term (8 weeks) and long-term
(> 14 weeks) high dosages of AAS administration markedly reduced HDL cholesterol. The
1113
suppressive effects of AAS administration on HDL plasma levels are dose dependent and
depending on the type of AAS and route of administration can result in decrement of 40-70
percent. The adverse effects of high AAS dosages on plasma levels of LDL cholesterol have
been shown in animal and human studies. Lipid profile impairment is causally implicated in
vascular wall injury by promoting inflammatory processes in the arterial wall, macrophage
recruitment, and uptake of LDL and oxidized LDL by macrophages which results in foam cell
formation. The aforementioned processes, which contribute to establishment and
progression of atherosclerotic plaques, can be depicted by molecular imaging techniques.
18
F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) has been studied in
a notable number of investigations and has been shown to correlate with the macrophage
density in atherosclerotic plaques in humans and in animal models. Additionally, 18F-FDG
PET depicts MI subsequent to coronary atherosclerosis. Molecular targeting of oxidized LDL
and macrophage uptake of radiolabelled LDL has verified promising targets for visualizing
vulnerable atherosclerotic plaques. Moreover, a recent pilot study reported feasibility of
ultrasmall superparamagnetic particles of iron oxide (USPIO) in detecting inflammation in
endothelial cells during atherogenesis with magnetic resonance imaging (MRI) [12125].
Impaired vasodilatation
Although endogeneous testosterone has been shown to exert vasodilatory effects, AAS use
in hypogonadal men has been shown to result in paradoxical pro-atherogenic
vasoconstrictive effects. It was shown that testosterone therapy in hypogonadal men is
correlated with impaired vasodilation, independently from lipid profile measures.
Supraphysiological doses of AAS have also shown to exert similar effects on vasoreactivity
in human and animal studies. In a study on male bodybuilders who abused AAS for 3-4
years, vasodilatation was significantly lower than that of ex-abusers and controls. AAS abuse
in body builders independently of the other factors impaired endothelium-independent
vasodilator pathways. It was also shown that a 3-month period of abstinence results in a
degree of improvement in vascular function. Moreover, longterm therapy with
supraphysiological doses of AAS in female-to-male transsexuals has shown to result in
decreased vasodilation independent of the effects of age, lipid profile and vessel size. The
mechanisms through which AAS induces deleterious effects on vasodilatation are not
sufficiently investigated. However, endothelial injury as a result of lipid profile alterations and
establishment of atherosclerosis could explain the impairment in endothelium-dependent
pathway through decreased NO production [12125].
Stroke
Anabolic-androgenic steroids are synthetic substances derived from testosterone that are
employed for their trophic effect on muscle tissue, among other uses. Their consumption can
give trigger a series of adverse side effects on the body, including the suppression of the
hypothalamus-pituitary-gonadal axis as well as liver, psychiatric and cardiovascular
disorders. The most common effects are altered fat profiles and blood pressure values,
cardiac remodelling, arrhythmias or myocardial infarcts. It was reported a case of a young
male, with a background of anabolic-androgenic steroids abuse, who visited because of an
acute neurological focus in the right hemisphere related with an ischaemic stroke. The
aetiological study, including cardiac monitoring, echocardiograph and imaging studies
(magnetic resonance and arteriography) and lab findings (thrombophilia, serology,
autoimmunity, tumour markers) showed no alterations. Thus, the association between
consumption of anabolic-androgenic steroids and cardiovascular pathologies is known, but
its relation with cerebrovascular disease has not received so much attention from
researchers [13128].
Popliteal-artery entrapment syndrome

The popliteal-artery entrapment syndrome is a potentially serious but rare cause of ischemia
1114
of the legs. It occurs predominantly in young persons and is due to an abnormal anatomical
relation between the popliteal artery and the tendinous insertion of the gastrocnemius
muscle. Usually, symptoms arise when there is occlusion of the functional artery during
contraction of the calf muscle; arterial thrombosis is a rare cause. Abuse of anabolic steroids
has increased in frequency during the past decade and is associated with a documented risk
of acute coronary-artery and peripheral-artery thrombosis. It was described the occurrence of
thrombotic occlusion of the popliteal artery in an athlete with the popliteal-artery entrapment
syndrome who abused anabolic steroids. It was speculated that the abuse of anabolic
steroids, as a result of their prothrombotic action and promotion of muscle hypertrophy, may
have led to the popliteal-artery entrapment syndrome in this patient. Athletes and the medical
community should be aware of this potential complication of the use of anabolic steroids
[02041].
Summary of metabolic and vascular effects of anabolic steroids

In summary, metabolic and vascular adverse effects of AAS abuse can be classified as
[12125]:
- alterations in the lipid profile, especially decreased serum HDL levels and
hyperhomocysteinaemia contributing to endothelial damage
- increased platelets adhesion to vascular wall
- vasospastic effects and impaired vasodilatation
Experimental
The objectives of one study were to investigate the time-course and the cellular, ionic and
molecular processes underlying ventricular repolarization in rats chronically treated with
AAS. Male Wistar rats were treated weekly for 8 weeks with 10 mg/kg of nandrolone
decanoate (n=21) or vehicle (n=20). ECG was recorded weekly. Action potential and
transient outward potassium current (Ito) were recorded in rat hearts. Expression of KChIP2,
Kv1.4, Kv4.2, and Kv4.3 was assessed by real-time PCR. Hematoxylin/eosin and Picrosirius
red staining were used for histological analysis. QTc was greater in the DECA group. After
nandrolone treatment the left, but not right, ventricle showed a longer AP duration than did
the control. Ito current densities were 48 percent lower in the left but not in the right ventricle
after nandrolone. In the right ventricle the Ito inactivation time-course was slower than in the
control group. After nadrolone the left ventricle showed lower KChIP2 ( approximately 26 %),
Kv1.4 ( approximately 23 %) and 4.3 ( approximately 70 %) expression while the Kv 4.2
increased in 4 ( approximately 250 %) and diminished in 3 ( approximately 30 %) animals of
this group. In the right ventricle the expression of I(to) subunits was similar between the
treatment and control groups. Nandrolone-treated hearts had 25 percent fewer nuclei and
greater nuclei diameters in both ventricles. The results strongly suggest that supra-
physiological doses of AAS induce morphological remodeling in both ventricles. However,
the electrical remodeling was mainly observed in the left ventricle [10328].
Cardiovascular effects of low androgens
Interestingly, if androgen levels are too low, cardiac risk may increase. Androgen-deprivation
therapy (ADT) is a widely used treatment for prostate cancer, and several studies have
reported an association between ADT and an increased risk of myocardial infarction and
cardiovascular mortality. Antiandrogens (e.g. flutamide, bicalutamide) block the binding of
androgen to its receptor, and they are often coupled with gonadotropin-releasing hormone
(GnRH) agonists (e.g. leuprolide, goserelin, triptorelin). In one population-based Medicare
study, the use of a GnRH agonist in men with prostate cancer for at least 1 to 4 months was
1115
associated with an increased risk of incident coronary heart disease (adjusted hazard ratio,
HR, 1.16), myocardial infarction (adjusted HR), and sudden cardiac death or life-threatening
ventricular arrhythmia (adjusted HR 1.16). Another population based study noted that the use
of ADT was associated with a 20 percent higher risk of cardiovascular morbidity (HR 1.20)
during 5 years of follow-up. In addition, androgen deficiency has been associated with
cardiovascular risk factors by causing increased serum total cholesterol, low-density
lipoprotein cholesterol, high-density lipoprotein cholesterol, and triglycerides. There is also an
association between low androgen levels and an adverse metabolic profile (insulin
resistance, metabolic syndrome, and diabetes) [12119].
The increasing prevalence of obesity adds another dimension to the pathophysiology of

testosterone (TEST) deficiency (TD) and potentially impairs the therapeutic efficacy of
classical TEST replacement therapy. We investigated the therapeutic effects of selective
androgen receptor modulation with trenbolone (TREN) in a model of TD with the metabolic
syndrome (MetS). Male Wistar rats (n=50) were fed either a control standard rat chow
(CTRL) or a high-fat/high-sucrose (HF/HS) diet. After 8 weeks of feeding, rats underwent
sham surgery or an orchiectomy (ORX). Alzet miniosmotic pumps containing either vehicle,
2-mg/kg·d TEST or 2-mg/kg·d TREN were implanted in HF/HS+ORX rats. Body composition,
fat distribution, lipid profile, and insulin sensitivity were assessed. Infarct size was quantified
to assess myocardial damage after in vivo ischaemia reperfusion, before cardiac and
prostate histology was performed. The HF/HS+ORX animals had increased sc and visceral
adiposity; circulating triglycerides, cholesterol, and insulin; and myocardial damage, with low
circulating TEST compared with CTRLs. Both TEST and TREN protected HF/HS+ORX
animals against sc fat accumulation, hypercholesterolaemia, and myocardial damage.
However, only TREN protected against visceral fat accumulation, hypertriglyceridaemia, and
hyperinsulinaemia and reduced myocardial damage relative to CTRLs. TEST caused
widespread cardiac fibrosis and prostate hyperplasia, which were less pronounced with
TREN. We propose that TEST replacement therapy may have contraindications for males
with TD and obesity-related MetS. TREN treatment may be more effective in restoring
androgen status and reducing cardiovascular risk in males with TD and MetS [150186].
Abuse of anabolic androgenic steroids is linked to a variety of cardiovascular complications.

The aim of our study was to investigate the possible cardiovascular effects of nandrolone
decanoate on young rabbits using echocardiography, histology and monitoring of telomerase
activity, oxidative stress and biochemical markers. Fourteen rabbits were divided into three
administration groups and the control group. Doses of 4 mg/kg and 10 mg/kg of nandrolone
decanoate, given intramuscularly and subcutaneously, two days per week for six months
were applied. A 4-months wash-out period followed. Focal fibrosis and inflammatory
infiltrations of cardiac tissue were observed in the high dose groups. Thiobarbituric acid-
reactive species (TBARS) levels were significantly increased in the high dose groups, while
catalase activity decreased. Myocardial Performance Index (MPI) is the main
echocardiographic index primarily affected by nandrolone administration in rabbits. Despite
the preserved systolic performance, histological lesions observed associated with distorted
MPI values, point to diastolic impairment of the thickened myocardium due to nandrolone
treatment. Oxidative stress accumulates and telomerase activity in cardiac tissue rises.
Subcutaneous administration seems to be more deleterious to the cardiovascular system, as
oxidative stress, telomerase activity and biochemical markers do not appear to return into
normal values in the wash-out period [150187].
Anabolic androgenic steroids lead to cardiac complications and have been shown to exhibit
proapoptotic effects in cardiac cells; however, the mechanism involved in those effects is
unclear. The aim of one study was to assess whether apoptosis and the activation of
caspase-3 (Casp-3) induced by testosterone in high concentrations involves increments in
1116
tumor necrosis factor-alpha (TNF-alpha) concentrations and angiotensin-converting enzyme
(ACE) activity in cardiomyocytes (H9c2) cell cultures. Cardiomyocytes were treated with
testosterone (5 × 10-6 mol/L), doxorubicin (9.2 × 10-6 mol/L), testosterone + etanercept (Eta;
6.67 × 10-5 mol/L), testosterone + losartan (Los; 10-7 mol/L), and testosterone + AC-DEVD-
CHO (10-5 mol/L; Casp-3 inhibitor). Apoptosis was determined by flow cytometry and by the
proteolytic activity of Casp-3. It was demonstrated that incubation of H9c2 cells for 48 h with
testosterone causes the apoptotic death of 60-70 percent of the cells and co-treatments with
Eta, Los, or AC-DEVD-CHO reduced this effect. Testosterone also induces apoptosis
(concentration dependent) and increases the proteolytic activity of Casp-3, which were
reduced by co-treatments. TNF-alpha and ACE activities were elevated by testosterone
treatment, while co-treatment with Los and Eta reduced these effects. It was concluded that
an interaction between testosterone, angiotensin II, and TNF-alpha induced apoptosis and
Casp-3 activity in cultured cardiomyocytes, which contributed to the reduced viability of these
cells induced by testosterone in toxic concentrations [150188].
Anabolic steroids used to improve muscular strength and performance in athletics. Its long-
term consumption may induce cardiovascular adverse effects. It was assessed the risk of
ventricular arrhythmias in rats which subjected to chronic nandrolone plus high-intensity
endurance exercise. Animals were grouped as; control (CTL), exercise (Ex): 8 weeks under
exercise, vehicle group (Arach): received arachis oil, and Nan group: received nandrolone
decanoate 5mg/kg twice a week for 8w eeks, Arach+Ex group, and Nan+Ex. Finally, under
anesthesia, arrhythmia was induced by infusion of 1.5 microg/0.1mL/min of aconitine IV and
ventricular arrhythmias were recorded for 15min. Then, animals' hearts were excised and
tissue samples were taken. Nandrolone plus exercise had no significant effect on blood
pressure but decreased the heart rate and increased the RR and JT intervals of
electrocardiogram. Nandrolone+exercise significantly increased the ventricular fibrillation
(VF) frequency and also decreased the VF latency. Combination of exercise and nandrolone
could not recover the decreasing effects of nandrolone on animals weight gain but, it
enhanced the heart hypertrophy index. In addition, nandrolone increased the level of
hydroxyproline (HYP) and malondialdehyde (MDA) but had not significant effect on
glutathione peroxidase of heart. Exercise only prevented the effect of nandrolone on HYP.
Nandrolone plus severe exercise increases the risk of VF that cannot be explained only by
the changes in redox system. The intensification of cardiac hypertrophy and prolongation of
JT interval may be a part of involved mechanisms [150189].
Summaries of effects of anabolic steroids on the heart
To sum up, the myocardial effects of AAS abuse can be summarized in three different
categories including [125]:
- myocardial hypertrophy as result of

o levated muscle sympathetic nerve activity
o direct anabolic effects of AAS
o renin-angiotensin system activity induced collagen deposition and interstitial
fibrosis
- left ventricular dysfunction as result of
o AAS-induced myocardial hypertrophy
o mitochondrial damage and apoptosis as consequences of Ca2+ signaling
o rennin-angiotensin system activity and fibrosis
- cardiac arrhythmias as result of increased myocardial mass and reduction
Some physiological effects of anabolic-androgen steroids were summarized [12119]:
1117
System Physiological effect
Musculoskeletal skeletal muscle hypertrophy and formation of new muscle cells, especially
in muscles of thorax, neck, shoulders, and upper arms. Increased
bone remodeling and growth, closure of the epiphyseal growth
centers
Hematological stimulation of bone marrow with increased production of red blood
cells
Dermatological hirsutism (i.e. increased growth of androgen-sensitive hair: pubic,
beard, chest, limb
Sebaceous gland stimulation, resulting in acne
Ear, nose, throat vocal cord hypertrophy (deepening of the voice)
Genitourinary hypertrophy of clitoris and penis. Testicular atrophy (oligospermia,
decreased ejaculatory volume). Prostate hypertrophy
Cardiovascular retention of sodium and fluid
Endocrine gynecomastia
Increased risk of diabetes
Anabolic steroids decrease glucose tolerance and increase insulin resistance, which lead to
hyperinsulinism and secondary diabetes mellitus with type II symptoms [07058].
The case of a 36-year-old male professional bodybuilder was reported. He presented to the
accident and emergency department with right upper quadrant pain. This was on the
background of a 15-year history of anabolic steroid and growth hormone misuse.
Examination revealed mild hepatomegaly and a random blood sugar of 30.2 mmol/L. There
was no evidence of ketonuria or acidosis. Biochemical evidence of hepatitis was found, and
the patient was in acute renal failure. He was given a sliding scale of insulin and an
intravenous infusion of crystalloid. The hepatitis and hyperglycaemia settled with
conservative treatment. It is believed that this is the first reported case of frank diabetes
precipitated by supraphysiological recreational growth hormone misuse [07069].
A 33-year-old male presented to the emergency department with complaints of polydipsia,

polyuria, nausea, headaches, blurry vision and malaise. Lab work revealed a serum glucose
level of 1166 mg/dl (64.8 mmol/L). The patient admitted to completing a cycle of androgenic
anabolic steroids (AASs) for bodybuilding. His regimen consisted of supraphysiologic
intramuscular injections of a bovine growth hormone, trenbolone acetate and testosterone.
The patient received intravenous fluids and insulin to restore metabolic balance. Previously
healthy with a non-contributory family history, he was diagnosed with new onset diabetes.
Discussion: It has been demonstrated that AAS use, specifically growth hormone, can affect
glucose homeostasis through increasing cellular insulin resistance and reducing glucose
uptake. Excess growth hormone has been shown to cause symptoms of acromegaly which
predisposes up to 40% of patients to diabetes. As trenbolone acetate is not indicated for
human use and athletes are known to use supraphysiologic doses of this underground,
performance enhancing drug, the correlation of the timing of events and the use of this
veterinary growth hormone likely exacerbated an underlying condition or caused this new
onset diabetes. It was reported a case of a young bodybuilder with no significant past
medical history who was diagnosed with new onset diabetes associated with
supraphysiologic self-injections of the bovine growth hormone, trenbolone acetate, combined
with testosterone. AAS have the potential to induce or exacerbate diabetic conditions due to
decreased glucose tolerance and increased insulin resistance [11094].
1118
Effects on the brain, cognition abnormalities and psychiatric effects
AAS are universally recognized to have psychoactive effects. Although some spared studies
have reported their therapeutic use in depression to improve mood and anergia, most
evidence points toward the association of AAS with depression, mania, psychosis, suicide
and increased aggression leading to violence and, in extreme cases, to homicide. Indeed,
suicide and homicide have been shown to be the main cause of premature deaths among
steroid users and, in particular, in the teen population. Although this does not imply that all
steroid users will suffer crippling depression or homicidal rage, steroids appear to strongly
contribute to psychiatric dysfunctions in susceptible individuals [150003].
Psychological motivations contributing to anabolic steroid use and abuse have received little
attention in psychiatric literature. Clinical studies demonstrate that steroids are used in part to
deal with an earlier trauma, such as childhood physical or sexual abuse [150004].
Psychological motivations contributing to anabolic steroid use and abuse have received little
attention in psychiatric literature. Clinical studies demonstrate that steroids are used in part to
deal with an earlier trauma, such as childhood physical or sexual abuse [150004].
Some adolescent and young males are engaged in misuse of anabolic-androgenic steroids
(AASs) in connection with multiple drug use, in order to become intoxicated and brave, apart
from currently known motives connected to sports performance and physical appearance.
Recent studies suggest that alterations in neurobiological circuits implicated in the regulation
of reward-related learning, aggression and motoric behavior underlie the behavioral changes
associated with AAS misuse. We have previously shown that AASs induce alterations in
dopamine receptor densities. The aim of the present study was to investigate if these effects
could be attributed to altered mRNA content for tyrosine hydroxylase, L-amino acid
decarboxylase, dopamine D1- and dopamine D2-receptor as measured by in situ
hybridisation. Male Sprague-Dawley rats were subjected to 2 weeks of treatment with daily
intramuscular injections of the AAS nandrolone decanoate at three different doses (1, 5 and
15 mg/kg/day). Results of the in situ hybridization showed that the mRNA content of the
dopamine D1-receptor subtype was significantly reduced at all doses in the caudate putamen
and at the highest doses in the nucleus accumbens shell. The mRNA expression of the
dopamine D2-receptor was significantly increased at the two lowest doses in the caudate
putamen and the nucleus accumbens shell. In conclusion, nandrolone has been shown to
affect the expression of gene transcripts of dopaminergic receptors possibly implicated in
underlying mechanisms of reward-related behavioral changes among AAS misusers [03061].
Supraphysiologic doses of testosterone are associated with increased aggression that is

hypothesized to be a function of testosterone serum concentrations, mood, and personality.
The study attempted to characterize this relationship among weightlifters who were users
(n=10) and nonusers (n=18) of anabolic steroids. Participants were interviewed using the
Modified Mania Rating Scale and Hamilton Rating Scale for Depression to assess mood, the
Buss-Durkee Hostility Inventory (BDHI) and Point Subtraction Aggression Paradigm (PSAP)
to assess aggression, and the Personality Disorder Questionnaire (PDQ-R) to assess
personality. Blood samples were obtained for the determination of total, free, and weakly
bound testosterone. Comparisons of continuous variables between testosterone users and
non-users were performed with a parametric (unpaired t-test) or non-parametric (Mann-
Whitney) test where appropriate. Correlations with testosterone were examined separately
for testosterone users and non-users, using Spearman rank correlation. The subjective
(BDHI) and objective (PSAP) assessments of aggression found that supranormal
testosterone concentrations were associated with increased aggression. However, the PDQ-
R results suggest that this finding was confounded by the personality disorder profile of the
1119
steroid users, because steroid users demonstrated Cluster B personality disorder traits for
antisocial, borderline, and histrionic personality disorder [03062].
Generally, athletes taking AS for more prolonged periods are likely to suffer from more
severe medical consequences. Those who do discontinue the AS treatment discover that the
improvements made with the steroids disappear and have little to show for years of
exhausting training beyond the psychological scars inherent in steroid use. The abuse of AS
may also cause psychiatric effects such as irritability, aggression, mood swings, dimini-shed
awareness of fatigue and depression [03002].
Chronic anabolic-androgenic steroid (AAS) treatment during adolescence facilitates offensive

aggression in male Syrian hamsters (Mesocricetus auratus). The current study assessed
whether adolescent AAS exposure influenced the immunohistochemical localization of
glutamic acid decarboxylase (GAD65), the rate-limiting enzyme in the synthesis of gamma-
aminobutyric acid (GABA), in areas of hamster brain implicated in aggressive behavior.
Hamsters were administered high dose AAS throughout adolescence, scored for offensive
aggression, and then examined for differences in GAD65 puncta to regions of the hamster
brain important for aggression. When compared with control animals, aggressive AAS-
treated hamsters showed significant increases in the area covered by GAD65
immunoreactive puncta in several of these aggression regions, including the anterior
hypothalamus, ventrolateral hypothalamus, and medial amygdala. Conversely, aggressive
AAS-treated hamsters showed a significant decrease in GAD65-ir puncta in the lateral
septum when compared with oil-treated controls. However, no differences in GAD65 puncta
were found in other aggression areas, such as the bed nucleus of the stria terminalis and
central amygdala. Together, these results support a role for altered GAD65 synthesis and
function in adolescent AAS-facilitated offensive aggression [03063].
Recent reports suggest that anabolic-androgenic steroids (AAS) may cause mood disorders
or dependence syndromes and may help to introduce some individuals to opioid abuse. At
present, however, little is known about prior AAS use among men entering inpatient
substance abuse treatment. It was assessed lifetime AAS use in 223 male substance
abusers admitted to a substance abuse treatment unit primarily for treatment of alcohol,
cocaine, and opioid dependence. Subjects reporting definite or possible AAS use were then
asked to participate in a detailed semistructured interview that covered demographics, drug
use history, and symptoms experienced during AAS use and withdrawal, and whether AAS
use had helped introduce the subject to other classes of drugs. Twenty-nine men (13 %)
reported prior AAS use, but this history was documented on physicians' admission
evaluations in only 4 cases. Among 88 men listing opioids as their drug of choice, 22 (25 %)
acknowledged AAS use, versus only 7 (5 %) of the other 135 men. Twenty-four (83 %) of the
29 AAS users were interviewed in detail. Seven (29 %) of the men interviewed, all with opioid
dependence, reported that they first learned about opioids from friends at the gym and
subsequently first obtained opioids from the same person who had sold them AAS. Eighteen
(75 %) of the men interviewed reported that AAS were the first drugs that they had ever self-
administered by injection, 4 (17 %) reported severe aggressiveness or violence during AAS
use, 1 (4%) attempted suicide during AAS withdrawal, and 5 (21 %) described a history of
AAS dependence. Thus, prior AAS use appears to be common but underrecognized among
men entering inpatient substance abuse treatment, especially those with opioid dependence.
AAS use may serve as a "gateway" to opioid abuse in some cases and may also cause
morbidity in its own right [03064].
Affective disorders and impulsivity are quite common when using anabolic substances, in this
case-study one of the rather rare cases of a psychotic disorder following the abuse of
androgenic steroids is described. A 30-year old formerly healthy white male was admitted as
1120
inpatient to psychiatric hospital showing symptoms of anxiety and paranoid ideation. In the
last 1.5 years he had consumed androgenic steroids, directly before the onset of the first
psychotic symptoms 8 weeks before admission he had received an i.m.-injection of
nandrolone. Under therapy with neuroleptics the patient recovered completely within 2
months [03065].
The misuse of anabolic androgenic steroids has in several reports been associated with
effects resulting in altered behavior. One study used the Morris water maze task to
investigate the effect of high doses of the anabolic androgenic steroid nandrolone on spatial
learning and memory in male rats. During the experiment, it was observed a significantly
impaired Morris water maze performance in the nandrolone-treated rats compared with
controls. The hippocampus, a brain region associated with cognitive function, was analyzed
for mRNA expression of prodynorphin, the precursor of dynorphinergic peptides. The results
indicated that the transcription levels of prodynorphin were significantly elevated in the
animals treated with nandrolone compared with controls. Thus, the findings suggest that
administration of nandrolone to male rats impairs memory function, possibly via
dynorphinergic actions [09059].
Anabolic androgenic steroids and high testosterone doses have been reported to induce
impulsive behavior in man and behavioral disinhibition in rats. The purpose of the present
study was to investigate whether aromatization of testosterone to estradiol is of importance
for the behavioral disinhibiting effect of a high testosterone dose in adult male rats.
Testosterone administered via five testosterone-filled silastic capsules implanted
subcutaneously to non-castrated, group-housed rats for six days induced behavioral
disinhibition in a modified Vogel's drinking conflict model and yielded supraphysiological
serum levels of testosterone and increased accessory sex organ weights. Moreover,
concurrent administration of the aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD;
60 mg/kg/day s.c.) decreased behavioral disinhibition in testosterone-treated rats (without
affecting accessory sex organ weights) while behavior was not significantly affected in sham-
treated animals. Since some reports indicate that ATD, in addition to inhibit aromatase, also
may affect the binding of testosterone to the androgen receptor, the effect of the non-
steroidal androgen receptor antagonist flutamide was investigated. Flutamide treatment did
not affect disinhibited behavior in testosterone-treated rats. However, in sham-treated
animals, flutamide (50 mg/kg/day) produced behavioral disinhibition. These results suggest
that estradiol is of importance in the mechanisms underlying behavioral disinhibition in non-
castrated rats treated with a high testosterone dose. Speculatively, aromatization may be
involved in pro-impulsive effects of high testosterone doses in humans [09060].
Several case reports associated chronic AAS abuse with the development of seizures,
ischemic midbrain lesions with residual dysfunction (hemiparesis, aphasia, and dysarthria),
and thrombotic strokes, but the causal link between cerebrovascular accidents and chronic
AAS abuse is unproven. A 34-year old bodybuilder developed an acute right hemipares is a
dysarthria after using various AASs for 4years. He had a simple partial seizure. At discharge,
he had mild motor weakness in the right upper extremity with no sensory changes. A 21-year
old man developed a generalized tonic-clonic seizure, left-sided hemiparesis after using 6-8
mg ethylestrenol daily for 6 weeks. Angiography demonstrated an occlusion of the right
anterior cerebral and right middle cerebral arteries. In a retrospective cohort study of 248
patients who tested positive for the presence of AAS in connection with receiving medical
care, the incidence of unspecified convulsions was increased in the AAS-positive group (RR
54) when compared with controls (i.e. patients testing negative for AAS at the same
institution). One of the AAS-positive patients with seizures died. However, there are few data
from cohort or case-control studies to confirm the causal link between AAS abuse ands
eizures [13003].
1121
One of the first reports investigating the effects of AAS on psyche and behaviour in athletes
was performed by Lindstrom et al. In their survey, male bodybuilders reported mood changes
and an increased libido when on steroids. Many articles have been published since then;
self-reporting of athletes as well as survey studies indicate that AAS users seem to be
subject to an increase of aggression and/or hostility. Conversely, some investigations did not
observe such a relationship and in one study strength athletes were found to demonstrate
even lower hostility with anabolic steroids [04002].
Neurotoxicity
Recent evidence suggests that supraphysiologic levels of testosterone and other AAS exhibit
apoptotic effects in a variety of cell types, including human neuronal cells. Two subsequent
studies have now also demonstrated neurotoxic effects of supraphysiologic AAS in
mammalian neuronal cells. A recent animal study found spatial memory deficits, as assessed
by the Morris water maze, in rats following supraphysiologic AAS exposure. Collectively,
these findings raise the ominous possibility that long-term users of high-dose AAS might
develop potentially irreversible cognitive deficits. In a pilot study exploring this possibility
among 31 AAS user and 13 nonuser weightlifters, one group of investigators found
significant deficits in visuospatial memory among AAS users as opposed to nonusers; and
within the AAS-users group these deficits were significantly associated with total lifetime
burden of AAS exposure. Thus the possibility of AAS-induced neurotoxicity clearly demands
further study [14017].
Recent evidence suggests that supraphysiologic levels of testosterone and other AASs
exhibit apoptotic effects in a variety of cell types, including human neuronal cells. Two
subsequent studies have now also demonstrated neurotoxic effects of supraphysiologic AAS
in mammalian neuronal cells. A recent animal study found spatial memory deficits, as
assessed by the Morris water maze, in rats after supraphysiologic AAS exposure.
Collectively, these findings raise the ominous possibility that long-term users of high-dose
AAS might develop potentially irreversible cognitive deficits. In a pilot study exploring this
possibility among 31 AAS user and 13 nonuser weightlifters, one group of investigators found
significant deficits in visuospatial memory among AAS users as opposed to nonusers; and
within the AAS-users group, these deficits were significantly associated with a total lifetime
burden of AAS exposure. Thus, the possibility of AAS-induced neurotoxicity clearly demands
Anabolic-androgenic steroids (AAS) are synthetic substances derived from testosterone that
are largely employed due to their trophic effect on muscle tissue of athletes at all levels.
Since a great number of organs and systems are a target of AAS, their adverse effects are
primarily on the following systems: reproductive, hepatic, musculoskeletal, endocrine, renal,
immunological, infectious, cardiovascular, cerebrovascular, and hematological.
Neuropsychiatric and behavioral effects as a result of AAS abuse are well known and
described in the literature. Mounting evidence exists suggesting that in addition to psychiatric
and behavioral effects, non-medical use of AAS carries neurodegenerative potential.
Although, the nature of this association remains largely unexplored, recent animal studies
have shown the recurrence of this AAS effect, ranging from neurotrophin unbalance to
increased neuronal susceptibility to apoptotic stimuli. Experimental and animal studies
strongly suggest that apoptotic mechanisms are at least in part involved in AAS-induced
neurotoxicity. Furthermore, a great body of evidence is emerging suggesting that increased
susceptibility to cellular oxidative stress could play a pivotal role in the pathogenesis of many
neurodegenerative disorders and cognitive impairment. As in other drug-evoked
1122
encephalopathies, the key mechanisms involved in AAS – induced neuropathology could
represent a target for future neuroprotective strategies. Progress in the understanding of
these mechanisms will provide important insights into the complex pathophysiology of AAS-
induced neurodegeneration, and will pave the way for forthcoming studies. Supplementary to
abandoning the drug abuse that represents the first step in reducing the possibility of
irreversible brain damage in AAS abusers, neuroprotective strategies have to be developed
and implemented in future [150193].
Brain trauma
Basic science has also largely overlooked the potential interaction of AASs and traumatic
brain injury. For many neurologic conditions, estrogen is neuroprotective in females. This is
particularly true for response to hypoxic-ischemic brain damage, as occurs with stroke.
Whether testosterone at physiologic levels reduces or exacerbates neuronal injury in males
remains unresolved. One emerging hypothesis is that endogenous androgens may be
harmful during the acute phase of ischemic brain injury but can have beneficial effects during
recovery. Even so, it is unclear how this may translate to the elevated levels of androgens
characteristic of AAS use. Under these circumstances, the cellular targets and mechanisms
of action may be substantially different from the effects at normal physiologic levels [14426].
Brain development
Puberty is a critical period for brain maturation that is highly dependent on gonadal sex
hormones. Modifications in the gonadal steroid environment, via the use of anabolic
androgenic steroids (AAS), have been shown to affect brain development and behavior.
Studies in both humans and animal models indicate that AAS exposure during adolescence
alters normal brain remodeling, including structural changes and neurotransmitter function.
The most commonly reported behavioral effect is an increase in aggression. Evidence has
been presented to identify factors that influence the effect of AAS on the expression of
aggression. The chemical composition of the AAS plays a major role in determining whether
aggression is displayed, with testosterone being the most effective. The hormonal context,
the environmental context, physical provocation and the perceived threat during the social
encounter have all been found to influence the expression of aggression and sexual
behavior. All of these factors point toward an altered behavioral state that includes an
increased readiness to respond to a social encounter with heightened vigilance, and
enhanced motivation. This AAS-induced state may be defined as emboldenment. The
evidence suggests that the use of AAS during this critical period of development may
increase the risk for maladaptive behaviors along with neurological disorders [12133].
Cerebral infarction
Anabolic androgenic steroid (AAS) abuse has increased among athletes in recent years.
However, AAS abuse can increase hypercoagulopathy and cause cerebrovascular disease.
We report a case of a 27-year-old man who had right hemiparalysis, hemianopia, dysarthria,
and double vision in the middle of muscle training. He suspected acute disseminated
encephalomyelitis at first, because of a preceding respiratory infection. However, extensive
work-up was performed, including brain magnetic resonance imaging, transcranial Doppler
and transesophageal echocardiography, confirming the final diagnosis of cardioembolic
stroke. Physicians should be aware that cerebrovascular disease may be a side effect of
AAS, even in younger populations [12134].
Cerebral venous thrombosis
1123
Androgen was reported to cause cerebral venous thrombosis (CVT) during replacement
therapy for aplastic anemia. Oxymetholone, a synthetic androgen analogue, has been widely
used in the treatment of aplastic anemia. A 40-year-old woman with aplastic anemia visited
our hospital because of severe headache, nausea, vomiting, blurred vision and diplopia for a
period of 1 month. She had taken oxymetholone for 2 years. Neurological examination
revealed bilateral papilledema and bilateral sixth nerve palsies. Brain magnetic resonance
imaging (MRI), performed at the time of admission, demonstrated left-sided tentorial SDH,
and focal cerebral thrombosis of the left superficial sylvian vein and sigmoid sinus. MR
venography revealed multiple irregularities in the superior sagittal sinus and left transverse
sinus. CVT with tentorial subdural hematoma (SDH) caused by oxymetholone was strongly
suggested. Oxymetholone was immediately discontinued, and her symptoms and signs
disappeared. Because of the thrombocytopenia, anticoagulation was not started. She was
discharged and visited the outpatient clinic without neurological symptoms for 6 months. This
report supports the cautions given about the risk of CVT with oxymetholone supplementation
in aplastic anemia. To the best of our knowledge, this is the first report of CVT associated
with tentorial SDH that was probably caused by oxymetholone [01058].
Calcification of the basal ganglia matter of the cerebellum (Fahr's disease)
A 33-year-old male black student suddenly died during a basketball game. His previous
medical history, including his neurological status, was unremarkable, but he was known to
take anabolic steroids for several years. At autopsy, the cause of death was due to a fresh
myocardial infarction. On neuropathological examination, there was extensive bilateral
symmetrical calcification involving the basal ganglia as well as the dentate nuclei and the
white matter of the cerebellum (Fahr's disease). A possible correlation between anabolic
steroid-induced hypercalcemia and brain calcification is discussed [01054].
Brain nerve growth factor
Anabolic androgenic steroids (AASs) are synthetic androgen-like compounds which are
abused in sport communities despite their side effects. Nerve growth factor (NGF) influences
neuronal differentiation and survival, and also mediates higher brain functions such as
learning and memory. Changes in NGF expression have been implicated in neurode-
generative disorders, including Alzheimer's disease. Hence, we decided to study the effect of
chronic AASs exposure on brain NGF profile, NGF-dependent cholinergic function and
related behavioral performance. Male Wistar rats were injected for 4 weeks with either
nandrolone or stanozolol at daily doses (5.0 mg/kg, s.c.) that are considered equivalent to
those abused by humans. NGF levels and NGF receptor (TrkA and p75NTR) expression
were measured in the hippocampus and in the basal forebrain. Choline acetyltransferase
expression was evaluated in basal forebrain. Spatial learning and memory were assessed
using the Morris water maze. AASs treatment caused region-specific changes in the
expression of NGF and its receptors. Both nandrolone and stanozolol increased NGF levels
in the hippocampus and reduced NGF levels in the basal forebrain, reduced p75NTR
expression in the hippocampus, and failed to affect TrkA expression in the basal forebrain.
Finally, AASs treatment reduced the expression of choline acetyltransferase in the basal
forebrain and impaired the behavioral performance in the Morris water maze. The evidence
that supraphysiological doses of AASs cause neurotrophic unbalance and related behavioral
disturbances, raises the concern that AASs abuse in humans may affect mechanisms that lie
at the core of neuronal plasticity [12132].
Decreased memory
1124
Chronic exposure to the anabolic androgenic steroids (AAS) nandrolone decanoate (ND) in
supra-physiological doses is associated with learning and memory impairments. Given the
well-known beneficial effects of voluntary exercise on cognitive functions, it was examined
whether voluntary exercise would improve the cognitive deficits induced by chronic
administration of ND. It was also investigated the effects of ND and voluntary exercise on
hippocampal BDNF levels. The rats were randomly distributed into 4 experimental groups:
the vehicle-sedentary group, the ND-sedentary group, the vehicle-exercise group, and the
ND-exercise group. The vehicle-exercise and the ND-exercise groups were allowed to freely
exercise in a running wheel for 15days. The vehicle-sedentary and the ND-sedentary groups
were kept sedentary for the same period. Vehicle or ND injections were started 14days prior
to the voluntary exercise and continued throughout the 15days of voluntary exercise. After
the 15-day period, the rats were trained and tested on a water maze spatial task using four
trials per day for 5 consecutive days followed by a probe trial two days later. Exercise
significantly improved performance during both the training and retention of the water maze
task, and enhanced hippocampal BDNF. ND impaired spatial learning and memory, and this
effect was not rescued by exercise. ND also potentiated the exercise-induced increase in
hippocampal BDNF levels. These results seem to indicate that voluntary exercise is unable
to improve the disruption of cognitive functions by chronic ND. Moreover, increased levels of
BDNF may play a role in ND-induced impairments in learning and memory. The harmful
effects of ND and other AAS on learning and memory should be taken into account when
athletes decide to use AAS for performance or body image improvement [12135].
Anabolic-androgenic steroids (AAS) are used in the medical treatment of many disorders.
Erythropoietin (EPO) is a hematopoietic cytokine that has anti-apoptotic, anti-oxidative, and
anti-inflammatory effects. The aim of one study was to investigate the neuroprotective effects
of EPO in the hippocampus, parietal cortex and prefrontal cortex, in brain damage due to
nandrolone decanoate. Thirty-five Wistar male rats were randomly divided into: (1) control
group, (2) sham group, (3) nandrolone decanoate group (ND, intramuscular, 10mg/(kgweek),
8 weeks), (4) ND+low dose EPO treated group (ND+L-EPO) and (5) ND+high dose EPO
treated group (ND+H-EPO). EPO was administrated by intraperitoneal injection at a dose of
100U/(kgday) for L-EPO treatment and at a dose of 500 U/(kg/day) for H-EPO treatment
during 8 weeks. The number of neurons of CA1, CA2, CA3 and dentate gyrus of
hippocampus, parietal cortex and prefrontal cortex were significantly less in the ND group
compared with the control group. Treatment with H-EPO significantly preserved the number
of neurons in hippocampus when compared with ND administrated. Besides, H-EPO
treatment decreased the number of TUNEL-positive and active caspase-3 positive cells and
MDA levels and increased GPx levels when compared to ND group. In conclusion, abuse of
AAS causes reduction in the number of neurons in hippocampus, parietal cortex and
prefrontal cortex regions and increases oxidative damage and therefore H-EPO may be
useful as a neuroprotective agent in brain injury [12136].
Long-term effects of pubertal steroid exposure on aggressive behaviors.
One study examined acute and long-term effects of anabolic-androgenic steroid (AAS)
exposure during puberty on copulation, vocalizations, scent marking, and intermale
aggression, both with and without tail pinch, in intact male rats. Animals received 5 mg/kg of
testosterone, nandrolone, stanozolol, or vehicle, beginning at puberty. After 5 weeks,
behavior tests were performed while continuing AAS injections. AAS treatment was then
discontinued. Behaviors were tested during 3-5 weeks, 9-11 weeks, and 15-17 weeks of
withdrawal. During AAS administration, stanozolol males showed significant reductions in all
behaviors compared with controls, except aggression with tail pinch. Nandrolone treatment
significantly reduced vocalizations and scent marking, and testosterone had no significant
effect on behavior. During withdrawal, behaviors in stanozolol males recovered to control
1125
levels at variable rates: aggression at 4 weeks; mounts, vocalizations, and scent marking at
9 weeks; and ejaculations at 15 weeks of withdrawal. Stanozolol males showed significantly
higher levels of tail pinch-induced aggression during every withdrawal test. Nandrolone-
treated males scent-marked at control levels by 9 weeks withdrawal but displayed
significantly fewer vocalizations and significantly more tail pinch-induced aggression than
controls for the entire study. Testosterone-treated males scent-marked significantly below
controls at 3 weeks withdrawal and showed significantly more tail pinch-induced aggression
at 5 weeks withdrawal. All three AAS significantly increased tail pinch-induced aggression
compared with corresponding nontail pinch tests, even at study endpoint. These results
suggest that alterations in androgen-dependent behaviors by pubertal AAS exposure can
persist long after drug exposure, and some effects may even be permanent [04063].
The behavioral effects of AAS may influence training intensity, thus indirectly increasing
muscle size and strength. ARs are widely distributed throughout the brain, and testosterone
exhibits diverse effects on several central nervous system neurotransmitters. High-dose AAS
administration in normal volunteers increases euphoria, energy, and sexual arousal, and the
cerebrospinal fluid of testosterone-treated men contains higher levels of 5-hydroxyindole-
acetic acid that correlates significantly with AAS-related effects [04018].
Some researchers could not demonstrate any effects on mood while others observed mood
disturbances in AAS users, although the alterations may be very subtle sometimes. Mood
changes associated with AAS abuse included depression, paranoia, (hypo)-mania and
psychotic features. Some studies indicated that the occurrence and seriousness of mood
disturbances are dose dependent, and the effect may be not uniform across individuals since
only few individuals will be affected; most will show only little psychological alterations while
only a few may develop prominent changes [002].
Anabolic-androgenic steroid (AAS) use is associated with psychiatric symptoms including

increased aggression as well as with cognitive dysfunction. The brain effects of long-term
AAS use have not been assessed in humans. One multimodal magnetic resonance imaging
study of the brain compared 10 male weightlifters reporting long-term AAS use with 10 age-
matched weightlifters reporting no AAS exposure. Participants were administered
visuospatial memory tests and underwent neuroimaging. Brain volumetric analyses were
performed; resting-state fMRI functional connectivity (rsFC) was evaluated using a region-of-
interest analysis focused on the amygdala; and dorsal anterior cingulate cortex (dACC)
metabolites were quantified by proton magnetic resonance spectroscopy (MRS). AAS users
had larger right amygdala volumes than nonusers and reduced rsFC between right amygdala
and frontal, striatal, limbic, hippocampal, and visual cortical areas. Left amygdala volumes
were slightly larger in AAS users but few group differences were detected in left amygdala
rsFC. AAS users also had lower dACC scyllo-inositol levels and higher glutamine/glutamate
ratios, possibly reflecting increased glutamate turnover. On a visuospatial cognitive task,
AAS users performed more poorly than nonusers, with the difference approaching
significance. Long-term AAS use is associated with right amygdala enlargement and reduced
right amygdala rsFC with brain areas involved in cognitive control and spatial memory, which
could contribute to the psychiatric effects and cognitive dysfunction associated with AAS use.
The MRS abnormalities we detected could reflect enhanced glutamate turnover and
increased vulnerability to neurotoxic or neurodegenerative processes, which could contribute
to AAS-associated cognitive dysfunction [150200].
The relationship between serum testosterone levels and mental state/behavior
When AAS abuse by athletes first began the medical society (and others) were rightly
1126
concerned about the untoward physical effects. Later it became obvious that these agents
exert profound effects on psyche and behaviour. From animal and human studies the
relationship between endogenous male sex hormones on one side and psychological
function and/or behaviour on the other has been assessed thoroughly. In several animal
species a relationship between endogenous testosterone levels and aggressive behaviour
was likely to be present; however, the findings in humans were less consistent. From clinical
studies it appeared that aggressive behaviour and endogenous testosterone levels did not
correlate very well [04002].
The first observations of psychological function alterations due to AAS abuse were reported
in case studies describing athletes abusing huge amounts of such steroids. For example,
self-administration of AAS was associated with the occurrence of schizophrenia, steroid
dependence, affective and psychotic symptoms, and homicide and near-homicide. Moreover,
many other changes of mental health and behaviour due to AAS abuse have been reported
in the literature, including hypomanic episodes, violent murder, child abuse and spouse
battery [04002].
Anti-social behaviors associated with anabolic-androgenic steroid

Anabolic-androgenic steroids (AAS) have been linked to a range of problematic behaviors,
but AAS use is still sometimes portrayed as more benign than other forms of classical drug
abuse. To address this issue, it was compared the prevalence of anti-social behaviors
among adolescent AAS users, non-AAS illicit drug users, and drug non-users. It was
examined 3 waves (2004, 2008, and 2012) of self-reported cross-sectional data from a
secondary school survey conducted in Stockholm, Sweden (total n=19,773; response
percentage, 79.6 %). Across all survey years, the risk ratios for virtually all measured anti-
social behaviors were significantly higher among AAS users compared to non-AAS illicit drug
users and to drug non-users [150199].
Sex and exercise interact to alter steroid-induced anxiety-like behaviors
Anabolic androgenic steroids (AAS) are taken by both sexes to enhance athletic
performance and body image, nearly always in conjunction with an exercise regime.
Although taken to improve physical attributes, chronic AAS use can promote negative
behavior, including anxiety. Few studies have directly compared the impact of AAS use in
males versus females or assessed the interaction of exercise and AAS. It was shown that
AAS increase anxiety-like behaviors in female but not male mice and that voluntary exercise
accentuates these sex-specific differences. It was also shown that levels of the anxiogenic
peptide corticotrophin releasing factor (CRF) are significantly greater in males, but that AAS
selectively increase CRF levels in females, thus abrogating this sex-specific difference.
Exercise did not ameliorate AAS-induced anxiety or alter CRF levels in females. Exercise
was anxiolytic in males, but this behavioral outcome did not correlate with CRF levels. Brain-
derived neurotrophic factor (BDNF) has also been implicated in the expression of anxiety. As
with CRF, levels of hippocampal BDNF mRNA were significantly greater in males than
females. AAS and exercise were without effect on BDNF mRNA in females. In males,
anxiolytic effects of exercise correlated with increased BDNF mRNA, however AAS-induced
changes in BDNF mRNA and anxiety did not. In sum, we find that AAS elicit sex-specific
differences in anxiety and that voluntary exercise accentuates these differences. In addition,
our data suggest that these behavioral outcomes may reflect convergent actions of AAS and
exercise on a sexually differentiated CRF signaling system within the extended amygdala
[14639].
1127
Behavioural manifestations
AASs have been negatively associated with depression, mania, psychosis, and aggression
but have also been used therapeutically to improve mood and alleviate depression. Placebo-
controlled trials indicate that at least 5 percent of AAS users will have manic or hypomanic
reactions, and the likelihood of psychiatric effects are increased with prior psychiatric history,
alcohol, and other drug abuse. A withdrawal syndrome has been described on
discontinuation of AAS that can persist for several months. Withdrawal-type symptoms
including reduced muscle size and strength, fatigue, depressed mood, and reduced libido
can affect up to 88 percent of AAS users. Such symptoms generate a strong desire to
resume AAS administration (craving), leading to drug habituation [04018].
The use of anabolic androgenic steroids (AAS) for gains in strength and muscle mass is
relatively common among certain subpopulations, including athletes, bodybuilders,
adolescents and young adults. Adverse physical effects associated with steroid abuse are
well documented, but more recently, increased attention has been given to the adverse
psychiatric effects of these compounds. Steroids may be used in oral, 17alpha-alkylated, or
intramuscular, 17beta-esterified, preparations. Commonly, steroid users employ these
agents at levels 10- to 100-fold in excess of therapeutic doses and use multiple steroids
simultaneously, a practice known as “stacking”. Significant psychiatric symptoms including
aggression and violence, mania, and less frequently psychosis and suicide have been
associated with steroid abuse. Long-term steroid abusers may develop symptoms of
dependence and withdrawal on discontinuation of AAS. Treatment of AAS abusers should
address both acute physical and behavioural symptoms as well as long-term abstinence and
recovery. To date, limited information is available regarding specific pharmacological
treatments for individuals recovering from steroid abuse. One paper reviewed the published
literature concerning the recognition and treatment of behavioural manifestations of AAS
abuse [05053].
Significance of body image

The relationship between body image and AAS use was subject of a small number of
studies. These investigations demonstrated that AAS users are often dissatisfied with their
body and have low self-esteem. This may lead to the so-called “reverse anorexia syndrome”
that was later defined as “muscle dysmorphia” as an expression of a form of body
dysmorphic disorder. This syndrome refers to athletes (in general, bodybuilders, although it
may refer to others as well) who believe they have a small and disproportionate body and are
pathologically preoccupied with their degree of muscularity. Because of their pathological
preoccupation, these subjects have been suggested to be more susceptible to taking AAS.
Furthermore, AAS-using bodybuilders and weightlifters were found to possess a more
narcissistic personality compared with non-users. It has been introduced the term “Adonis
complex” for athletes who experience such personality changes [04002].
Mania
Anabolic androgenic steroids (AASs have been associated with several major psychiatric
symptoms and disorders such as violence, aggression, suicidal tendency, psychotic
deterioration, cognitive impairment, depression, and mania. Among these, manic symptoms
were considered to be the most frequent, as shown by a clinical trial with testosterone
cypionate and a controlled study that analyzed a population of AAS-using athletes. The
synthetic male sex hormone mesterolone has even been tested as an antidepressant in
clinical studies in the 1980s. These studies, however, yielded inconsistent results. Here it
1128
was reported the development of a manic episode subsequent to oral intake of mesterolone
in a previously mentally healthy person. The 38-year-old white patient was admitted to a
psychiatric ward because of a maniform syndrome that was present for approximately 2
weeks. On admission, he demonstrated with disturbed contact behavior, logorrhea,
incoherent thinking with flight of ideas, reduced attention and appetite, increase of impulse,
euphoric mood, and reduced need to sleep. There were no overt psychotic phenomena in
terms of megalomania or delusion. Physical examination was unremarkable (body weight, 96
kg; height, 178 cm; body mass index, 30.3 kg/m2) apart from hypertrophic upper body
musculature. Evidence for previous psychiatric disorders, especially history of an addictive
disorder or abuse of psychotropic substances, was absent. Family history revealed major
depressive disorder of the patient’s sister, mother, and aunt on the mother’s side. The patient
was working as a car mechanic and had a stable partnership without children. On admission,
routine blood examination including thyroid tests and urine drug screening (amphetamines,
benzodiazepines, cannabinoids, cocaine, 3,4-methylenedioxymethamphetamine, opiates,
tricyclic antidepressants) were unremarkable. Blood glucose levels as well as glycated
hemoglobin (HbA1c) were within reference limits. Four days later, the patient suddenly
reported to have used Proviron (mesterolone 25 mg/d) for muscle gain during the last 21
days before admission until the day of admission. He reported that he had been bodybuilding
for more than 15 years and that he had never used any illicit substances, that is, from the
group of AAS to enhance training effects before the use of mesterolone. As the initial urine
samples were already discarded, urine was collected again (4 days after the last self-
reported mesterolone intake) and analyzed for mesterolone metabolites (1alpha-methyl-
androsterone, 1alpha-methyl-5alpha-androstane-3alpha, 17beta-diol), but as expected in
view of the short window for drug detection, these substances could no longer be detected
by means of gas chromatography combined with high-resolution mass spectroscopy and
liquid chromatography combined with mass spectroscopy. In addition, pituitary hormones
were measured normal. To rule out an underlying somatic disease affecting the central
nervous system (eg, encephalitis), electroencephalogram, magnetic resonance imaging of
the brain, and examination of the cerebrospinal fluid were performed without pathological
findings. Hence, a diagnosis of (putative) mesterolone-induced mania was made. Initially, the
patient was treated with olanzapine that was gradually increased up to 30 mg/d. Because of
a remarkable increase in liver enzymes, olanzapine was discontinued, and amisulpride was
administered. Because the patient developed parkinsonism under amisulpride at a dosage of
800 mg/d and the antimanic effect was still insufficient, it was reduced amisulpride and
administered lithium carbonate. Under treatment with lithium carbonate (lithium carbonate
1800 mg/d) and amisulpride (400 mg/d), the patient developed complete remission within 2
months of in-patient treatment. At follow-up 6 months after discharge, the patient was still
without psychopathological findings. Mesterolone (1-methyl-dihydrotestosterone) is a
relatively weak androgen with only partial androgenicity. Thus, it is rarely used for oral
testosterone replacement therapy in male patients with hypogonadism associated with
androgen deficiency. Although its application in medicine is rather infrequent, mesterolone is
still used in professional as well as in amateur sports to enhance training success,
particularly regarding muscle hypertrophy, and thus, it can require medical treatment for the
development of adverse effects, as in the case presented. To our knowledge, this is the first
case report that describes the development of a manic episode related to the use of
mesterolone. Considering the absence of other conclusive etiologic factors and the
chronological coherence between the onset of AAS use and the development of psychiatric
symptoms, mesterolone is the most plausible causal agent in the development of the manic
episode. Taking into account the short elimination half-life of mesterolone (drug levels in
serum decrease with a terminal half-life of 12-13 hours and 50 % of the orally applied dose is
excreted in urine within 24 hours), absent proof of metabolites in the urine 4 days after the
last intake does not speak against the use of mesterolone, rather being in full agreement with
the patient’s reports. He was also able to show the Proviron medicine box in detail. He
1129
maintained his statements after remission of mania and came from the bodybuilding scene,
so we had no reasons to believe that the patient had not told the truth about taking
mesterolone; although, unfortunately, we were not able to prove its use. Apart from that, the
patient’s positive family history of mood disorders certainly has posed an additional individual
risk factor for the development of mania related to mesterolone use. Another aspect is the
comparatively low dosage of mesterolone (175 mg/wk) and its short duration of use that was
associated with the development of mania in our case, especially when compared to clinical
and field studies that described effects of AAS on mood after significantly higher dosages (up
to 600 mg testosterone cypionate per week or even up to 1000 mg testosterone per week)
and longer application intervals (up to 6 weeks) [12137].
Effects on GABA

therapeutic purposes, but now taken predominantly as drugs of abuse. The most common
behavioral effects associated with anabolic androgenic steroid use are changes in anxiety,
aggression and reproductive behaviors, including the onset of puberty and sexual receptivity.
GABAergic circuits in the forebrain underlie these behaviors and are regulated by gonadal
steroids. Work from one laboratories has shown that the expression and function of GABAA
receptors in the rat and mouse forebrain varies between the sexes and across the estrous
cycle. It was also shown that there are significant changes in GABAA receptor expression
that occur with the progression through puberty to adulthood. Because GABAergic systems
are both steroid-sensitive and critical for the expression of behaviors altered with anabolic
androgenic steroid use, forebrain GABA(A) receptors are an attractive candidate to assess
how molecular actions of anabolic androgenic steroids may be translated to known
behavioral outcomes. The studies demonstrate that anabolic androgenic steroids elicit both
acute modulation of GABAA receptor-mediated currents, as well as chronic regulation of
GABAA receptor expression and forebrain GABAergic transmission. Because anabolic
androgenic steroid use has now become prevalent not only among adolescent boys, but in
an increasing number of adolescent girls, we have also been particularly interested in
determining age- and sex-specific effects of anabolic androgenic steroids. The data show
that the effects of chronic anabolic androgenic steroid exposure can be greater for
adolescent than adult animals and are more marked in females than in males. These data
have particularly important implications with respect to studies we have done demonstrating
that chronic anabolic androgenic steroid exposure alters the onset of puberty, estrous
cyclicity and sexual receptivity [06076].
Age-, sex, and dose-dependent changes in GABAA receptor

Chronic exposure to anabolic androgenic steroids (AAS) has deleterious effects on
reproductive health in both human and animal subjects. Neurotransmission mediated by the
gamma-aminobutyric acid type A (GABAA) receptor in the medial amygdala (MeA), the
medial preoptic area (mPOA), and the ventromedial nucleus (VMN) of the hypothalamus
plays a critical role in mediating sexual behaviors. Here it was used semi-quantitative reverse
transcription-polymerase chain reaction (RT-PCR) to examine levels of alpha1, alpha2,
alpha5, gamma1, gamma2, and epsilon subunit mRNAs in these three regions of the brain.
Our results demonstrate that chronic exposure to either a high or a moderate dose of the
AAS, 17alpha-methyltestosterone (17alpha-MeT), significantly decreased the levels of
specific alpha and gamma subunit mRNAs in a manner that depended on the dose of AAS
and age and sex of the animals. Specifically, the moderate dose of AAS elicited significant
changes only in pubertal females and the majority of changes observed in pubertal animals
with the high dose also occurred in females. In contrast, the moderate dose of AAS induced
no significant changes in adult mice of either sex, while the high dose had effects in both
1130
males and females. In addition to determining the effects of chronic AAS treatment, a
developmental analysis of drug-naïve animals demonstrated that GABAA receptor subunit
mRNA levels in these regions of the forebrain undergo significant changes as animals
proceed through puberty. These data demonstrate that the effects of AAS exposure on
GABAA receptor expression are superimposed upon dynamic developmental changes that
accompany the transition from puberty to adulthood [02045].
Modulation of alpha1 beta3 gamma2L GABAA receptors

Modulation of GABAA receptors induced by both anabolic androgenic steroids (AAS) and the
benzodiazepine (BZ) site agonist, zolpidem, show equivalent dependence upon gamma
subunit composition suggesting that both compounds may be acting at a shared allosteric
site. Here we have characterized modulation induced by the AAS, 17alpha-
methyltestosterone (17alpha-MeT), for responses elicited from alpha1 beta3 gamma2L GABAA
receptors and compared it to modulation induced by the BZ site agonists, zolpidem and
diazepam. For responses elicited by brief pulses of 20 microM GABA, both the AAS and the
BZ site compounds significantly increased the peak current amplitudes and total charge
transfer, although 17alpha-MeT was an appreciably weaker agonist than either diazepam or
zolpidem at alpha1 beta3 gamma2L receptors. Neither class of modulator enhanced peak
current amplitudes for responses elicited by mM concentrations of GABA. BZ site
compounds altered time constants of deactivation, desensitization, and recovery from
desensitization, however 17alpha-MeT had no overall effect on these parameters.
Experiments in which 17alpha-MeT and BZ site ligands were applied concomitantly indicated
that potentiation elicited by 17alpha-MeT and zolpidem were additive and that potentiation by
17alpha-MeT could be elicited in the presence of concentrations of flumazenil that blocked
BZ potentiation. Finally, kinetic modeling suggests that while effects of 17alpha-MeT can be
simulated by altering receptor affinity, the data for these alpha(1)beta(3)gamma(2L)
receptors were best fitted by simulations in which 17alpha-MeT increases transitions into the
singly liganded open state. Taken together, our results suggest that 17alpha-MeT does not
act at the high-affinity BZ site, but may elicit some of its effects at the low affinity BZ site or at
a novel site [02046].
GABAergic neuroactive steroids

Neurosteroids are synthesized in the brain and modulate brain excitability. There is
increasing evidence of their sedative, anesthetic and antiseizure properties, as well as their
influence on mood. Currently neurosteroids are classified as pregnane neurosteroids
(allopregnanolone and allotetrahydrodeoxycorticosterone), androstane neurosteroids
(androstanediol and etiocholanone) or sulfated neurosteroids (pregnenolone sulfate and
dehydroepiandrosterone sulfate). Both preclinical and clinical findings indicate that
progesterone derivative neurosteroids such as allopregnanolone and allotetrahydrodeoxy-
corticosterone play a role in mood disorders. Clozapine and olanzapine, which were shown
to be effective in stabilizing bipolar disorder, elevate pregnenolone levels in rat hippocampus,
cerebral cortex, and serum. In lithium-treated mice, the blood levels of allopregnanolone and
pregnenolone were elevated compared to control levels. Women diagnosed with bipolar
disorder typically show symptomatic exacerbation in relation to the menstrual cycle, and
show vulnerability to the onset or recurrence of mood disorders immediately after giving birth,
when the levels of neurosteroid derivatives of progesterone drop. Whereas in women who
had recovered from bipolar disorder, the plasma concentration of allopregnanolone was
elevated compared to either healthy controls or women with major depressive disorder
during the premenstrual period. During depressive episodes, blood level of allopregnanolone
is low. Treatment with fluoxetine tends to stabilize the levels of neurosteroids in depression.
These findings converge to suggest that these steroids have significant mood-stabilizing
effect. This hypothesis is consistent with the observation that a number of anticonvulsants
are effective therapies for bipolar disorder, a finding also consistent with the antiseizure
1131
properties of neurosteroids. Further exploration of action of neuroactive steroids is likely to
open new frontiers in the investigation of the etiology and treatment of mood disorders,
particularly bipolar disorders [12140].
Rewarding systems
Research findings regarding androgen abuse in people and hedonic effects of androgens in
laboratory rats are reviewed. Androgens, like other steroids, can have traditional actions via
cognate intracellular steroid receptors, as well as other substrates. Results indicate that
testosterone (T) metabolites may have actions in part via gamma-aminobutyric acid
(GABAA)/benzodiazepine receptor complexes (GBRs) and/or dopaminergic neurons in the
nucleus accumbens, to mediate T's positive hedonic states. This may provide the basis for
positive reinforcing effects of androgen seeking and use behavior. Following a
comprehensive review of the background literature, findings are presented that have
explored the extent to which metabolites of T mediate euphorogenic effects of androgens by
acting in the nucleus accumbens. Then results regarding whether GBRs are necessary
substrates for androgens' positive hedonic effects are discussed. Lastly, research that
addresses if dopaminergic neurons in the nucleus accumbens are necessary for these
effects of androgens are discussed. This review provides a comprehensive examination of
the hedonic properties and abuse/addiction potential of androgens and the putative
mechanisms underlying these effects [06077].
Androgens are mainly prescribed to treat several diseases caused by testosterone

deficiency. However, athletes try to promote muscle growth by manipulating testosterone
levels or assuming androgen anabolic steroids (AAS). These substances were originally
synthesized to obtain anabolic effects greater than testosterone. Although AAS are rarely
prescribed compared to testosterone, their off-label utilization is very wide. Furthermore,
combinations of different steroids and doses generally higher than those used in therapy are
common. Symptoms of the chronic use of supra-therapeutic doses of AAS include anxiety,
depression, aggression, paranoia, distractibility, confusion, amnesia. Interestingly, some
studies have shown that AAS elicited electroencephalographic changes similar to those
observed with amphetamine abuse. The frequency of side effects is higher among AAS
abusers, with psychiatric complications such as labile mood, lack of impulse control and high
violence. On the other hand, AAS addiction studies are complex because data collection is
very difficult due to the subjects' reticence and can be biased by many variables, including
physical exercise, that alter the reward system. Moreover, it has been reported that AAS may
imbalance neurotransmitter systems involved in the reward process, leading to increased
sensitivity toward opioid narcotics and central stimulants. The goal of this article is to review
the literature on steroid abuse and changes to the reward system in preclinical and clinical
studies [150004].
The data in literature highlight the potential for AAS addiction in humans. Nevertheless, it is
difficult to separate the direct rewarding effects of AAS from the psychological dependence of
users on their physical appearance, muscular strength, and athletic performance. Hence,
studies in animal models are a useful tool when examining androgen-reinforcing properties in
conditions where anabolic effects and athletic performance are not relevant. Conditioned
place preference (CPP) and self-administration are relevant experimental paradigms used to
study reward in an experimental condition. Several studies in adult rodents have reported
that systemic testosterone injections induced CPP in male rats and mice. In another animal
model, it has been demonstrated that 15 days of administration of an AAS cocktail consisting
of testosterone cypionate, nandrolone decanoate, and boldenone undecylenate, increased
the rate of self-administration and enhanced the sensitivity to amphetamine challenge.
1132
However, in the same study, a 2 week treatment with MT had no effect on reward or
performance of intracranial self-stimulation. In this light, it was reported that in animals
drostanolone and nandrolone tend to be self-administered and can cause CPP. Moreover,
such effects can be prevented by dopaminergic antagonists indicating that dopaminergic
pathways are necessary for these behavioral outcomes. Indeed, the mesocorticolimbic
circuitry, such as nucleus accumbens (NAc) and ventral tegmental area (VTA) are crucial for
the reward system [150004].
It was investigated the rewarding effects of three different types of synthetic androgens
differing in chemical structure and metabolism by using the CPP test in adult mice. They
found that systemic injection of testosterone propionate and nandrolone decanoate, but not
17alpha-methyltestosterone, produced a dose-dependent shift in CPP suggesting that the
rewarding properties of AAS might depend on their interaction with different pathways.
Recently, the same research group has demonstrated nandrolone's failure to reward in
adolescent mice. Although the literature reports that the adolescent brain is more sensitive to
the reinforcing effects of drugs of abuse, this study suggests that such sensitivity may be
drug dependent. It was also reported that testosterone induced CPP when directly injected
into NAc. Similarly, it was shown that direct implants of testosterone or its metabolites
(dihydrotestosterone, 3alpha-androstanediol) in the NAc shell induced a preference for the
androgen-associated compartment, while no effect was observed with androgenic stimulation
of the NAc core, suggesting a sub-region-specific functional role in reinforcement and reward
pathway [150004].
A growing body of evidence has shown the reinforcing effects of AAS using the experimental
self-administration (oral, intravenous iv, intracerebroventricular icv) paradigm, which is
considered as a model of addiction with the greatest face validity. It was demonstrated that
gonadally intact adult male hamsters preferentially self-administer testosterone orally by
using a food-induced drinking model. Although oral self-administration resembles oral AAS
intake in humans, potential effects of taste solution or gut fill might present an inherent
limitation on AAS oral consumption. Thus, it was used an operant chamber to train animals
with chronic jugular cannulae and demonstrated an increase in testosterone iv self-
administration compared to controls. Moreover, Syrian hamsters voluntarily consume
testosterone through icv self-administration, suggesting that testosterone-reinforcing effects
are centrally mediated. In this extention of the research study on androgens and compared
icv self-administration of four commonly abused AAS (nandrolone, drostanolone,
oxymetholone, stanozolol) that differ in their method of administration, duration of action and
metabolism. Results from this study showed that male hamsters preferentially self-
administered nandrolone or drostanolone, which are two of the mostly used injectable
androgens in humans. Conversely, animals failed to self-administer the orally active
androgens oxymetholone or stanozolol, suggesting that injectable androgens may be more
reinforcing than orally active steroids [150004].
To better understand the behavioral outcomes described above, various neurochemical

studies have examined AAS effects on the monoaminergic system by measuring
neurotransmitter and metabolite levels or by detecting receptors and enzyme alterations in
key brain areas linked to the reward pathway. It has been reported that CPP induced by
testosterone was blocked when adult male rats were directly injected into NAc with a D1-like
or D2-like dopamine receptor antagonist (SCH23390 or sulpiride, respectively). Sub-chronic
administration of high AAS doses reduced dopamine D1-like receptor protein and mRNA
levels in the NAc core and shell and increased D4-receptor mRNA expression in NAc, while
D2-like receptors were up-regulated in the NAc core but down-regulated in the shell. An up-
regulation of the dopamine transporter (DAT) protein was observed in vivo by a binding study
using positron emission tomography (PET), in the striatum of male rat brain after chronic
1133
treatment with nandrolone. Interestingly, it was in 2015 observed no difference of D1-receptor
protein expression in adolescent mice suggesting that the mesolimbic dopaminergic system
during adolescence is immature or not sensitive to the rewarding response induced by
nandrolone. Studies in Syrian hamsters suggested that testosterone reduced dopamine (DA)
release in NAc. Likewise, our research group showed a reduction in DA content in NAc of
rats treated for 4 weeks with nandrolone, changes which were accompanied by reduced
hedonic-related behavior. Furthermore, in a microdialysis study, it was demonstrated that
sub-chronic nandrolone decreased extracellular levels of DA metabolites (DOPAC and HVA)
in rat NAc shell without affecting the release of DA. In line with these results, nandrolone was
shown to reduce type A and B activity of monoamine oxidase (MAO) although a previous
study reported no effects of the drug on these enzymes activity in rats. Further confirming the
role of dopaminergic system in AAS effects on reward pathway, subchronic nandrolone has
been shown to significantly down-regulate D1 receptors in the NAc and caudate putamen of
rats, and to up-regulate D2-like receptors in the NAc core and VTA. In this regard, D1 and D2
receptors have been implicated in the reinforcing effects of drugs, as D1 is necessary for the
acquisition of the effect and D2 crucial in mediating positive reinforcement. On the other
hand, we have previously reported that stanozolol had no effect on DA content in NAc
[0004].
Contradictory neurochemical results have been reported regarding AAS effects on the
serotonergic system. In particular, intranasal administration of testosterone has been shown
to increase dopaminergic and serotonergic systems in rat neostriatum and NAc. Accordingly,
nandrolone decanoate and oxymethenolone treatment enhanced 5-HT and 5-HIAA
concentrations in rat cerebral cortex and hypothalamus, while decreased levels of 5-HT and
5-HIAA were observed in the striatum of nandrolone-treated rats. Moreover, it has been
shown that AAS affects 5-HT receptor expression. In particular, sub-chronic nandrolone
administration down-regulates 5-HT1B and up-regulates 5-HT2 receptor density in rat brain. In
addition, it was demonstrated that serotonin transporter (SERT) mRNA-expressing cells in
the dorsal raphe nucleus, as well as the density of SERT sites increase after sub-chronic
treatment with testosterone. On the other hand, several studies have associated the
endogenous opioid system to behaviors linked to reward and reinforcement. Thus, a number
of experimental investigations have been carried out to ascertain whether AAS treatment
modifies the levels of opioid peptides and their receptors in brain areas mediating reward. In
particular, beta-endorphin levels have been reported to significantly increase in the
paraventricular thalamic nucleus and VTA of rats treated with AAS cocktails or nandrolone
decanoate, respectively. In line with previous reports, chronic exposure to nandrolone
decanoate has been linked to enhanced my-, delta-, and kappa-receptor binding in the
hypothalamus, striatum, and midbrain periaqueductal gray. However, in the NAc shell and
central amygdala of rats treated with the higher dose of nandrolone regimen, a down-
regulation of kappa-receptor binding, as measured by autoradiography has been
demonstrated. Moreover, an increase in dynorphin converting enzyme-like activity was found
only in the NAc of rats exposed to chronic nandrolone, suggesting an increased biosynthesis
of dynorphin peptides, which, in turn, might affect basal DA levels in the NAc [150004].
Monocygotic twins
Anabolic androgenic steroids (AAS) are derived by chemical manipulation of the testosterone
molecule. The specified category of drugs produces anabolic, androgenic and psycho-active
effects including elevated aggressive, hostile, violent and anti social behavior. The objective
of this case report observational study was to evaluate the possible psychological
consequences of AS use in the twin user of each pair, compared with the non-user twin. It
was studied two pairs of male monozygotic twins: one pair 24 years old and the other 31
1134
years old, with absolute genome and phenotype similarity. One of the twins of each pair used
AAS while the other did not. Both pairs lived in Hellenic provincial towns and followed a
common training and nutrition regime. The psychometric instruments used were the
Symptoms Check List-90 (SCL-90) and the Hostility and Direction of Hostility Questionnaire
(HDHQ). The psychometric evaluations took place within a time interval of 6 months. The
study found high levels of aggressiveness, hostility, anxiety and paranoid ideation in the
twins who used AS. The non-user twins showed no deviation from their initial status. The use
of AAS induced several important psychiatric changes in monozygotic twins which were not
present in the twin who did not use AAS [06078].
Anabolic androgenic steroid (AAS) use is associated with aggressive and violent behaviour,
suggested risk factors for violence. In 2005, all Swedish-born male twins aged 20-47 years
were invited to participate in the Swedish Twin Adults: Genes and Environment (STAGE)
survey of the Swedish Twin Register (response rate 60 %). A total of 10,365 male survey
participants with information on AAS use were included. Data on self-reported use of AAS,
alcohol and other substances, attention deficit hyperactivity disorder (ADHD) and personality
disorder symptoms were linked to nation-wide, longitudinal register information on criminal
convictions, IQ, psychological functioning and childhood socio-economic status (SES)
covariates. Any life-time use of AAS was associated strongly with conviction for a violent
crime (2.7 versus 0.6 % in convicted and non-convicted men, respectively; odds ratio 5.0).
However, this link was substantially reduced and no longer significant when controlling for
other substance abuse (OR 1.6). Controlling for IQ, psychological functioning, ADHD,
personality disorder symptoms and childhood SES did not reduce the risk further. It was
concluded that in the general population, co-occurring polysubstance abuse, but not IQ,
other neuropsychological risks or socio-economic status, explains most of the relatively
strong association between any anabolic androgenic steroid use and conviction for a violent
crime [150196].
Sleeping pattern
Anabolic androgenic steroid (AAS) abuse has become a public health problem in many
countries, and is associated with many psychiatric disorders. Epidemiological studies have
also found increasing numbers of sleep disorders reported by individuals using AASs. The
purpose of this study was to evaluate sleep patterns and disorders in anabolic androgenic
users who practice resistance exercise. The sample comprised 58 males divided into three
groups: 20 current AAS users aged 26, and 21 controls with no history of AAS use, aged 26,
and 17 sedentary men with no sleep disorders aged 27. The volunteers spent a night in the
sleep laboratory for polysomnography. Comparing the three groups, the user group showed
significantly reduced sleep efficiency and more wakings after sleep onset than the sedentary
group. The sedentary group showed a higher percentage of stage 4 than the non-users
group. It was suggested that using of anabolic steroids reduced sleep efficiency and alters
sleep architecture [07075].
Other psychologic and psychiatric effects
Numerous field studies have described psychiatric symptoms associated with illicit AAS use,
including major mood disorders. These psychological studies have included interview studies
assessing psychiatric history in AAS users, on-drug vs off-drug, comparisons of AAS users
versus nonusers using interviews or psychological rating scales; and/or longitudinal
assessments of AAS users over intervals of AAS use versus intervals of nonexposure. In
1135
general, these field studies have suggested that some AAS users exhibit hypomanic or
manic symptoms during AAS exposure (characterized by irritability, aggressiveness,
exaggerated self-confidence, hyperactivity, reckless behavior, and occasional psychotic
symptoms) and depressive symptoms during AAS withdrawal (characterized by depressed
mood, loss of interest in usual activities, hypersomnia, anorexia, loss of libido, and
occasional suicidality). However, these psychiatric effects appear to be idiosyncratic, with a
majority of users displaying few such symptoms, and only a small minority showing severe or
disabling symptoms. Tentative evidence suggests that mood disorders are more common in
individuals using higher doses of AAS, especially at levels equivalent to more than 1,000 mg
of testosterone per week. However, there are no clear predictors of AAS-induced psychiatric
effects, and it appears that there are wide variations in individual sensitivity to both androgen
excess, and androgen withdrawal or deprivation. Certainly, psychosocial factors account for
many of the differences in psychiatric vulnerability observed among AAS users. However,
these factors alone cannot fully explain the variation among AAS users, because a similar
variation has been observed with blinded administration of supraphysiologic doses of AAS to
normal volunteers, and also in the behavior of laboratory animals that were given AAS.
Occasional field observations have also documented strikingly aggressive or violent behavior
in some AAS users who had no prior history of such behaviors. These have included cases
of previously normal individuals committing murder or attempted murder, or displaying other
uncharacteristically aggressive behavior while using AAS. Although the causal relationships
between AAS use and aggressive behaviors may vary, and AAS are not necessarily the
proximal trigger to violence, the phenomenon of AAS-induced aggression is sufficiently
established that it likely meets the American “Daubert” standard for admissibility as legal
testimony (i.e. it may be regarded by the court as a phenomenon that is testable, subject to
peer review and publication, and generally accepted in the relevant scientific community).
Although the discussion has involved primarily field studies of illicit AAS users, some
controlled laboratory studies have also examined the psychiatric effects of AAS. However, a
majority of these studies have used a maximum dose of only 300 mg of testosterone
enanthate or equivalent per week, a dose much lower than generally self-administered by
illicit users, who typically use at least 500 mg per week and often well over 1,000 mg per
week. Thus it is inappropriate to use these low dose laboratory studies to gauge the
experience of illicit users. However, there have now been four additional laboratory studies
that have assessed psychiatric symptoms in individuals receiving the equivalent of at least
500 mg of testosterone per week. Of 109 men treated under blinded conditions in these
studies, five (5 %) displayed hypomanic or manic syndromes on AAS versus none on
placebo. These latter studies offer clear evidence for a biologically mediated psychiatric
effect of supraphysiologic doses of AAS, although they still likely underestimate the
prevalence of such effects among illicit users, who may ingest much higher doses. Also, in
human subjects, studies have reported increased aggressive responsiveness to provocation
[14017].
assessing psychiatric history in AAS users, on-drug vs off-drug, comparisons of AAS users
versus nonusers using interviews or psychological rating scales; and/or longitudinal
majority of users displaying few such symptoms, and only a small minority showing severe or
1136
individuals using higher doses of AAS, especially at levels equivalent to more than 1,000 mg
excess, and androgen withdrawal or deprivation. Certainly, psychosocial factors account for
normal volunteers, and also in the behavior of laboratory animals that were given AAS.
in some AAS users who had no prior history of such behaviors. These have included cases
of previously normal individuals committing murder or attempted murder, or displaying other
uncharacteristically aggressive behavior while using AAS. Although the causal relationships
between AAS use and aggressive behaviors may vary, and AAS are not necessarily the
established that it likely meets the American “Daubert” standard for admissibility as legal
Although the discussion has involved primarily field studies of illicit AAS users, some
illicit users, who typically use at least 500 mg per week and often well over 1,000 mg per
week. Thus it is inappropriate to use these low dose laboratory studies to gauge the
experience of illicit users. However, there have now been four additional laboratory studies
that have assessed psychiatric symptoms in individuals receiving the equivalent of at least
500 mg of testosterone per week. Of 109 men treated under blinded conditions in these
studies, five (5 %) displayed hypomanic or manic syndromes on AAS versus none on
placebo. These latter studies offer clear evidence for a biologically mediated psychiatric
effect of supraphysiologic doses of AAS, although they still likely underestimate the
prevalence of such effects among illicit users, who may ingest much higher doses. Also, in
human subjects, studies have reported increased aggressive responsiveness to provocation
[14017].
Human
assessing psychiatric history in AAS users, on-drug versus off-drug; comparisons of AAS
users versus nonusers using interviews or psychological rating scales; and/or longitudinal
majority of users displaying few such symptoms and only a small minority showing severe or
individuals using higher doses of AAS, especially at levels equivalent to more than 1000 mg
excess and androgen withdrawal or deprivation. Certainly, psychosocial factors account for
1137
normal volunteers and also in the behavior of laboratory animals that were given AAS
[14426].
in some AAS users who had no history of such behaviors. These have included cases of
previously normal individuals committing murder or attempted murder or displaying other
uncharacteristically aggressive behavior while using AASs. Although the causal relationships
between AAS use and aggressive behaviors may vary, and AASs are not necessarily the
established that it likely meets the American Daubert standard for admissibility as legal
Although our discussion has involved primarily field studies of illicit AAS users, some
illicit users, who typically use at least 500 mg per week and often well over 1000 mg per
week. Thus, it is inappropriate to use these low-dose laboratory studies to gauge the
experience of illicit users. However, there have now been 4 additional laboratory studies that
have assessed psychiatric symptoms in individuals receiving the equivalent of at least 500
mg of testosterone per week. Of 109 men treated under blinded conditions in these studies,
5 (4.6 %) displayed hypomanic or manic syndromes on AAS vs none on placebo. These
latter studies offer clear evidence for a biologically mediated psychiatric effect of
supraphysiologic doses of AAS, although they still likely underestimate the prevalence of
such effects among illicit users, who may ingest much higher doses. Also, in human subjects,
studies have reported increased aggressive responsiveness to provocation [14426].
It was reviewed the literature from human and animal studies on the neurochemical and
pathological psychiatric effects of supraphysiological doses of anabolic-androgenic steroids
(AAS) and discuss the AAS use and abuse patterns, additional drug use patterns, and
personality and behavioral characteristics of AAS abusers [05055].
Observations by health-care professionals suggest that the use of anabolic androgenic

steroids (AAS) may be associated with lethal complications, but this has not yet been
confirmed by controlled epidemiological studies. Here, it was investigated the diagnoses (in
the Swedish patient care records) and mortality rate among patients who tested positively for
the presence of AAS (n=248) in connection with receiving medical care. Patients who had
tested negatively (n=1215) were used for comparison. The proportions of patients who had
received institutionalized care for substance abuse, psychiatric disorder or central thoracic
pain were significantly higher in the AAS-positive subjects (RR 2.2, 2.1, and 3.5,
respectively). Furthermore, unspecified convulsions were highly over-represented in the
AAS-positive group (RR 53.9) and one of these patients died during a seizure. The
standardized mortality ratios (SMR) in the AAS-positive patients and -negative patients were
20.43 and 6.02, respectively. The relatively higher SMR in the AAS-positive patients was
observed irrespective of what type clinic had referred the patients for AAS testing. In
conclusion, use of AAS appears to be an indicator of increased risk for premature death in
several categories of patients. However, the nature of the association between AAS and
premature death remains unclear and additional research on this question is urgently
required [05056].
1138
The objective of one study was to evaluate the psychological consequences of real-world
AAS use in athletes abusing such agents, in comparison with a placebo and control group of
comparable athletes, while correlating the severity of abuse with the side effects observed.
The hypothesis tested by the study was that the use of AAS induces a wide range of
psychological side effects whose impact and emergence is dependent upon the severity of
the abuse. The study includes a substantial group of AAS abusing athletes and two more
groups demographically similar to the first, one composed of athletes not using any
substance and a placebo group. All athletes were stratified according to the severity of AAS
abuse. Psychometric instruments were applied to all athletes in specific time intervals,
dependent to the AAS abusers' regimens, providing us with a final psychological profile that
was to be compared to the pre-study profile. All results were comparable (within and
between groups) for statistically significant differences and correlated to the severity of the
abuse. Homogeneity of all groups was safeguarded by random doping controls, monitoring of
drug levels and analysis of all self obtained drugs by method of liquid chromatography/mass
spectrometry. All athletes were provided with a common exercise and dietary regime, so
common training and nutritional conditions were achieved. It was studied a cohort of 320
body-building, amateur and recreational athletes, of whom 160 were active users of AAS
(group C), 80 users administering placebo drugs (group B) and 80 not abusing any
substance (Group A). Group C athletes were stratified according to AAS abuse parameters,
thus providing us with three subgroups of "light, medium and heavy abuse". Athletes of
groups A and B were included in a "no abuse" subgroup. The psychometric instruments used
were the Symptoms Check List-90 (SCL-90) and the Hostility and Direction of Hostility
Questionnaire (HDHQ). The psychometric evaluations took place within a time interval of 13
months. Statistical analysis was performed by using the Mann-Whitney/Wilcoxon two-sample
non-parametric test (Kruskal-Wallis test for two groups) for data that were not normally
distributed and Linear regression analysis was used to ascertain the correlation between
severity of use and escalation of side effects. The study showed a statistically significant
increase in all psychometric subscales recorded in group C, and no statistically significant
difference in group C and A. There was a significant increase in the scorings of group C for
all subscales of SCL-90 and HDHQ. Correlation of abuse severity and side effects showed
that there was a statistical significant increase in Delta values of all SCL-90 and HDHQ
subscales that escalated from light abuse to medium and heavy abuse/consumption
patterns. The results of the study suggest that the wide range of psychiatric side effects
induced by the use of AAS is correlated to the severity of abuse and the force of these side
effects intensifies as the abuse escalates [05054].
The objective of one study was to evaluate the psychological consequences of real-world
AAS use in athletes abusing such agents, in comparison with a placebo and control group of
comparable athletes, while correlating the severity of abuse with the side effects observed.
The hypothesis tested by the study was that the use of AAS induces a wide range of
psychological side effects whose impact and emergence is dependent upon the severity of
the abuse. The study includes a substantial group of AAS abusing athletes and two more
groups demographically similar to the first, one composed of athletes not using any
substance and a placebo group. All athletes were stratified according to the severity of AAS
abuse. Psychometric instruments were applied to all athletes in specific time intervals,
dependent to the AAS abusers' regimens, providing us with a final psychological profile that
was to be compared to the pre-study profile. All results were comparable (within and
between groups) for statistically significant differences and correlated to the severity of the
abuse. Homogeneity of all groups was safeguarded by random doping controls, monitoring of
drug levels and analysis of all self obtained drugs by method of liquid chromatography/mass
spectrometry. All athletes were provided with a common exercise and dietary regime, so
common training and nutritional conditions were achieved. It was studied a cohort of 320
body-building, amateur and recreational athletes, of whom 160 were active users of AAS
1139
(group C), 80 users administering placebo drugs (group B) and 80 not abusing any
substance (Group A). Group C athletes were stratified according to AAS abuse parameters,
thus providing us with three subgroups of "light, medium and heavy abuse". Athletes of
groups A and B were included in a "no abuse" subgroup. The psychometric instruments used
were the Symptoms Check List-90 (SCL-90) and the Hostility and Direction of Hostility
Questionnaire (HDHQ). The psychometric evaluations took place within a time interval of 13
months. Statistical analysis was performed by using the Mann-Whitney/Wilcoxon two-sample
non-parametric test (Kruskal-Wallis test for two groups) for data that were not normally
distributed and Linear regression analysis was used to ascertain the correlation between
severity of use and escalation of side effects. The study showed a statistically significant
increase in all psychometric subscales recorded in group C, and no statistically significant
difference in group C and A. There was a significant increase in the scorings of group C for
all subscales of SCL-90 and HDHQ. Correlation of abuse severity and side effects showed
that there was a statistical significant increase in Delta values of all SCL-90 and HDHQ
subscales that escalated from light abuse to medium and heavy abuse/consumption
patterns. The results of the study suggest that the wide range of psychiatric side effects
induced by the use of AAS is correlated to the severity of abuse and the force of these side
effects intensifies as the abuse escalates [06075].
Muscle dysmorphia has been described as a disorder in which individuals are pathologically
preoccupied with their muscularity. One study was designed to further investigate the
symptom characteristics and psychiatric conditions associated with the disorder. Weight
lifting males meeting current criteria for muscle dysmorphia (n=15), past muscle dysmorphia
(n=8), and no history of muscle dysmorphia (n=28) responded to advertisements placed in
gymnasium and nutrition stores. Structured and semistructured interviews were
administered, as well as survey measures. Relative to controls, males with current muscle
dysmorphia experienced more aversive symptoms related to the appearance of their bodies,
including more often thinking about their muscularity, dissatisfaction with appearance,
appearance checking, bodybuilding dependence, and functional impairment. Higher rates of
mood and anxiety disorders were found among individuals with a history of muscle
dysmorphia relative to individuals with no history of muscle dysmorphia. The findings suggest
that muscle dysmorphia can be distinguished from normal weight lifting on a number of
clinical dimensions. Muscle dysmorphia appears to be comorbid with other psychiatric
conditions [08127].
The risks from chronic administration of anabolic steroids may appear relatively low when
compared with the use of socially acceptable drugs such as tobacco and alcohol. The
literature tends to rely heavily on specific case reports, identifying psychiatric or
psychological disorder, because of the private and personal nature of the abuse of this class
of drug [08128, 08129]. Anabolic androgen steroids self-administration have increased over
the last decade, in the wake of the demonstration that steroids increase muscle mass and
strength in healthy adult males, over and above resistance training [08130]. However, there
may be other reasons why individuals abuse anabolic substances. Thirteen percent of 75
female weightlifters, who admitted to abusing androgenic anabolic steroids to gain muscle
mass, had increased their weightlifting activities to be better able to defend themselves
against men and also reported that they were previously sexually abused [08131]. It is
believed that compulsive weightlifting and steroid abuse may represent a form of response to
the trauma of sexual assault and also may assist in raising self-esteem. The psychiatric
evaluation of dedicated female athletes also demonstrated the exhibition of ergogenic
polysubstance dependence, often with significant co-morbidity [08132]. Fifty six percent
demonstrated hypomania during the “administration-phase” and 40 percent reported
depressive symptoms during withdrawal. These athletes also displayed several psychiatric
syndromes, previously ill-defined, such as obsessive compulsive disorder, rigid dietary
1140
practice, nontraditional gender roles, and chronic dissatisfaction and preoccupation with their
physiques (muscle dysmorphia) [08133].
Pagonis et al [08134] has shown in a study of 160 steroid-abusing athletes compared with
160 placebo and controls that the wide range of psychiatric side effects induced by the abuse
of anabolic steroids is correlated to the severity of abuse. The force of these side effects
intensifies as the abuse escalates. They have a distorted body image and reported the
condition of “reverse anorexia”, where they believed they were small and weak, despite
being large and muscular [08135]. The reasons for use of these anabolic agents appear to
be based not only on peer review, but also scientific research [08042]. Low self-esteem and
unrealistic, muscular male body ideals, puts individuals at risk for negative body images and
unhealthy eating and exercise habits. These individuals resort to drug-taking to counteract
their altered body images [08133].
The psychiatric effects of anabolic-androgenic steroids (i.e. testosterone and its derivatives)
have been less well studied than their physical effects but are reported to include depression,
mania, psychosis, and aggression. Dependence can also occur, with withdrawal involving
psychiatric and physical symptoms [07031].
The psychiatric effects of anabolic-androgenic steroids are hard to study, for several
reasons. Many of the available studies are, by necessity, observational. But because the
substances are illicit, users have no way to verify their exact nature or amounts taken.
Moreover, many steroid users concomitantly take a multitude of other performance-
enhancing drugs and dietary supplements that also may have psychiatric effects.
Prospective studies are hard to carry out because of the ethical issues inherent in testing a
potentially dangerous substance. Because many users belong to a subculture of
bodybuilders, weightlifters, or elite athletes, study results are hard to extrapolate to the
general public. Most studies to date have evaluated dosages lower than most users report
taking. Further, users of anabolic-androgenic steroids tend to use them for prolonged and
repeated cycles over many years, which is hard to recreate in clinical trials. More studies are
needed on a larger scale with dosing that is compatible with the supraphysiologic dosages
used in the community. In addition, pre-existing personality traits that might predispose
people to use steroids may significantly confound assessing any psychiatric effects of drug
use. Suspected risk factors for men include antisocial personality traits, low self-esteem, and
poor body image (body dysmorphia). It was found that weightlifters and bodybuilders who
used anabolic steroids had significantly higher scores on dimensions of pathologic
narcissism and lower scores on ratings of empathy. Another study found that up to 50
percent of steroid users had worked as bouncers and described themselves as aggressive
regardless of their drug use [07031].
Uncontrolled, observational trials in the 1930s and 1940s found that men with refractory
depression responded favorably to testosterone treatment. However, randomized, placebo-
controlled studies conducted in the 1980s were equivocal. Observational studies show
hypomania, mania, and depression. It wasretrospectively studied 164 weightlifters and
bodybuilders who used anabolic- androgenic steroids and found that about 10 percent had
hypomania. Depression occurred when steroids were stopped in about 10 percent. In
another study it was interviewed 41 bodybuilders and football players taking anabolic-
androgenic steroids and found that 9 displayed full affective syndromes and 5 showed
psychotic symptoms. In a later study, it was compared 88 athletes who were using anabolic-
androgenic steroids with 68 nonusers and found that 23 percent of the steroid users reported
major mood symptoms (including mania, hypomania, and depression) versus only 6 percent
of the nonusers, and several users reported aggressive thoughts. The higher the steroid
dosage, the more severe were the psychiatric symptoms. It was conducted a similar study of
1141
weightlifters and found more symptoms of depression and mania among users of anabolic-
androgenic steroids, although formal diagnoses were not made. In controlled studies, high
dosages led to mood changes in some users Studies with supraphysiologic doses of
anabolic-androgenic steroids found minimal or no changes in mood in most users, but a
minority of users had significant mood changes. In another randomized, placebo-controlled
crossover trial, it was given injections of testosterone cypionate (Depotest®) to 56 men,
gradually increasing the dosage to 600 mg/week. Most of the men showed no significant
manic symptoms, but 6 (12 %) had mild hypomania and 2 (4 %) had marked hypomania. In
yet another placebo-controlled, crossover prospective trial, oral methyltestosterone (Virilon®)
40 or 240 mg/day was given to 20 normal men. Those on the high dose had increased
positive mood changes (euphoria, increased energy, and sexual arousal) as well as negative
mood changes (irritability, violent feelings, hostility, and distractibility). One man developed
mania at the high dosage, and another developed hypomania. Physiologic doses have
minimal mood effects. Studies of the effects of low or near-physiologic doses of anabolic-
androgenic steroids found minimal effects on mood [07031].
Studies in mice have found aggressive behavior correlating with increasing dosages and
duration of anabolic-androgenic steroid treatment, culminating in females killing their
offspring. Observational studies of aggressive behavior changes in people taking steroids
have been equivocal. Placebo-controlled studies using supraphysiologic doses of anabolic-
androgenic steroids have also been equivocal. Physiologic doses do not enhance
aggression. However, serum testosterone level correlates with aggressiveness [07031].
AAS use has been associated with self-reported changes in mood and behavior. One study
identified psychiatric syndromes in weightlifters using AASs. Twenty-three percent of AAS
users experienced major mood changes of mania, hypomania, or major depression. Also
common in AAS users was aggressive behavior resulting in fights, domestic disturbances,
assaults, and arrests. Data from the National Household Survey on Drug Abuse have
demonstrated a strong association between AAS use and self-acknowledged acts of violence
against people and crimes against property. In general, the behavioral effects of AASs are
variable, short-lived on discontinuation, and seem to be related to the type and dosage of
AAS. The potential for physical dependence upon AASs does exist. In one study of AAS
users, 50 percent of them met the Diagnostic and Statistical Manual of Mental Disorders,
Fourth Edition criteria for dependence or abuse of steroids. Deeply entrenched body
dissatisfaction and body dysmorphic disorder may underlie a psychologic dependence.
Clearly, the addictive potential of AASs cannot be [07008].
AASs induce a state of euphoria and diminished fatigue that enables prolongation of training
sessions by users. Recent data may explain how AASs exert these psychoactive effects on
the brain. It has been proposed that AAS-mediated acute and chronic changes in the
gamma-aminobutyric acid (GABA) receptor system cause many of the known behavioral
effects. For instance, the immediate effects of decreased anxiety and enhanced sense of
well-being that are experienced by AAS users likely arise from enhancement of forebrain
GABAergic circuits. In contrast, anxiety and aggression are the result of a down-regulation of
GABA receptor expression secondary to chronic AAS exposure. Further study may reveal
that expression of these behaviors is influenced by the age and gender of the AAS user and
the particular chemical composition of the AAS administered [07008].
In a study in middle and high school students, 5 percent of boys and 3 percent of girls had
used steroids in the previous year. Use in boys was associated with higher rates of
depressed mood, prior suicide attempts, greater substance abuse, and lower self-esteem.
Another study of adolescents suggested that steroid use was associated with other high-risk
behaviors and was less likely to be an isolated behavior. Many case reports describe
1142
psychiatric symptoms in patients using AASs. Reports of suicide include at least one patient
who did not have a personal or family history of depression or suicidal behaviors. In one
series of eight suicides in AAS users from Sweden, collateral information was sought, and
when possible, “psychologic autopsies” were performed. Retrospectively, psychiatric
symptoms, such as irritability, aggressiveness (“roid rage”), mood swings, decreased impulse
control, and increased energy were noted during AAS use; however, the series included men
who had prior psychiatric syndromes, personality disorders, and other substance abuse.
Another report showed homicidal or near homicidal behavior in three men during AAS use.
None had a history of psychiatric illness or violence before AAS use, and all met the
Diagnostic and Statistical Manual of Mental Disorders, Revised Third Edition criteria for
manic episode during use. Two of the three men experienced depression and suicidal
ideation upon the abrupt discontinuation of use. Although it is tempting to attribute this
behavior to AAS use, this is a small set of case studies and as the investigators note, legal
ramifications for the patients may have led to exaggeration in their reports. A series of 34
deaths of AAS users revealed nine victims of homicide, 11 suicides, 12 accidental deaths,
and 2 deaths of indeterminate cause. The homicide victims showed high levels of
aggression; in most of the suicide and accidental deaths, impulsive and violent behaviors
had been noted by family or physicians. Most of the cases of accidental death were related
to polysubstance overdose; four of the victims were heroin addicts who had histories of only
sporadic or moderate AAS use. One study identified seven AAS users and evaluated them
every 2 weeks for as long as 44 weeks. During clinic visits, subjects reported their AAS use,
and assessments, including the Beck Depression Inventory (BDI), Profile of Mood States
Questionnaire, and Buss-Durkee Hostility Scale, were administered. Scores fluctuated over
time, but the fluctuations were not associated with AAS use. Additionally, most of the
subjects had a history of major depression, and five reported abusing other substances. A
larger study (n=160) comparing AAS-using athletes with nonusing athletes revealed that far
more AAS users displayed mood disorders compared with nonusers and AAS users during
periods of no use. Another study describing 41 AAS-using athletes reported that 22 percent
displayed mood syndromes during use, which was significantly higher than the rate observed
during periods of no exposure. Additionally, this study reported that 12 percent displayed
psychotic symptoms during use compared with 0 percent during AAS-free periods. Yet
another naturalistic study comparing weight-lifting AAS users with nonusers correlated
supranormal testosterone levels with subjective and objective measures of aggression.
Cluster B personality traits, including antisocial, borderline, and histrionic, were more
prominent in AAS users [07058].
Attempts have been made to study the effects of AASs in humans in prospective laboratory-
controlled settings. One double-blind study administered placebo followed by low-dose (40
mg/d) and then high-dose (240 mg/d) methyltestosterone to 20 normal healthy men without
psychiatric disease or history of AAS use. During the high-dose period (3 days), distractibility,
irritability, and energy level increased significantly, and there was a trend for an increase in
anger and violent feelings. One subject developed acute mania, and another developed
hypomania. Subtle, but significant, elevations in the BDI, Hamilton Depression Rating Scale,
Brief Psychiatric Rating Scale, and hostility, anxiety and somatization on the Symptom
Checklist (SCL-90) were observed. In follow-up studies, an increase in aggressiveness
correlated with an increase in free T4, an increase in forgetfulness and distractibility
correlated with total testosterone levels, and an increase in activation symptoms (energy,
sexual arousal, and diminished sleep) correlated with cerebrospinal fluid 5-hydroxyindole
acetic acid levels. In another placebo-controlled cross-over study of 50 men free of
substance abuse or psychiatric illness, increasing levels of testosterone cypionate were
administered over 6 weeks. Aggressive responses on the Point Subtraction Aggression
Paradigm and increased manic scores on the Young Mania Rating Scale were
demonstrated; 84 percent showed minimal psychiatric symptoms, 12 percent became mildly
1143
hypomanic, and 4 percent became markedly hypomanic. An additional study evaluated
testosterone cypionate over 14 weeks at levels up to 500 mg/week in healthy men free of
psychiatric illnesses and personality disorders; it found minimal psychologic effects in most
men, but one adverse psychiatric effect resembled mania. Additionally, some studies showed
no changes in psychometric measures in healthy men who were administered AASs. All of
these studies used doses lower than those typical in AAS use, so they likely underestimated
the psychiatric consequences of AAS [07058].
Several case reports and survey studies have indicated that abuse of anabolic androgenic
steroids (AAS) often leads to increased aggressiveness and feelings of hostility that may
occasionally trigger violent behaviour. Other observations indicate that many users of AAS
also abuse alcohol and/or various illegal substances. Since substance abuse is a well-known
risk factor for violent behaviour, it could be that violence committed by AAS users might, at
least in many cases, actually be caused by abuse of other drugs. In order to examine this
possibility further here, the criminal histories (in terms of incidences of convictions) of
deceased users of AAS with (AASpos-subst.pos) and without (AASpos-subst.neg) signs of
abuse of other illegal substances were compared to the corresponding histories of deceased
users of illicit substances testing negatively for AAS (subst.pos-AASneg) at the time of
autopsy. The risk of being convicted for a crime against property was significantly higher in
the subst.pos-AASneg group than in either the AASpos-subst.neg or AASpos-subst.pos
groups (RR=0.048 versus 0.408). At the same time, the risk of being convicted for a crime of
violence was at least as high for the two AAS-positive groups as for the AAS-negative group.
Furthermore, when compared with the first 3 years after the first criminal conviction, a
pronounced increase in the proportion of incidence of violent crimes and a marked reduction
in the proportion of incidence of crime against property was observed during the 3-year
period immediately preceding death only among the AASpos-subst.neg subjects. In
conclusion, the incidence of violent crime among users of AAS without signs of other drug
abuse was comparable to the corresponding incidences for drug addicts without AAS use.
This observation suggests that the violent criminality observed among AAS users is not
confounded in any systematic fashion by abuse of other drugs. The findings also indicate
that use of AAS in certain predisposed individuals might cause a high rate of violent crimes,
especially if the use of AAS is combined with the use of other illegal substances [07076].
Psychiatric effects in humans preclinical models

Animal studies have provided important insights into the specific neurochemical changes and
the mechanisms underlying the various behaviors associated with AAS use. Many of the
central nervous system (CNS) effects and behaviors observed in humans in association with
AAS use at high doses are related to brain circuits that function similarly in other mammalian
species. Indeed several studies carried out in animal models confirm that changes in
defensive and offensive aggression, dominant behavior, anxiety, and sensitivity to other
abusing drugs often mimic what has been observed in human subjects abusing AAS. For
example, AAS has been shown to increase the expression of opioid tolerance in mice.
Although it is difdoi: ficult to precisely scale androgen doses from rodents to humans, when
adjusted according to body surface area (BSA) using the US Federal Drug Administration
guidelines, the doses tested in animal studies (up to 7.5 mg/kg) appear to fall within the
range of human AAS use [14017].
Lack of energy in hypogonodanal men
To investigate the association between hypogonadal symptoms and total serum

testosterone levels in young men (<40 years of age) with an attempt to determine whether
there exists a clear-cut discriminatory threshold of total testosterone below which
1144
hypogonadal symptoms become more prevalent it was retrospectively reviewed the charts of
352 men who presented to an outpatient Men's Health Clinic with chief complaint of "low
testosterone". Sexual, psychological and physical symptoms were evaluated using the
Androgen deficiency in Aging Male (ADAM) questionnaire. Serum levels of total
testosterone were collected on the same day that men completed their ADAM
questionnaires. The probability of hypogonadal symptoms increased at a serum total
testosterone level of 400 ng/dL. A cluster of symptoms: two psychological (decreased
energy, sadness), and three physical (decreased strength and endurance, decreased ability
to play sports, and deterioration in work performance) were most strongly associated with
total serum testosterone levels of <400 ng/dL. On multivariable analysis, only “lack of
energy” predicted a total testosterone of less than 400 ng/dL. It was concluded that
hypogonadal symptoms in men <40 years of age can be associated with a total
testosterone level of less than 40 0ng/dL. Of the hypogonadal symptoms evaluated with the
ADAM questionnaire, lack of energy appears to be the most important symptom that predicts
a total testosterone level < 400 ng/dL [14755].
Dependence in humans
Anabolic-androgenic steroids (AAS) are mainly used to treat androgen deficiency syndromes
and, more recently, catabolic states such as AIDS-associated wasting. There is no evidence
in the reviewed literature that AAS abuse or dependence develops from the therapeutic use
of AAS. Conversely, 165 instances of AAS dependence have been reported among
weightlifters and bodybuilders who, as part of their weight training regimens, chronically
administered supraphysiologic doses, often including combinations of injected and oral AAS
as well as other drugs of abuse. A new model is proposed in which both the "myoactive" and
psychoactive effects of AAS contribute to the development of AAS dependence. The adverse
consequences of AAS are reviewed, as well as their assessment by means of a history and
physical, mental status examination, and laboratory testing. When patients with AAS use
disorders are compared with patients with other substance use disorders, both similarities
and differences become apparent and have implications for treatment [02037].
It appears that about 30 percent of AAS users may develop AAS dependence, which in some
instances may be part of a larger pattern of dependence on PEDs, involving additional
agents such as hGH and CNS stimulants. Unlike most dependence-inducing drugs, which
typically deliver an immediate reward of intoxication, AASs produce few intoxicating effects
and are instead taken primarily for the delayed reward of increased muscle mass and
decreased body fat. Despite these differences, AAS dependence may nevertheless become
a chronic and potentially dangerous disorder. One group has suggested that AAS
dependence may develop via any or all of 3 different pathways, namely a body image
pathway, a neuroendocrine pathway, and a hedonic pathway. The body image pathway
refers to the observation that many individuals initiate AAS use because they exhibit
symptoms of muscle dysmorphia, a form of body dysmorphic disorder where individuals
develop severe preoccupations that they are not adequately muscular. Muscle dysmorphia
appears closely associated with AAS use. Individuals with such concerns often become
extremely anxious if they stop AAS use and lose even a little muscular size. Thus, they often
quickly resume AAS, which contributes to the AAS dependence syndrome [14426].
Neuroendocrine factors also contribute to AAS dependence. Because exogenous AAS

suppresses hypothalamic-pituitary-testicular (HPT) function, users will gradually develop
suppressed testosterone levels and may become hypogonadal upon discontinuation of AAS
use. Although illicit AAS users employ various techniques to minimize hypogonadism
associated with AAS withdrawal (e.g. self-administration of clomiphene and/or human
1145
chorionic gonadotropin at the end of a cycle of AAS use), many will display profound
hypogonadism for weeks or months after discontinuing use. The associated symptoms of
fatigue, loss of libido, and depression may prompt some users to quickly resume using AAS
to treat these dysphoric symptoms [14426].
Long-term effects on mental health

The knowledge concerning the long-term effect of former anabolic androgenic steroids
(AAS)-use on mental health is sparse. One study aims to investigate whether previous AAS-
use affects mental health, present sociodemographic data, sport activity and substance
abuse in a retrospective 30-year follow-up study of former elite athletes. Swedish male-elite
power sport athletes (n=683) on the top 10 national ranking lists during any of the years
1960-1979 in wrestling, Olympic lifting, powerlifting and the throwing events in track and field
answered a questionnaire. At least 20 percent of the former athletes admitted previous AAS-
use. They had more often sought professional expertise for mental problems and had used
illicit drugs compared to those not having used AAS. The AAS-users also differed in former
sport activity pattern compared to non AAS-users. It is clear that a relationship exists
between use of AAS and mental-health problems. Further studies need to be done in order
to clarify this relationship [13149].
Rat
It was previously showed that 14 days of daily intramuscular injections of the anabolic
androgenic steroid nandrolone decanoate (15 mg/kg) reduced the extracellular levels of the
dopaminergic metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid
(HVA) in the nucleus accumbens shell using microdialysis. The aim of one study was to
investigate whether the same dose regimen of nandrolone decanoate may affect the
activities of the dopamine-metabolizing enzymes monoamine oxidases A and B (MAO-A and
MAO-B). A radiometric assay was used to determine the activities of MAO-A and MAO-B in
rat brain tissues after 14 days of daily i.m. nandrolone decanoate injections at the doses 3
and 15 mg/kg. Gene transcript contents of MAO-A, MAO-B and cathecol-O-
methyltransferase (COMT) were measured with quantitative real-time reverse transcription
PCR. 3 mg/kg of nandrolone decanoate significantly reduced the activity of both MAO-A and
-B in the caudate putamen. 15 mg/kg of nandrolone decanoate significantly reduced the
activity of MAO-A in the amygdala and increased the gene transcript level of MAO-B in the
substantia nigra. In conclusion, imbalanced MAO activities may contribute to explain the
impulsive and aggressive behaviour often described in AAS abusers. The reduced MAO
activities observed are in line with previously presented findings of decreased extracellular
levels of DOPAC and HVA in the rat brain, indicating decreased monoaminergic activity
following repeated AAS administration [08136].
It has been shown that male rats pubertally exposed to anabolic androgenic steroids (AAS)
displayed aggression towards females in response to physical provocation. This experiment
examined two factors that may modulate AAS-induced behavior towards females: olfactory
cues and frustration. Gonadally intact males began one of three AAS treatments at puberty
(D40): testosterone propionate (T), stanozolol (S), T+S, or vehicle control. To test for the
relevance of olfactory cues in the elicitation of behavior toward females, a hidden neighbor
paradigm was used. The proximal stimulus was an ovariectomized (OVX) female, estrogen
plus progesterone (E+P) female, or an E+P female with tape-obstructed vagina (OBS). Distal
olfactory cues from a hidden neighbor were delivered from a separate cage connected to the
testing arena. The vaginally obstructed, sexually receptive female (OBS) was used to
determine the effects of frustration on behavior by AAS males. Both sexual and aggressive
behaviors were measured. The presence of distal olfactory cues had no effect on either
sexual or aggressive behavior. In the presence of E+P and OBS females, all males displayed
sex behaviors, not aggression. However, AAS males displayed significantly more aggression
1146
towards proximal OVX females than controls. AAS males mounted OBS females significantly
more than controls, indicating a persistence of once rewarded behavior. These results
suggest that proximal cues of the conspecific female are more salient than distal olfactory
cues in determining behavior and that AAS males display frustration-induced persistence in
response to vaginally obstructed receptive females [06081].
Human studies suggest that anabolic androgenic steroid (AAS) users are aggressive towards
women. This study used a rat model to evaluate whether AAS potentiated aggression
towards females and the conditions under which this occurs. Gonadally intact pubertal male
rats received one of the following AAS treatments (5 mg/kg s.c. 5 days/week for nine weeks):
testosterone (T), stanozolol (S), testosterone + stanozolol (T + S), or vehicle control. Each rat
was tested with 3 conspecific stimuli: ovariectomized females (OVX), estrogen only females
(E), and estrogen + progesterone females (E + P). The response to physical provocation was
tested under three conditions: without physical provocation, provocation of the experimental
male, and provocation of the conspecific female. Provocation was a mild tail pinch. Both
aggressive and sexual behaviors were measured during each test. In the absence of
physical provocation, AAS males were not aggressive towards females. However,
provocation significantly increased aggression in males treated with testosterone but only
towards OVX females. In the presence of E or E + P females, all animals displayed sex
behavior, not aggression. Thus, factors such as the nature of the AAS and the hormonal
status of the females are important in determining whether male rats will be aggressive
towards females. However, the most salient factor determining aggression towards females
is the presence of provocation in combination with high levels of testosterone [06082].
Illicit use of anabolic androgenic steroids (AAS) has become a prevalent health concern not
only among male professional athletes, but, disturbingly, among a growing number of women
and adolescent girls. Despite the increasing use of AAS among women and adolescents, few
studies have focused on the effects of these steroids in females, and female adolescent
subjects are particularly underrepresented. Among the hallmarks of AAS abuse are changes
in reproductive behaviors. Here, it was discussed work from laboratories on the actions of
AAS on the onset of puberty and sexual behaviors in female rodents, AAS interactions and
sex- and age-specific effects of these steroids on neural transmission mediated by gamma-
aminobutyric acid receptors within forebrain neuroendocrine control regions that may
underlie AAS-induced changes in these behaviors [06083].
Anabolic androgenic steroid (AAS) abuse is increasing in teenagers. It was examined the
effects of stacked AAS in adolescent male rats. Stacking, in which multiple AAS are taken
simultaneously, is commonly employed by humans. Beginning at puberty gonadally intact
male rats received testosterone, nandrolone, or stanozolol. Additional groups received
stacked AAS: testosterone + stanozolol, nandrolone + stanozolol, or nandrolone +
testosterone. Injections continued during tests for sexual behavior, vocalizations, scent
marking, partner preference, aggression and fertility. Body and reproductive tissue weights
were taken. Sexual and aggressive behaviors were increased by testosterone yet inhibited
by stanozolol; nandrolone had no effect. Stacking testosterone with stanozolol prevented the
inhibitory effects of stanozolol. Body weight was decreased by testosterone and all stacked
AAS. Cell nuclear androgen receptor binding in brain was significantly increased in
nandrolone males and decreased in stanozolol males; testosterone males were slightly
higher than controls. Androgen receptors in stacked groups were intermediate between
individual AAS suggesting that stanozolol competed with other AAS for androgen receptors
despite its low affinity. The results indicate that stacking AAS influences the effects of
individual AAS on behavioral and endocrine measures, and levels of androgen receptor
occupation are not directly correlated with AAS effects on behavior [06084].
1147
Mouse
Animal studies have provided important insights into the specific neurochemical changes and
the mechanisms underlying the various behaviors associated with AAS use. Many of the
central nervous system (CNS) effects and behaviors observed in humans in association with
AAS use at high doses are related to brain circuits that function similarly in other mammalian
species. Indeed, several studies carried out in animal models confirm that changes in
defensive and offensive aggression, dominant behavior, anxiety, and sensitivity to other
abused drugs often mimic what has been observed in human subjects abusing AASs. For
example, AAS has been shown to increase the expression of opioid tolerance in mice.
Although it is difficult to precisely scale androgen doses from rodents to humans, when
adjusted according to body surface area using the US Federal Drug Administration
guidelines, the doses tested in animal studies (up to 7.5 mg/kg) appear to fall within the
range of human AAS use. The effect of AAS on aggressive behavior has been studied
extensively in many laboratories. A recent article reviewing the impact of AAS exposure on
brain circuits crucial for the expression of anxiety and aggressive behavior compared these
effects in relation to different classes of AAS; the study examined potential signaling
mechanisms as well as aspects of their action in relation to age and sex. The study revealed
that these steroids induce profound effects on aggression as well as the signaling molecules
and receptors in pathways related to aggression [14426].
Anabolic androgenic steroid abuse triggers impulsive aggression, anxiety, and depression,
which suggests a dysfunction of GABAergic neurotransmission. Socially isolated female mice
that have received testosterone propionate (1.45 micromol/kg) treatment for 3 weeks during
social isolation express aggression, neurosteroid downregulation, and changes in the cortical
mRNA expression of several gamma-aminobutyric acid type A receptor subunits (alpha1,
alpha2, gamma2 are decreased by 30-40 %, and alpha4 and alpha5 are increased by 50 %).
Administration of allopregnanolone or the potent selective brain steroidogenic stimulant S-
norfluoxetine, in doses (1.8-3.6 micromol/kg) that fail to inhibit 5-hydroxytryptamine reuptake,
normalizes olfactory bulb neurosteroid level downregulation and abolishes aggression. This
work underscores the role of neurosteroids in the regulation of aggression elicited by
testosterone propionate in socially isolated female mice [06080].
Anabolic androgenic steroids effects vary according to chemical structure and metabolism,
route of administration, and AAS regimen. In one study, male mice were systemically
exposed to testosterone propionate, nandrolone or 17alpha-methyltestosterone, type I, type
II and type III AAS, respectively, in order to determine the hedonic or aversive properties of
each drug. For this purpose, the conditioned place preference test was employed at three
different AAS doses (0.075, 0.75 and 7.5 mg/kg). Other behavioral domains monitored were
light-dark transitions (side changes) and general activity. Testosterone propionate shifted
place preference at all doses tested, and nandrolone shifted place preference at 0.75 and 7.5
mg/kg, but not at 0.075 mg/kg, the lower dose tested. Conversely, mice receiving 17alpha-
methyltestosterone did not show alteration in the preference score. The lower dose of
nandrolone did modify exploratory-based anxiety showing a decrease in light-dark transitions
if compared to vehicle-treated animals, while mice treated with testosterone propionateor
17alpha-methyltestosteronewere not affected. The data suggest that when studying hedonic
and rewarding properties of synthetic androgens, distinction has to be made based on type
of AAS and metabolism [08137].
Hamster
Repeated exposure to anabolic/androgenic steroids (AAS) during adolescence stimulates
high levels of offensive aggression in Syrian hamsters. The current study investigated
whether adolescent AAS exposure activated neurons in areas of hamster forebrain
implicated in aggressive behavior by examining the expression of FOS, i.e., the protein
1148
product of the immediate early gene c-fos shown to be a reliably sensitive marker of neuronal
activation. Adolescent AAS-treated hamsters and sesame oil-treated littermates were scored
for offensive aggression and then sacrificed 1 day later and examined for the number of FOS
immunoreactive (FOS-ir) cells in regions of the hamster forebrain important for aggression
control. When compared with non-aggressive, oil-treated controls, aggressive AAS-treated
hamsters showed persistent increases in the number of FOS-ir cells in select aggression
regions, namely the anterior hypothalamus and lateral septum. However, no differences in
FOS-ir cells were found in other areas implicated in aggression such as the ventrolateral
hypothalamus, bed nucleus of the stria terminals, central and/or medial amygdala or in non-
aggression areas, such as the samatosensory cortex and the suprachiasmatic nucleus.
These results suggest that adolescent AAS exposure may constitutively activate neurons in
select forebrain areas critical for the regulation of aggression in hamsters. A model for how
persistent activation of neurons in one of these brain regions (i.e., the anterior hypothalamus)
may facilitate the development of the aggressive phenotype in adolescent-AAS exposed
animals is presented [06079].
Chronic treatment with anabolic-androgenic steroids during adolescence alters the activity of
various neurotransmitter systems in male Syrian hamsters (Mesocricetus auratus). One
study was conducted to determine whether glutamatergic cells in the lateral anterior
hypothalamus, a sub-region of the anterior hypothalamus, have lasting activation following
adolescent AAS exposure, and to examine AAS-induced alterations in the connections
between the lateral anterior hypotalamus and the ventrolateral hypothalamus governed by
glutamate. Hamsters were administered AAS during adolescence and then examined for
changes in FOS (protein product of the immediate early gene c-fos) and phosphate activated
glutaminase (PAG; the rate-limiting enzyme in the synthesis of glutamate) immunoreactivity
(FOS/PAG-IR) using double-label immunohistochemistry. In a second experiment, a
retrograde tracing study was conducted using a red fluorescent tracer microinjected into the
ventrolateral hypothalamus . Then brains were processes for PAG immunofluorescence and
examined for AAS-induced changes in the number of PAG positive cells containing the tracer
(PAG/Tracer) in the lateral anterior hypothalamus. When compared to oil-treated controls,
AAS-treated hamsters showed significant increases in PAG-IR and FOS/PAG-IR in the
lateral anterior hypothalamus, decreases in afferent innervation from the lateral anterior
hypothalamusto the ventrolateral hypothalamus and decreases in the total number of
glutamate cells in the lateral anterior hypotalamus projecting to the ventrolateral
hypothalamus. Together with previous research from our lab showing increased AAS-
induced expression of PAG in the AH and increased glutamate receptor expression in the
ventrolateral hypothalamus, the current results suggest that adolescent AAS exposure leads
to alterations in the function and expression of the glutamatergic system as well as changes
in hypothalamic neural connections. In addition, the current results further strengthen the
notion that a specific nucleus in the lateral anterior hypothalamusis a critical hypothalamic
sub-region particularly sensitive to AAS-induced neurodevelopmental effects [08138].
Anabolic/androgenic steroid (AAS) use remains high in both teens and adults in the U.S. and
worldwide despite studies showing that AAS use is associated with a higher incidence of
aggression and anxiety. Recently we showed that chronic exposure to AAS through
adolescence increases aggression and decreases anxious behaviors, while during AAS-
withdrawal aggression is lowered to species-normative levels and anxiety increases. AAS
exposure is known to differentially alter behaviors and their underlying neural substrates
between adults and adolescents and thus the current study investigated whether exposure to
AAS during adulthood affects the relationship between aggression and anxiety in a manner
similar to that previously observed in adolescents. Male hamsters were administered a
moderate dose of AAS (5.0 mg/kg/day×30 days) during adolescence (P27-56) or young
adulthood (P65-P94) and then tested for aggression and anxiety during AAS exposure (i.e.,
1149
on P57 or P95) and during AAS withdrawal (i.e. 30d ays later on P77 or P115). Adolescent
exposure to AAS increased aggressive responding during the AAS exposure period and
anxiety-like responding during AAS withdrawal. Neither behavior was similarly influenced by
adult exposure to AAS. Adult AAS exposure produced no difference in aggressive
responding during AAS exposure (P95) or AAS withdrawal (P115); however, while AAS
exposure during adulthood produced no difference in anxiety-like responding during AAS
exposure, adult hamsters administered AAS were less anxious than vehicle control animals
following AAS withdrawal. Together these data suggest that the aggression and anxiety
provoking influence of AAS are likely a developmental phenomenon and that adult exposure
to AAS may be anxiolytic over the long term [150194].
Syrian hamsters exposed to anabolic/androgenic steroids (AAS) during adolescence

consistently show increased aggressive behavior across studies. Although the behavioral
and anatomical profiles of AAS-induced alterations have been well characterized, there is a
lack of data describing physiological changes that accompany these alterations. For
instance, behavioral pharmacology and neuroanatomical studies show that AAS-induced
changes in the vasopressin (AVP) neural system within the latero-anterior hypothalamus
(LAH) interact with the serotonin (5HT) and dopamine (DA) systems to modulate aggression.
To characterize the electrophysiological profile of the AAS aggression circuit, it was recorded
LAH neurons in adolescent male hamsters in vivo and microiontophoretically applied
agonists and antagonists of aggressive behavior. The interspike interval (ISI) of neurons from
AAS-treated animals correlated positively with aggressive behaviors, and adolescent AAS
exposure altered parameters of activity in regular firing neurons while also changing the
proportion of neuron types (i.e. bursting, regular, irregular). AAS-treated animals had more
responsive neurons that were excited by AVP application, while cells from control animals
showed the opposite effect and were predominantly inhibited by AVP. Both DA D2
antagonists and 5HT increased the firing frequency of AVP-responsive cells from AAS
animals and dual application of AVP and D2 antagonists doubled the excitatory effect of AVP
or D2 antagonist administration alone. These data suggest that multiple DA circuits in the
LAH modulate AAS-induced aggressive responding. More broadly, these data show that
multiple neurochemical interactions at the neurophysiological level are altered by adolescent
AAS exposure [150195].
Other animal models

The administration of testosterone propionate has been shown to significantly increase
aggressive behavior in cynomolgus monkeys; similar observations were later recorded in
rodents. The type of aggression, which we record in our experimental animal models, is
characterized as defensive aggression, measured by means of specific approaches to
provoke the animals. Chronic exposure to testosterone has also been shown to increase
male aggressive response patterns without altering the male sexual behavior or body weight.
Additional studies have confirmed that high doses of AASs could elicit aggressive behavior in
both rats and hamsters. However, different steroids may exhibit different potency in this
regard. Furthermore, AASs can induce both offensive and defensive behaviors, and various
strains of rats exhibited different responses to provocation. A variety of signaling pathways
are involved in mediating the effects of AASs on aggressive behaviors observed in rodents.
The brain pathways associated with aggression include neural circuits that use signaling by
excitatory amino acid systems and monoaminergic and peptidergic neurotransmitters. The
changes within each neurotransmitter system within different neural circuits are specific for
the type of AAS used. The key brain regions involved in aggressive behavior include the
anterior hypothalamus, periaqueductal gray, and amygdaloid nuclei (particularly the central
and medial amygdala). For instance, a tachykinin (substance P) pathway originating in the
central amygdala and innervating the hypothalamus and the periaqueductal gray is activated
in rats chronically treated with supraphysiologic doses of AAS, whereas an enkephalinergic
1150
pathway was downregulated. All these events were consistent with increased sensitivity
toward provocation. AAS exert additional effects on the glutamate system, also known to be
involved in aggressive behavior [14426].
Another amino acid of interest with respect to aggressive behavior is gamma-aminobutyric

acid (GABA). AASs elicit both acute modulation of GABA(A) receptor-mediated currents and
chronic regulation of the expression of the GABA(A) receptor and forebrain GABAergic
transmission. The serotonergic system also may have an important function in the control of
the aggressive dominance induced by AAS. The serotonergic 5-hydroxytryptamine (5HT)1B
or 5HT2 receptors may play a role in the mediation of emotional states and behavioral
changes that we see among human AAS users. A role of dopaminergic pathways in AAS-
induced aggression has also been suggested. AAS exposure affects dopamine receptors in
brain areas included in the functional anatomy of aggression. A typical feature seen in
individuals taking steroids seems to be a competitive and dominant behavior. Studies have
used experimental animal models to better understand the relationship between and AAS
use and competitive behavior under various conditions. For instance, researchers have
studied competition and locomotor activity response to a sedative dose of ethanol after AAS
exposure in rats. The rats treated with AASs exhibited enhanced dominant behavior in the
competition test compared with controls. Ethanol did not affect the AAS groups' locomotor
activity, whereas the controls showed decreased locomotor activity. Also, AAS animals had
significantly lower levels of serotonin in basal forebrain and dorsal striatum compared with
controls. These results have led to the hypothesis that AAS use may constitute a risk factor
for disinhibitory behavior, partly by affecting the serotonergic system. An additional study on
dominant behavior assessed pair-housed male rats for dominance status based on their
behavior and alterations in body weights. Throughout the study, the rats had limited social
interactions on a daily basis. After 1 week, rats received nandrolone or placebo, and their
behavior was observed over 2 months. Dominant AAS-treated rats spent more time on highly
aggressive behaviors than the dominant placebo-treated rats. In addition, the probability for
highly aggressive behaviors was maintained for the AAS-treated rats throughout the study,
whereas it was decreased for the placebo-treated rats. These observations are similar to the
relatively long-term behavioral changes we see in humans after AAS use [14426].
Anabolic androgenic steroids (AAS) are taken by both sexes to enhance athletic
performance and body image, nearly always in conjunction with an exercise regime.
Although taken to improve physical attributes, chronic AAS use can promote negative
behavior, including anxiety. Few studies have directly compared the impact of AAS use in
males versus females or assessed the interaction of exercise and AAS. It was show that
AAS increase anxiety-like behaviors in female but not male mice and that voluntary exercise
accentuates these sex-specific differences. We also show that levels of the anxiogenic
peptide corticotrophin releasing factor (CRF) are significantly greater in males, but that AAS
selectively increase CRF levels in females, thus abrogating this sex-specific difference.
Exercise did not ameliorate AAS-induced anxiety or alter CRF levels in females. Exercise
was anxiolytic in males, but this behavioral outcome did not correlate with CRF levels. Brain-
derived neurotrophic factor (BDNF) has also been implicated in the expression of anxiety. As
with CRF, levels of hippocampal BDNF mRNA were significantly greater in males than
females. AAS and exercise were without effect on BDNF mRNA in females. In males,
anxiolytic effects of exercise correlated with increased BDNF mRNA, however AAS-induced
changes in BDNF mRNA and anxiety did not. In sum, we find that AAS elicit sex-specific
differences in anxiety and that voluntary exercise accentuates these differences. In addition,
our data suggest that these behavioral outcomes may reflect convergent actions of AAS and
exercise on a sexually differentiated CRF signaling system within the extended amygdale
[14254].
1151
Dependence in preclinical models
Finally, animal studies have provided strong support for a third, hedonic pathway to AAS
dependence, likely mediated by nongenomic pathways via membrane receptors rather than
by the classical genomic effects of AASs. Reports that AAS abusers often experience mental
effects within 15 to 20 minutes of AAS administration also favor the nongenomic effects
through membrane receptors rather than the classical androgen receptor-mediated genomic
effects. In fact, studies have reported steroid binding sites on both GABA and the N-methyl-
d-aspartate neurons. Studies have also reported interaction of AAS with omega-receptors.
The function of these receptors remains poorly understood, although there is some overlap
with the opioid system. These sites are recognized by neurosteroids produced endogenously
in the brain. AASs also may interact with enzymes involved in neurosteroid metabolism,
thereby modulating the action of these neurosteroids, which are known to produce effects on
various behaviors. Rats and mice display conditioned place preference to testosterone, and
male hamsters will self-administer testosterone to the point of death. AASs enhance beta-
endorphin in the ventral tegmental area and may thereby activate the brain reward system.
Interestingly, the opioid antagonist naltrexone can block testosterone self-administration in
hamsters. These observations, combined with others, suggest that opioidergic mechanisms
may be involved in the hedonic pathway to AAS dependence [14426].
Influence on serotonin in the brain
The goal of this study was to assess the interactive effects of chronic anabolic androgenic
steroid (AAS) exposure and brain serotonin (5-hydroxytryptamine, 5-HT) depletion on
behavior of pubertal male rats. Serotonin was depleted beginning on postnatal day 26 with
parachlorophenylalanine (PCPA 100 mg/kg, every other day); controls received saline. At
puberty (P40), half the PCPA-treated rats and half the saline-treated rats began treatment
with testosterone (T, 5 mg/kg, 5 days/week). Behavioral measures included locomotion,
irritability, copulation, partner preference, and aggression. Animals were tested for
aggression in their home cage, both with and without physical provocation (mild tail pinch).
Brain levels of 5-HT and its metabolite, 5-hydroxyindoleacetic acid (5-HIAA), were
determined using HPLC. PCPA significantly and substantially depleted 5-HT and 5-HIAA in
all brain regions examined. Chronic T treatment significantly decreased 5-HT and 5-HIAA in
certain brain areas, but to a much lesser extent than PCPA. Chronic exposure to PCPA
alone significantly decreased locomotor activity and increased irritability but had no effect on
sexual behavior, partner preference, or aggression. T alone had no effect on locomotion,
irritability, or sexual behavior but increased partner preference and aggression. The most
striking effect of combining T+PCPA was a significant increase in attack frequency as well as
a significant decrease in the latency to attack, particularly following physical provocation.
Based on these data, it can be speculated that pubertal AAS users with low central 5-HT
may be especially prone to exhibit aggressive behavior [06085].
Serotonin modulates offensive attack in steroid-treated hamsters

Chronic anabolic-androgenic steroid (AAS) treatment during adolescence facilitates offensive
aggression in male Syrian hamsters (Mesocricetus auratus). The current study assessed
whether adolescent AAS-facilitated offensive attack was modulated by serotonin (5-HT) and
if AAS exposure during this developmental period influenced 5-HT innervation to areas of
hamster brain implicated in aggressive behavior. In a first experiment, hamsters were
administered high-dose AAS throughout adolescence, and then scored for offensive attack
following the systemic administration of saline or fluoxetine, a selective 5-HT reuptake
inhibitor. Saline-treated hamsters showed high levels of offensive attack, while treatment with
fluoxetine attenuated the AAS-facilitated aggressive response. In a second experiment,were
administered high-dose AAS or sesame oil throughout adolescence, tested for offensive
1152
attack and then examined for differences in 5-HT innervation to areas of the hamster brain
important for aggression. Aggressive AAS-treated hamsters showed significant reductions in
the number of 5-HT immunoreactive (5-HT-ir) varicosities and fibers in several of these
areas, most notably the anterior hypothalamus (AH), ventrolateral hypothalamus (VLH) and
medial amygdala (MeA). However, no differences in 5-HT afferent innervation were found in
other aggression areas, such as the bed nucleus of the stria terminalis (BNST) and lateral
septum (LS). Together, these results support a role for altered 5-HT innervation and function
in adolescent AAS-facilitated offensive aggression [02047].
Agression and violence
There have been numerous case studies and press reports of individuals committing acts of
extreme violence while taking large doses of Anabolic-Androgenic Steroids (AAS). However,
research using psychometric measures of aggression has tended to use small numbers of
subjects which makes generalizing results difficult. In one study the State Trait Anger
Expression Inventory (STAXI) was administered to 50 AAS users and 40 non AAS-using
control subjects. Subjects also underwent a semistructured interview focusing on AAS's
effects on levels of aggression. Results showed that AAS users reported being significantly
less in control of their aggression than controls. The semi-structured interview findings
showed that elevations in aggression due to AAS use were reported by 60 per cent of AAS
users. However, these elevations appeared more related to irritability and bad temper than
acts of physical violence. The study also found that more AAS users than controls had
worked as doormen/bouncers. This highlights the issue of whether AAS use causes
aggression or whether aggressive individuals are attracted to AAS use. Future research
should investigate this question [01061].
Behavioral human studies linking AAS abuse and aggression have confounding factors, such
as regimen (multiple steroids over a cycle of use), co-administration with other drugs of
abuse and inaccurate measures of behavior simulated by subjective reports. Conversely,
experimental designs in animals that correlate AAS exposure and aggression are less
equivocal [150004].
suggested risk factors for violence through a cross-sectional study of a population-based
sample. In 2005, all Swedish-born male twins aged 20-47 years were invited to participate in
the Swedish Twin Adults: Genes and Environment (STAGE) survey of the Swedish Twin
Register (response rate 60 %). A total of 10,365 male survey participants with information on
AAS use were included in the study. Data on self-reported use of AAS, alcohol and other
substances, attention deficit hyperactivity disorder (ADHD) and personality disorder
symptoms were linked to nation-wide, longitudinal register information on criminal
crime (2.7 versus 0.6 % in convicted and non-convicted men, respectively; odds ratio 5.0, 95
% confidence interval 2.7 to 9.3). However, this link was substantially reduced and no longer
significant when controlling for other substance abuse (OR 1.6). Controlling for IQ,
psychological functioning, ADHD, personality disorder symptoms and childhood SES did not
reduce the risk further. In the general population, co-occurring polysubstance abuse, but not
IQ, other neuropsychological risks or socio-economic status, explains most of the relatively
crime [14705].
1153
The resident-intruder test is a common paradigm for assessing aggression. Initial studies on
animal models have reported that long-term exposure to high doses of testosterone raised
levels of aggression in gonadally intact rats and re-established aggression in castrated rats.
However, indices of aggressive responses depend on environmental context, social cues,
sex and hormonal status of the intruder, age of exposure, physical provocation, and type of
AAS administered. Hence, studies in rats showed that AAS-treated males demonstrated a
different predisposition for aggression when tested in three different environments (home
cage, opponent cage, or neutral cage). Adult male rats receiving high doses of AAS are more
aggressive toward the intruder in their home cage and displayed lower levels of aggression
in either opponents or neutral cages. Investigators extended their interest to other
experimental factors demonstrating that AAS-treated rats are typically more aggressive
toward intact rather than castrated rats, as well as toward ovariectomized rather than
sexually receptive females. It was also shown that 12 weeks of testosterone propionate
exposure enhanced inter-male aggression in adult rats after physical provocation in the form
of a mild tail pinch. Moreover, the environmental and social discriminating cues described
above failed to alter testosterone-induced aggressive responses to physical provocatio.
While testosterone clearly increases aggression, conflicting results have been reported in the
literature concerning other commonly abused AAS (stanozolol, nandrolone decanoate,
boldenone undecylenate) tested either in combination or individually. It was also tested
whether a 2-week administration of an AAS cocktail containing testosterone cypionate,
nandrolone decanoate, and boldenone undecylenate had dissimilar behavioral
consequences when drug exposure occurred during adolescence or adulthood. Higher
aggression levels were observed in male Syrian hamsters exposed to an AAS cocktail
compared to controls, regardless of age treatment. On the other hand, stanozolol failed to
induce aggressive behavior in gonadectomized and intact rats and mice. More conflicting
results have been reported by using nandrolone decanoate. It was shown increased levels of
aggression in Sprague-Dawley rats receiving chronic nandrolone decanoate, while no effect
has been evidenced in Wistar rats. Accordingly, adult rats exposed to mild physical
provocation demonstrated decreased inter-male aggression when treated with stanozolol,
while no effects of nandrolone have been reported. Regardless of the experimental
methodologies employed to assess aggression, these findings suggest that strain, AAS
chemical composition and regimen reflect the diversity of supra-therapeutic AAS exposure
on behavioral responses in animals [150004].
Several studies in preclinical models of aggression have investigated the AAS effects on the
neurochemical changes in specific brain areas related to this behavior. High aggression is
often associated to decreased serotonin (5-HT) neurotransmission. Although this may
account for high aggression as an individual feature, it has been suggested that serotonergic
activity is probably higher during performance of aggressive behavior. In particular,
testosterone propionate exposure decreased both 5-HT and 5-HT metabolite, 5-HIAA, in the
hippocampus but not in the striatum or in the frontal cortex of adult rats. Moreover, the
aggressive behavior of dominant rats was decreased by treatment with selective agonists of
5-HT1A, 5-HT1B, and 5-HT2A∕2C receptors. A significant decrease in 5-HT1A and 5-HT1B
receptors immunoreactive staining has been shown in the latero-anterior hypothalamus and
amygdala of hamsters treated with a mixture of AAS. However, no decrease in the number of
5-HT1A receptor-expressing neurons and an increase in 5-HT2A receptor immunoreactivity
have been reported in the hypothalamus. In 2009 it was reported reduced 5-HT1B mRNA
levels in the hippocampus, hypothalamus, amygdala, and prefrontal cortex of nandrolone-
treated mice suggesting that the serotonergic tone in these brain areas has a pivotal role for
AAS-induced aggression in rodents [150004].
The use of anabolic androgenic steroids (AASs) has escalated in teenagers and is
associated with increased violence. Adolescent exposure to chronic high levels of AASs is of
1154
particular concern because puberty is a hormonally sensitive period during which neural
circuitry for adult male patterns of behavior develop. Thus, teenage AAS use may have long-
term repercussions on the potential for displaying aggression and violence. Animal models
have contributed valuable information on the effects of AAS use. For example, studies in
rodents confirmed that exposure to the AASs testosterone and nandrolone, but not
stanozolol, does indeed increase aggression. A side effect of AAS use reported in humans is
"'roid rage," characterized by indiscriminate and unprovoked aggression. Results of animal
studies demonstrated that pubertal rats receiving AASs respond appropriately to social cues
as they are more aggressive toward intact males than are castrates. Also, testosterone-
treated males recognize appropriate environmental cues as they are most aggressive in their
home cage. Thus, adolescent AAS exposure increases aggressive behaviors, but does not
induce indiscriminate aggression. To assess whether AAS exposure increases aggression
after provocation, rats were tested following a mild tail-pinch. In adolescent males,
provocation increased aggression after withdrawal from testosterone, nandrolone, and
stanozolol, an effect which persisted for many weeks. The data suggest that AASs sensitize
animals to their surroundings and lower the threshold to respond to provocation with
aggression. Thus, in humans, pubertal AAS exposure may not cause violent behaviors, but
may increase the likelihood that aggressive acts will result in violence. This may persist into
adulthood [04064].
To scrutinize the criminal career among users of anabolic androgenic steroids (AAS) with
focus on a possible relationship between use of AAS and violent offences a prospective
longitudinal follow-up of police records concerning known users of AAS was performed. The
study described five young men who started to use AAS with the primary motive of gaining
muscle mass and strength and who subsequently got involved in criminal activities, including
violent offences. One person showed deviant behaviour suggestive of conduct disorder at an
early age. The other persons appeared to have low self-confidence, but had not been acting
out during early adolescence. It was concluded that use of AAS may constitute an increased
risk of developing an antisocial life style with involvement in criminal violence [02036].
The effect of AAS on aggressive behavior has been studied extensively in many laboratories.
An article reviewing the impact of AAS exposure on brain circuits crucial for the expression of
anxiety and aggressive behavior compared these effects in relation to different classes of
AAS; the study examined potential signaling mechanisms as well as aspects of their action in
relation to age and sex. The study revealed that these steroids induce profound effects on
aggression, as well as the signaling molecules and receptors in pathways related to
aggression. The administration of testosterone propionate has been shown to significantly
increase aggressive behavior in cynomolgus monkeys; similar observations were later
recorded in rodents. The type of aggression, which is recorded in experimental animal
models, is characterized as defensive aggression, measured by means of specific
approaches to provoke the animals. Chronic exposure to testosterone has also been shown
to increase male aggressive response patterns without altering the male sexual behavior or
body weight. Additional studies have confirmed that high doses of AAS could elicit
aggressive behavior in both rats and hamsters. However, different steroids may exhibit
different potency in this regard. Furthermore, AAS can induce both offensive and defensive
behaviors, and various strains of rats exhibited different response to provocation. A variety of
signaling pathways are involved in mediating the effects of AAS on aggressive behaviors
observed in rodents. The brain pathways associated with aggression include neural circuits
that utilize signaling by excitatory amino acid systems and monoaminergic and peptidergic
neurotransmitters. The changes within each neurotransmitter system within different neural
circuits are specific for the type of AAS used. The key brain regions involved in aggressive
behavior include the anterior hypothalamus, periaqueductal gray (PAG), and amygdaloid
nuclei (particularly the central and medial amygdala). For instance, a tachykinin (substance
1155
P) pathway originating in central amygdala and innervating hypothalamus and the PAG is
activated in rats chronically treated with supraphysiological doses of AAS, whereas an
enkephalinergic pathway was down-regulated. All these events were consistent with
increased sensitivity towards provocation. AAS exert additional effects on the glutamate
system, also known to be involved in aggressive behavior. Another amino acid of interest
with respect to aggressive behavior is gamma-aminobutyric acid (GABA). AAS elicit both
acute modulation of GABAA receptor-mediated currents and chronic regulation of the
expression of the GABAA receptor and forebrain GABAergic transmission. The serotonergic
system also may have an important function in the control of the aggressive dominance
induced by AAS. The serotonergic 5HT1B or 5HT2 receptors may play a role in the mediation
of emotional states and behavioral changes that we see among human AAS users. A role of
dopaminergic pathways in AAS-induced aggression has also been suggested. AAS exposure
affects dopamine receptors in brain areas included in the functional anatomy of aggression
[14017].
It was examined the effects of anabolic-androgenic steroid use on serious violent behavior.
Multivariate models based on data from the National Longitudinal Study of Adolescent Health
in the US (n=6823) were used to examine the association between lifetime and past-year
self-reported anabolic-androgenic steroid use and involvement in violent acts. Compared
with individuals who did not use steroids, young adult males who used anabolic-androgenic
steroids reported greater involvement in violent behaviors after it was controlled for the
effects of key demographic variables, previous violent behavior, and polydrug use [08147].
In humans and animals, anabolic-androgenic steroids (AAS) increase aggression, but the
underlying behavioral mechanisms are unclear. AAS may increase the motivation to fight.
Alternatively, AAS may increase impulsive behavior, consistent with the popular image of
'roid rage. To test this, adolescent male rats were treated chronically with testosterone (7.5
mg/kg) or vehicle and tested for aggressive motivation and impulsivity. Rats were trained to
respond on a nose-poke on a 10min fixed-interval schedule for the opportunity to fight in their
home cage with an unfamiliar rat. Although testosterone increased aggression (6.3 ± 1.3
fights/5 min vs 2.4 ± 0.8 for controls), there was no difference in operant responding (28.4 ±
1.6 nose-pokes/10 min for testosterone, 32.4 ± 7.0 for vehicle). This suggests that
testosterone does not enhance motivation for aggression. To test for impulsivity, rats were
trained to respond for food in a delay-discounting procedure. In an operant chamber, one
lever delivered one food pellet immediately, the other lever gave 4 pellets after a delay (0,
15, 30 or 45s). In testosterone- and vehicle-treated rats, body weights and food intake did not
differ. However, testosterone-treated rats chose the larger, delayed reward more often (4.5 ±
0.7 times in 10 trials with 4 5s delay) than vehicle controls (2.5 ± 0.5 times), consistent with a
reduction in impulsive choice. Thus, although chronic high-dose testosterone enhances
aggression, this does not include an increase in impulsive behavior or motivation to fight.
This is further supported by measurement of tyrosine hydroxylase (TH) by Western
immunoblot analysis in brain regions important for motivation (nucleus accumbens, Acb) and
executive function (medial prefrontal cortex, PFC). There were no differences in TH between
testosterone- and vehicle-treated rats in Acb or PFC. However, testosterone significantly
reduced TH (to 76.9 ± 3.1 % of controls) in the caudate-putamen, a brain area important for
behavioral inhibition, motor control and habit learning [12138].
Millions of individuals worldwide have used anabolic-androgenic steroids (AAS) to gain

muscle or improve athletic performance. Recently, in vitro investigations have suggested that
supraphysiologic AAS doses cause apoptosis of neuronal cells. These findings raise the
possibility, apparently still untested, that humans using high-dose AAS might eventually
develop cognitive deficits. It was administered five cognitive tests from the computerized
CANTAB battery (Pattern Recognition Memory, Verbal Recognition Memory, Paired
1156
Associates Learning, Choice Reaction Time, and Rapid Visual Information Processing) to 31
male AAS users and 13 non-AAS-using weightlifters age 29-55, recruited and studied in
2012 in Middlesbrough, UK. Testers were blinded to participants' AAS status and other
historical data. Long-term AAS users showed no significant differences from nonusers on
measures of response speed, sustained attention, and verbal memory. On visuospatial
memory, however, AAS users performed significantly more poorly than nonusers, and within
the user group, visuospatial performance showed a significant negative correlation with total
lifetime AAS dose. These were large effects: on Pattern Recognition Memory, long-term AAS
users underperformed nonusers by almost one standard deviation, based on normative
population scores (adjusted mean difference in z-scores=0.89; p=0.036), and performance
on this test declined markedly with increasing lifetime AAS dose. These results remained
stable in sensitivity analyses addressing potential confounding factors. These preliminary
findings raise the ominous possibility that long-term high-dose AAS exposure may cause
cognitive deficits, notably in visuospatial memory [12139].
The association between substance abuse, particularly alcohol abuse, and violence has
been well established. However, since substance abuse co-occurs with several other risk
factors for violence, the causal link between substance abuse and violence and the extent to
which the acute influence of alcohol, illicit drugs, benzodiazepines, and anabolic androgenic
steroids have a triggering effect on violent behavior are more uncertain. Case-crossover
design was used based on data from structured face to face interviews with remand
prisoners (n=194; 172 men, 22 women) suspected of violent crimes. Main outcome measure:
odds ratio (OR) for a violent crime, 24h after exposure to different substances, compared to
periods of no exposure was calculated using conditional logistic regression and a Mantel-
Haenszel estimator with confidence intervals for sparse data. Intake of alcohol (OR 6) and
large doses of benzodiazepines (OR 36) triggered interpersonal violence. Stratified analyses
of possible effect modifiers were sex, conduct/behavioral problems, trauma experiences;
psychiatric vulnerability did not reveal any substantial differences. Influences of alcohol and
unusually high doses of benzodiazepines are proximal risk factors for violent crime. Improved
knowledge of short-term (and dose-related) risk factors may contribute to treatment planning
and risk assessment of violence [13160].
In humans and animals, anabolic-androgenic steroids (AAS) increase aggression, but the
underlying behavioral mechanisms are unclear. AAS may increase the motivation to fight.
Alternatively, AAS may increase impulsive behavior, consistent with the popular image of
'roid rage. To test this, adolescent male rats were treated chronically with testosterone (7.5
mg/kg) or vehicle and tested for aggressive motivation and impulsivity. Rats were trained to
respond on a nose-poke on a 10 min fixed-interval schedule for the opportunity to fight in
their home cage with an unfamiliar rat. Although testosterone increased aggression (6.3 ±
1.3 fights/5 min vs 2.4 ± 0.8 for controls), there was no difference in operant responding
(28.4 ± 1.6 nose-pokes/10 min for testosterone, 32.4 ± 7.0 for vehicle). This suggests that
testosterone does not enhance motivation for aggression. To test for impulsivity, rats were
trained to respond for food in a delay-discounting procedure. In an operant chamber, one
lever delivered one food pellet immediately, the other lever gave 4 pellets after a delay (0,
15, 30 or 45 s). In testosterone- and vehicle-treated rats, body weights and food intake did
not differ. However, testosterone-treated rats chose the larger, delayed reward more often
(4.5 ± 0.7 times in 10 trials with 45 s delay) than vehicle controls (2.5 ± 0.5 times), consistent
with a reduction in impulsive choice. Thus, although chronic high-dose testosterone
enhances aggression, this does not include an increase in impulsive behavior or motivation
to fight. This is further supported by measurement of tyrosine hydroxylase (TH) by Western
immunoblot analysis in brain regions important for motivation (nucleus accumbens, Acb) and
executive function (medial prefrontal cortex, PFC). There were no differences in TH between
testosterone- and vehicle-treated rats in Acb or PFC. However, testosterone significantly
1157
reduced TH (to 76.9 ± 3.1 % of controls) in the caudate-putamen, a brain area important for
behavioral inhibition, motor control and habit learning [13206].
Twin studies
Individual behavioral and psychiatric responses to chronic AAS abuse are extremely
variable, depending on pre-existing psychiatric conditions, personality, and type and dose of
AAS. Case reports and some epidemiology studies associate chronic AAS use with changes
in behavior, mood, and somatic perceptions in a small subset of chronic AAS abusers.
Psychiatric complications associated with chronic AAS use include mania, aggression, and
agitation, but no large-scale epidemiologic studies have confirmed a causal relationship
between chronic AAS abuse and severe psychiatric complications (e.g. psychosis).
Depression may occur, particularly in the immediate period after cessation of use, along with
fatigue, decreased libido, insomnia, anorexia, andheadaches. Studies suggest that male
AAS users have more frequent and prolonged periods of anger, aggression, irritability, and
hostility than nonusers. Multivariate models based on data from the Nationa lLongitudinal
Study of Adolescent Health (n=6823) indicate that young men using anabolic-androgenic
steroids report greater involvement in violent behaviors than non-users after control for the
effects of key demographic variables, previous violent behavior, and polydruguse. In a study
of 2 sets of male monozygotic twins with only 1 twin using AASs, the use of AASs was
associated with anxiety, hostility, aggressiveness,and paranoid ideations as determined by
comparing responses to a Symptoms Checklist-90 and the Hostility and Direction of Hostility
Questionnaire. Behavioral abnormalities included are tractibility, delirium, delusions,
irritability, paranoia, impulsivity, insomnia, hostility, anxiety, agitation, aggression, violence,
and mood lability. These behavioral effects are dose dependent with effects ranging from
mild effects (increased confidence, enhancedself-esteem, and euphoria) to serious
behavioral abnormalities (mood swings, grandiose ethinking, paranoia, impulsivity, hostility,
violence, and antisocial behavior) [13003].
Associated of use of AAS with differences in inhibitory control and impulsivity

A growing translational literature suggests that adolescent exposure to anabolic-androgenic
steroids (AASs) leads to increased aggression and impulsivity. However, little is known
about the cognitive effects of AASs among AAS users or the differences between
adolescent- and adult-onset users. This study provides a test of the effects of acute
naturalistic AAS use and age of onset (adolescent vs adult) on measures of inhibitory control,
planning and attention, and decision making. Seventy-one active adult male AAS users
completed self-report measures of impulsivity and aggression, and a subsample (11
adolescent onset vs 11 adult onset) matched on current age were administered 4
computerized tests from the Cambridge Neuropsychological Test Automated Battery
(CANTAB) and the Iowa Gambling Task. Multiple regression analyses and a series of 2
(adolescent vs adult) × 2 (on-cycle vs off-cycle) analyses of variance (ANOVAs) were used to
examine the differential effects of age of onset and acute drug use on cognition and
behavior. Regression analyses revealed larger on-cycle effects for adolescent users than
adult users. Subsample analyses indicated that on-cycle users performed less well on
cognitive measures of inhibitory control and attention, but not on tests of planning or decision
making. Adolescent onset was associated with greater impulsivity and more acute sensitivity
to AAS effects on attention. These preliminary findings suggest the possibility that acute AAS
use is associated with some differences in inhibitory control and impulsivity and to a lesser
degree, aggression. These effects may be more potent for those initiating AAS use in
adolescence [14756].
Competitive and dominant behavior

A typical feature seen in individuals taking steroids seems to be a competitive and dominant
behavior. Studies have used experimental animal models to better understand the
1158
relationship between and AAS use and competitive behavior under various conditions. For
instance, researchers have studied competition and locomotor activity response to a sedative
dose of ethanol following AAS exposure in rats. The rats treated with AAS exhibited
enhanced dominant behavior in the competition test compared to controls. Ethanol did not
affect the AAS groups’ locomotor activity, whereas the controls showed decreased locomotor
activity. Also, AAS animals had significant lower levels of serotonin in basal forebrain and
dorsal striatum compared to controls. These results have led to the hypothesis that AAS use
may constitute a risk factor for disinhibitory behavior, partly by affecting the serotonergic
system. An additional study on dominant behavior assessed pair-housed male rats for
dominance status based on their behavior and alterations in body weights. Throughout the
study the rats had limited social interactions on a daily basis. After 1 week, rats received
nandrolone or placebo and their behavior was observed over 2 months. Dominant AAS-
treated rats spent more time on highly aggressive behaviors than the dominant placebo-
treated rats. In addition, the probability for highly aggressive behaviors was maintained for
the AAS-treated rats throughout the study, whereas it was decreased for the placebo-treated
rats [14017].
Enhances risk tolerance

Anabolic-androgenic steroids (AAS) increase impulsive and uncontrolled aggressive (“roid
rage”) in humans and enhance agonistic behavior in animals. However, the underlying
mechanisms for AAS-induced aggression remain unclear. Potential contributing elements
include an increase risk-taking and/or motor impulsivity due to AAS. This study addressed
the effects of chronic high-dose testosterone on risk tolerance using a risky decision-making
task (RDT) and motor impulsivity with a go/no-go task in operant chambers. Male Long-
Evans rats were treated for at least 4 weeks with testosterone (7.5 mg/kg) or vehicle
beginning in late adolescence. Testosterone was used because it is popular among human
AAS users. In RDT testing, one lever was paired with delivery of a small "safe" food reward,
while the other was paired with a large "risky" reward associated with an increasing risk of
footshock (0 %, 25 %, 50 %, 75 %, 100 %) in successive test blocks. Three shock intensities
were used: 1.0, 1.2, and 1.4 mA/kg. As shock intensity and risk of shock increased,
preference for the lever signifying a large reward significantly declined for both vehicle- and
testosterone-treated rats. There was also a significant effect of drug, where testosterone-
treated rats showed greater preference for the large reward, compared to vehicle-treated
controls. Increased preference for the large reward, despite risk of footshock, is consistent
with increased risk tolerance. In go/no-go testing, rats were trained to press a single lever if
the go cue was presented (stimulus light) or to refrain from pressing during the no-go cue
(tone). There was no effect of testosterone on pre-cue responses, or performance in go and
no-go trials. These results suggest that AAS may increase risk-tolerance without altering
motor impulsivity [14050].
Experimental
In the US and worldwide anabolic/androgenic steroid use remains high in the adolescent
population. This is concerning given that anabolic/androgenic steroid use is associated with a
higher incidence of aggressive behavior during exposure and anxiety during withdrawal. This
study uses pubertal Syrian hamsters (Mesocricetus auratus) to investigate the hypothesis
that an inverse behavioral relationship exists between anabolic/androgenic steroid-induced
aggression and anxiety across adolescent exposure and withdrawal. In the first experiment, it
was examined aggression and anxiety during adolescent anabolic/androgenic steroid
exposure and withdrawal. Adolescent anabolic/androgenic steroid administration produced
significant increases in aggression and decreases in anxiety during the exposure period
followed by significant decreases in aggression and increases in anxiety during anabolic/
androgenic steroid withdrawal. In a second experiment, anabolic/androgenic steroid exposed
animals were separated into groups based on their aggressive response during the exposure
1159
period and then tested for anxiety during exposure and then for both aggression and anxiety
during withdrawal. Data were analyzed using a within-subjects repeated measures predictive
analysis. Linear regression analysis revealed that the difference in aggressive responding
between the anabolic/androgenic steroid exposure and withdrawal periods was a significant
predictor of differences in anxiety for both days of testing. Moreover, the combined data
suggest that the decrease in aggressive behavior from exposure to withdrawal predicts an
increase in anxiety-like responding within these same animals during this time span.
Together these findings indicate that early anabolic/androgenic steroid exposure has potent
aggression- and anxiety-eliciting effects and that these behavioral changes occur alongside a
predictive relationship that exists between these two behaviors over time [13150].
Male Syrian hamsters (Mesocricetus auratus) treated with anabolic/androgenic steroids

(AAS) during adolescence (P27-P56) display highly escalated and mature forms of offensive
aggression correlated with increased gamma-aminobutyric acid (GABA) afferent
development as well as decreased GABAA receptors in the lateroanterior hypothalamus
(LAH) – an area of convergence for developmental and neuroplastic changes that underlie
offensive aggressive behaviors in hamsters. This study investigated whether microinfusion of
a GABAA receptor agonist (muscimol; 0.01-1.0 pmol/L) or antagonist (bicuculline; 0.04-
4.0 pmol/l) directly into the LAH modulate adolescent AAS-induced offensive aggression.
Activation of LAH GABAA receptors enhanced adolescent AAS-induced offensive
aggression, beginning at the 0.1 pmol/l dose, when compared with AAS-treated animals
injected with saline into the LAH. Importantly, GABAA receptor agonism within the LAH
significantly increased the frequency of belly/rear attacks, while simultaneously decreasing
the frequency of frontal attacks. These data identify a neuroanatomical locus where GABAA
receptor activation functions to enhance aggression in adolescent AAS-treated animals,
while also promoting the display of mature forms of aggression and suppressing juvenile play
behaviors [14757].
One study examined the effects of anabolic androgenic steroids (AAS) on aggression under
different social and environmental conditions. Three AAS were tested in gonadally intact
male rats: testosterone propionate (TP), nandrolone (ND), and stanozolol (ST). Doses of 5
mg/kg were given 5 times/week, with gonadally intact controls receiving vehicle only
(propylene glycol). Animals received six weekly tests under each condition in a
counterbalanced order. Results show that the three AAS differed in their ability to elicit
aggression. Males receiving TP were more aggressive than controls, ND males were similar
to controls, and ST males were less aggressive than controls. In the social and
environmental provocation tests TP-treated males were more aggressive than other groups,
but were able to discriminate between intact and castrated opponents and between their
home cage and a neutral cage. In the environmental provocation test, TP males were also
more aggressive against opponents when tested in the opponent's home cage. It is
suggested that chronic exposure to high levels of TP does not eliminate the ability to
discriminate between social or environmental cues, as might be expected if it induces a "
'roid rage." However, TP does increase the likelihood that the animal will respond with
aggression/dominance in a provoking situation. All three AAS variably affected serum
testosterone and LH levels, as well as testes, seminal vesicle, and prostate weights. No
effect on body weight was observed [01062].
Effect on ethanol response. One study investigated whether anabolic androgenic steroid
(AAS) treatment (daily subcutaneous injections during 2 weeks with nandrolone decanoate;
15 mg/kg) affects competitive behaviour, and locomotor activity response to a sedative dose
of ethanol (0.5 g ethanol/kg). In addition, levels of brain monoamines were assessed. The
results showed that AAS treated animals exhibited enhanced dominant behaviour in the
competition test compared to controls. The AAS groups' locomotor activity was not affected
1160
by ethanol in contrast to the controls who showed a sedative locomotor activity. AAS animals
had significant lower levels of serotonin in basal forebrain and dorsal striatum compared to
controls. These findings further strengthen the fact that AAS affects behaviour, as well as
biochemical parameters. Based on previous studies and results from the present study, it
was hypothesize that AAS abuse may constitute a risk factor for disinhibitory behaviour,
partly by affecting the serotonergic system [02042].
Physical provocation potentiates aggression in male rats receiving anabolic steroids Anabolic
androgenic steroids (AAS) have been linked to indiscriminant and unprovoked aggression
and violence. It was employed a brief tail pinch to examine the effects of different AAS on
intermale aggression in gonadally intact male rats in response to a mild physical provocation.
Animals received 5 mg/kg testosterone propionate (TP), nandrolone (ND), or stanozolol (ST)
5 days/week. Controls received vehicle injections. After 12 weeks, rats were tested for
aggression while treatments continued. Animals were paired with either gonadally intact or
castrated opponents and were tested in the subject rat's home cage, the opponents's home
cage, and a neutral cage. Aggression was tested during tail pinch of the subject rat and
during tail pinch of the opponent rat. In TP-treated males, tail pinch significantly enhanced
aggression in all social and environmental conditions compared to intact controls. TP
treatment also significantly enhanced aggression when the opponents were tail pinched. Tail
pinch did not increase aggression in ND-treated males, and aggression was significantly
lower than controls in ST-treated males. As expected, cell nuclear androgen receptor binding
was significantly elevated by the high dose of TP. The results show that while AAS alone
does not induce the indiscriminate and unprovoked aggression characteristic of 'roid rage
[02043].
Association with criminality
Sweden
Observations suggest that the use of anabolic androgenic steroids (AAS) may trigger
uncontrolled, violent rage. Other observations indicate that certain groups of criminals may
use AAS with the intention of being capable of committing crime more efficiently. To examine
the proposed association between the use of AAS and criminality a controlled retrospective
cohort study of registered criminal activity among individuals tested for AAS use during the
period of 1995 to 2001 was performed. All individuals in Sweden who were tested for AAS
use during this period were included. These individuals were referred for testing from both
inpatient and outpatient clinics as well as from centers for treatment of substance abuse:
individuals testing positive for AAS (n=241), with those testing negative for AAS during the
same period (n=1199) serving as the control group. The risk of having been convicted for a
weapons offense or fraud was higher among individuals testing positive for AAS than among
those testing negative (RR, 2.090 and 1.511, respectively) whereas there were no significant
differences with respect to violent crimes (RR, 1.116) or crimes against property (RR, 0.942).
When patients referred from substance abuse centers were excluded, a lower risk for crimes
against property was observed for the individuals who tested positive for AAS (RR, 0.761)
and the risk for fraud in the 2 groups was equalized (RR, 1.117). The increased risk for a
weapons offense among the individuals testing positive for AAS remained virtually
unchanged. It was concluded that in addition to the impulsive violent behavior previously
shown to be related to AAS use, such use might also be associated with an antisocial
lifestyle involving various types of criminality. However, the existence and nature of this
possible association remain unclear and call for further investigation [06086].
1161
suggested risk factors for violence. In 2005, all Swedish-born male twins aged 20-47 years
were invited to participate in the Swedish Twin Adults: Genes and Environment (STAGE)
survey of the Swedish Twin Register (response rate 60 %). A total of 10,365 male survey
participants with information on AAS use were included. Data on self-reported use of AAS,
alcohol and other substances, attention deficit hyperactivity disorder (ADHD) and personality
disorder symptoms were linked to nation-wide, longitudinal register information on criminal
crime (2.7 versus 0.6 % in convicted and non-convicted men, respectively; odds ratio 5.0).
However, this link was substantially reduced and no longer significant when controlling for
other substance abuse (OR 1.6). Controlling for IQ, psychological functioning, ADHD,
personality disorder symptoms and childhood SES did not reduce the risk further. It was
concluded that in the general population, co-occurring polysubstance abuse, but not IQ,
other neuropsychological risks or socio-economic status, explains most of the relatively
crime [150196].
Suicide risk
It was studied 62 professional weightlifters in Finland who were strongly suspected of using
anabolic-androgenic steroids. They were compared them with 1,094 population controls.
Over a 12-year period, 8 (13 %) of the weightlifters died versus 34 (3 %) of the controls,
which was a statistical significant difference. The causes of death in the weightlifters were
suicide (3 subjects), acute myocardial infarction (3), hepatic coma (1), and non-Hodgkin
lymphoma (1) [07031].
Not only are athletes at risk for psychiatric illness, but they are at risk of suicide. In an effort
to learn more about suicide in athletes and those connected to the sports arena, a review of
the medical literature from 1960 to 2000 was conducted through Medline, and a review of the
periodical literature from 1980 to 2000 was conducted through Infotrac. These reviews
revealed 71 cases of athletes who have either contemplated, attempted, or completed
suicide. In one article, these cases were analyzed by sport, gender, and age. Through
inference, an attempt to establish the etiologic basis for these behaviors is undertaken.
Intervention and prevention strategies are discussed, based on the available data [05384].
With the aim to characterize patterns in toxicological profile and manner of death in
deceased users of anabolic androgenic steroids (AAS), a retrospective autopsy protocol
study of 52 deceased users of AAS was undertaken. The AAS users were compared to 68
deceased users of amphetamine and/or heroin who were consecutively tested and found to
be negative for AAS. Use of AAS was in the majority of cases (79 %) associated with
concomitant use of psychotropic substances. AAS-related deaths differed in several respects
from deaths among users of heroin or amphetamine, most strikingly with regard to: (a) the
median age at death, which was significantly lower for AAS users (25 years) than for users of
heroin and/or amphetamine (34 and 40 years, respectively); (b) the manner of death, with
AAS users dying significantly more often from homicide or suicide than users of other drugs;
and (c) the body mass index (BMI), with AAS users exhibiting significantly higher BMI than
users of other drugs. These results support the earlier reported association between use of
AAS and use of other psychoactive substances. In addition, the data suggest that AAS users
are more likely to become involved in incidents leading to violent death and have a higher
risk of dying at a younger age than users of other drugs [05057].
1162
Addiction
An estimated 14 to 57 percent of anabolic-androgenic steroid users develop dependence.

How addiction develops is unknown, but psychological dependence is believed to play a
large role. Different substance abuse patterns exist in different populations that use anabolic-
androgenic steroids. In one study it was found that steroid-using weightlifters almost always
previously tried other illicit substances. On the other hand, others found that elite athletes,
weightlifters, and bodybuilders rarely abuse illicit drugs, reflecting their interest in optimizing
their physique and performance. Adolescents who abuse steroids are more likely to smoke
and use other illicit substances than are older users [07031]
Dependence and addiction potential

The data in the literature show no documented cases of dependence induced by AAS use at
therapeutic doses. This suggests that dependence is likely associated to the use of higher
doses of AAS. However, molecular mechanisms leading to AAS-induced dependence are
still unclear [150003].
A “withdrawal syndrome” induced by AAS abuse has been clearly described, consisting
mainly of depressed mood, fatigue, AAS craving, restlessness, anorexia, insomnia, and
decreased libido lasting for several weeks or months. In the 1980s, it was described a case
report on which a model of a biphasic course of withdrawal was proposed. The initial phase
of the AAS-induced withdrawal (lasting for about 1 week) seemed to be comparable to
opioid-induced withdrawal, while the second phase was mostly characterized by clear
depressive symptoms and craving. Considerable evidence suggests that AAS dependence
might share crucial mechanisms of opioid dependence in humans. In 1989, it was posited
that AAS dependence might partly arise via an opioidergic mechanism, through which AAS
might enhance the activity of central endogenous opioids, and AAS withdrawal would lead to
a decrease in this activity and a subsequent acute hyperadrenergic syndrome. This posited
link between AAS and opioids was later confirmed by a large number of observations
indicating that AAS users seem to be particularly at risk for developing opioid abuse or
dependence. Additional clinical studies provided evidence that AAS might decrease the
analgesic action of both metamizol and morphine. In 2009, it was added further evidence for
a relationship between AAS and opioids. In the population included in that study, opioid
abuse or dependence began either before or after the onset of AAS use, suggesting the
possibility that these forms of substance abuse might arise from a common molecular
pathway. However, in another study the authors could not detect any withdrawal phenomena
following administration of high doses of AAS [150004].
AAS seem to act through a more modest reinforcement mechanism compared to cocaine or
heroin and resembles the reinforcement mechanism described for caffeine, nicotine, and
benzodiazepines. In 2002, it was proposed a 2-stage model of steroid dependence. In Stage
1, anabolic effects of AAS provide the initial input and motivation for AAS consumption.
Stage 2 deals with consequent chronic use, following which physiological and psychological
dependence may develop, thereby making it increasingly difficult for users to quit.
Psychoactive effects, such as mood changes and increases in aggressive behavior,
characterize this stage of dependence. Diagnostic and Statistical Manual of Mental Disorders
criteria for AAS dependence are met and users are not able to stop or discontinue AAS
consumption. In Stage 2, addiction treatment may be required, especially when AAS abuse
is associated with other substance dependence, such as alcohol, opioids, or amphetamine
abuse. In 2000 it was investigated this phenomenon in a clinical study, including 227 patients
admitted to a private facility for dependence on heroin or other opioids. Results of this study
strongly suggested that these patients were introduced to opioids through AAS use and
bodybuilding physical activity. In particular, 81 percent of them first purchased opioids from
1163
the same drug dealer who had sold them AAS; 67 percent were introduced to opioids by a
fellow body-builder; 86 percent first used opioids to reduce insomnia and irritability induced
by AAS, and 67 percent used opioids to diminish depression feelings induced by withdrawal
from AAS [150004].
A second model, explaining mechanisms leading to AAS dependence, has also been
proposed. This model holds that AAS-dependence development occurs specifically in socio-
cultural contexts that are likely to motivate certain individuals, particularly men, to attain large
and strong muscles by frequent and intensive training sessions. These training sessions also
improve mood and self-esteem and are generally associated with very strict and controlled
dietary regimens. Thus, AAS-induced muscle-active effects might underlie the reinforcing
actions of these compounds and the compulsive features of AAS use seem to strengthen the
likely compulsive patterns of training and diet. Studies to elucidate mechanisms leading to
AAS dependence have also included surveys of current and former AAS users, recruited
from gyms, websites, and physicians. It was also reported numerous criteria for psychoactive
substance dependence in a survey of eight AAS abusers, including continued use despite
adverse side effects, and withdrawal symptoms [150004].
Specific dysfunctions of the various components of the brain reward system have been
described in clinical studies. For example, alterations in levels of monoamine metabolites,
neurohormones, and neuropeptides, which play a crucial role in the reward mechanism, have
been investigated in the cerebrospinal fluid of subjects who received methyltestosterone
(MT) with respect to placebo-treatment. Results showed that levels of 5-hydroxyindolacetic
acid (5-HIAA) increased while 3-metoxy-4-hydroxyphenylglycol (MHPG) levels decreased in
cerebrospinal fluid, following MT administration. In particular, changes in cerebrospinal fluid
5-HIAA significantly correlated with the activation of specific psychiatric symptom cluster
scores. In addition, according to this study, a decrease in cerebrospinal fluid MHPG may
derive from reduced norepinephrine clearance, even though authors did not detect any
significant correlations between changes in MHPG levels and the development of clear
psychiatric symptoms, suggesting a less crucial role for noradrenergic changes in this
process. An increase in substance P levels and vasopressin, as well as dysfunctions of the
central opioid system, have been proposed as playing a potential role in the development of
aggressive behavior after AAS abuse [150004].
Multiple factors have been associated with the induction of dependence in AAS users, such
as low endogenous levels of testosterone. Indeed, it has been demonstrated that women,
adolescents and elderly subjects have a lower probability of developing AAS dependence.
Among possible risk factors for dependence development, the most relevant appears to be
participation in competitive sports with intense and repetitive physical exercise. Some
investigators have also suggested that personality psychopathology may be a risk factor for
AAS abuse. In 1990 it was reported that AAS users and weight lifters had a higher
prevalence of histrionic, antisocial, and borderline personality traits than community controls.
Although a growing number of reports, current knowledge of molecular mechanisms leading
to AAS dependence in humans remains limited. In this regard, the reinforcing effects of AAS
may also be biased by intensive physical exercise and by increased narcissistic self-esteem
arising from the fulfillment of the desired body appearance. On the other hand, many users
practice “stacking” consumption, consisting in the contemporary mixed use of multiple
steroids [150004].
Since it has been reported that around 96 percent of users combine AAS with other drugs in
order to relieve non-medical steroid side effects, pharmacodynamics, and pharmacokinetic
interaction studies are surely warranted, although hardly feasible, in order to exclude further
bias [150004].
1164
Withdrawal effects
Users of high-dose AAS regimens report a withdrawal syndrome, including steroid craving,
depression, suicidality, irritability, muscle aches, and autonomic instability including hot
flashes, nausea and vomiting, tachycardia, and hypertension [07058].
Physical symptoms of withdrawal are similar to those seen during alcohol and opioid
withdrawal, including diaphoresis, myalgias, nausea, and increases in blood pressure and
heart rate. Withdrawal may also be characterized by depressive symptoms [07008].
Withdrawal of anabolics: effect on aggression

In gonadally intact male rats, chronic exposure to high levels of testosterone propionate (TP)
increases aggression, nandrolone (ND) has little effect and stanozolol (ST) suppresses
aggression. The present experiment tested whether the effects of TP, ND and ST on
aggression and reproductive tissues are reversed following anabolic androgenic steroid
(AAS) withdrawal. Gonadally intact males received TP, ND, ST or vehicle for 12 weeks.
Injections were then discontinued. Aggression was tested 3 weeks (short term) and 12
weeks (long term) after withdrawal of AAS treatment, with either a gonadally intact or a
castrated opponent in three different environments (home, opponent's and neutral cage).
After short-term withdrawal, some parameters of aggression were significantly above control
levels in TP males. There were no significant differences between ND or ST males and
controls, though ST males showed the lowest levels of aggression. No significant differences
between any of the groups were found after long-term withdrawal. Eighteen weeks after AAS
withdrawal, serum testosterone (T) and LH levels were comparable to controls in all groups.
Testes weights were at control levels in ST males, but significantly higher than controls in TP
and ND males. Seminal vesicle weights were significantly elevated in TP males, but similar
to controls in both ND and ST males. None of the prostate weights were significantly different
from controls. These results suggest that aggression gradually returns to normal following
withdrawal from AAS. Some, if not all, hormone levels and tissue weights return to normal,
suggesting possible long-lasting effects of chronic AAS exposure [02044].
Dependence and withdrawal effects

During the last decade, AAS dependence and the withdrawal effects of AAS have been
subject to research. In 1989, it was proposed the anabolic steroid addiction hypothesis and
suggested that a proportion of AAS abusers are prone to developing addictive disorders,
although their hypothesis was not proven at that time. Shortly thereafter, several
investigations exploring the relationship between AAS abuse and mental disorders appeared
in the scientific literature. It was also reported that more than half of the AAS users
demonstrated symptoms consistent with a diagnosis of dependence.However, more recent
research could not confirm such a high percentage of addictive AAS users, instead reporting
that AAS dependence may exist in approximately 25 percent of users. Risk factors for
dependency on AAS involve a perception of oneself as not being big or strong enough and
long-term abuse of high doses of AAS. On the other hand, withdrawal effects seem to occur
in only a small number of AAS users [04002].
Substance abuse is a significant problem in itself and can greatly complicate the
symptomatology and treatment of comorbid psychiatric disorders. In one article, the authors
review literature concerning the use of medication to prevent relapse to substance abuse or
decrease substance use. Five different general strategies are employed for this purpose: 1)
use of a drug with pharmacological properties similar to the substance of concern (i.e.,
agonist or substitution therapy); 2) use of a receptor antagonist to block or lessen the effects
1165
of the substance of concern; 3) use of a medication that produces a conditioned aversive
reaction to the substance of concern; 4) use of a medication to reduce the reinforcing
properties of the substance of concern; and 5) use of a substance to increase the
metabolism or clearance of the substance of concern from the body. The authors review
pharmacological treatments that have been studied for the treatment of dependence on the
following types of substances: alcohol, sedative-hypnotics, opioids, stimulants, nicotine,
hallucinogens, cannabis, inhalants, anabolic steroids, phencyclidine, and designer drugs.
There are no proven, specific pharmacological treatments for anabolic steroid abuse.
However, given the wide range of symptoms associated with intoxication and chronic use,
non-specific adjunctive therapies may be helpful. Although tricyclic antidepressants have
been reported to worsen symptoms, other reports show that antidepres antidepressants may
be helpful in reducing steroid use and craving. They may, however, be treating the
depression that is often seen as part of steroid withdrawal. There are no controlled studies
comparing various antidepressants for this use. The article ends with a brief discussion of the
importance of including psychosocial and behavioral interventions in any substance abuse
treatment program [01075].
Anabolic-androgenic steroid (AAS) withdrawal is established to be an important, though

poorly known medical problem, because of AAS potency to cause physical and psychological
dependence. Thus discontinuation of high-dose, long-term anabolic steroid use, apart from
endocrine dysfunction (hypogonadotropic hypogonadism), may lead to development of
withdrawal symptoms. They include mood disorders (with suicidal depression as the most
life-threatening complication), insomnia, anorexia, decreased libido, fatigue, headache,
muscle and joint pain, and desire to take more steroids. The withdrawal from anabolic
steroids usually requires treatment. Clinical management, as with other drugs of abuse,
consists of supportive therapy and pharmacotherapy. The goals of treatment are to restore
endocrine (hypothalamic-pituitary-gonadal, HPG) function and to alleviate withdrawal
symptoms. The endocrine medications that are targeted specifically to ameliorate HPG
function include testosterone esters, human chorionic gonadotropin, synthetic analogues of
gonadotropin-releasing hormone and antiestrogens. They are indicated in the presence of
persistent clinical symptoms or/and laboratory evidence of HPG dysfunction. Other
medications, that are targeted to provide symptomatic relief include antidepressants
(especially serotonin selective re-uptake inhibitors), nonsteroidal anti-inflammatory drugs and
clonidine. Notwithstanding, it should be remembered that many of the above mentioned
drugs have their own potential for abuse or side-effects, so their use must be carefully
weighted and optimal treatment strategies for AAS withdrawal must await further clinical
research [01076].
Treating psychiatric effects of steroid use
Steroid abusers rarely seek help, and many regard the psychiatric effects as beneficial,
especially for athletes in certain sports. Illicit use is compounded by mistrust of doctors, a
perception that medical people lack knowledge about these drugs, and fear of stigma or
negative consequences that may result from drug use being exposed. Adverse effects of
steroid abuse should be managed by discontinuing the drugs-by tapering if necessary-and by
treating the symptoms. Steroid abusers typically take doses 10 to 100 times higher than
physiologic doses, in cycles lasting 6 to 14 weeks, consisting of daily oral doses plus weekly
or monthly intramuscular injections. Treatment of psychiatric effects starts with stopping the
steroids. It is reasonable to substitute testosterone enanthate (Andro-Estro) and gradually
taper the dose. The short-term use of antipsychotic medications may help treat steroid-
induced mania and psychosis. Benzodiazepines may help control panic or anxiety in the
short term. Selective serotonin reuptake inhibitors or tricyclic antidepressants should be used
if long-term treatment is needed. Depression sometimes occurs when use is stopped.
1166
Fluoxetine (Prozac®) can be used in this situation. Anabolic-androgenic steroid abuse is no
longer confined to professional athletes; therefore physicians should be aware of its signs
and symptoms in order to address adverse effects and provide treatment [07031].
Cognitive deficits
Millions of individuals worldwide have used anabolic-androgenic steroids (AAS) to gain

muscle or improve athletic performance. Recently, in vitro investigations have suggested that
supraphysiologic AAS doses cause apoptosis of neuronal cells. These findings raise the
possibility, apparently still untested, that humans using high-dose AAS might eventually
develop cognitive deficits. It was administered five cognitive tests from the computerized
CANTAB battery (Pattern Recognition Memory, Verbal Recognition Memory, Paired
Associates Learning, Choice Reaction Time, and Rapid Visual Information Processing) to 31
male AAS users and 13 non-AAS-using weightlifters age 29-55, recruited and studied in May
2012 in the UK. Testers were blinded to participants' AAS status and other historical data.
Long-term AAS users showed no significant differences from nonusers on measures of
response speed, sustained attention, and verbal memory. On visuospatial memory, however,
AAS users performed significantly more poorly than nonusers, and within the user group,
visuospatial performance showed a significant negative correlation with total lifetime AAS
dose. These were large effects: on Pattern Recognition Memory, long-term AAS users
underperformed nonusers by almost one standard deviation, based on normative population
scores, and performance on this test declined markedly with increasing lifetime AAS dose.
These results remained stable in sensitivity analyses addressing potential confounding
factors. These preliminary findings raise the ominous possibility that long-term high-dose
AAS exposure may cause cognitive deficits, notably in visuospatial memory [13144].
Effects on learning
The illicit use of anabolic androgenic steroids (AAS) has gained popularity among
adolescents in the last decade. However, although it is known that exposure to AAS impairs
cognition in adult animal models, the cognitive effects during adolescence remain
undetermined. An inhibitory avoidance task (IAT) was used to assess the effect of AAS
(17alpha-methyltestosterone; 17alpha-meT-7.5 mg/kg) in male and female periadolescent
rats. A single injection of 17alpha-meT immediately before the footshock produced significant
impairment of inhibitory avoidance learning in males but not females. Generalized anxiety,
locomotion, and risk assessment behaviors (RAB) were not affected. The results show that
exposure to a single pharmacological dose of 17alpha-meT during periadolescence exerts
sex-specific cognitive effects without affecting anxiety. Thus, disruption of the hormonal
milieu during this early developmental period might have negative impact on learning and
memory [13145].
Brain and cognition abnormalities in long-term anabolic-androgenic steroid users

Anabolic-androgenic steroid (AAS) use is associated with psychiatric symptoms including
increased aggression as well as with cognitive dysfunction. The brain effects of long-term
AAS use have not been assessed in humans. This multimodal magnetic resonance imaging
study of the brain compared 10 male weightlifters reporting long-term AAS use with 10 age-
matched weightlifters reporting no AAS exposure. Participants were administered
visuospatial memory tests and underwent neuroimaging. Brain volumetric analyses were
performed; resting-state fMRI functional connectivity (rsFC) was evaluated using a region-of-
interest analysis focused on the amygdala; and dorsal anterior cingulate cortex (dACC)
metabolites were quantified by proton magnetic resonance spectroscopy (MRS). AAS users
had significantly larger right amygdala volumes than nonusers and reduced rsFC between
right amygdala and frontal, striatal, limbic, hippocampal, and visual cortical areas. Left
amygdala volumes were slightly larger in AAS users but few group differences were detected
1167
in left amygdala rsFC. AAS users also had lower dACC scyllo-inositol levels and higher
glutamine/glutamate ratios, possibly reflecting increased glutamate turnover. On a
visuospatial cognitive task, AAS users performed more poorly than nonusers, with the
difference approaching significance. It was concluded that long-term AAS use is associated
with right amygdala enlargement and reduced right amygdala rsFC with brain areas involved
in cognitive control and spatial memory, which could contribute to the psychiatric effects and
cognitive dysfunction associated with AAS use. The MRS abnormalities that was detected
could reflect enhanced glutamate turnover and increased vulnerability to neurotoxic or
neurodegenerative processes, which could contribute to AAS-associated cognitive
dysfunction [150192].
Impairment of set-shifting and reversal learning in male rats

Anabolic-androgenic steroid (AAS) abuse is prevalent not only among elite athletes, but is
increasingly common in high school and collegiate sports. AAS are implicated in maladaptive
behaviors such as increased aggression and risk taking, which may result from impaired
cognition. Because they affect dopamine function in prefrontal cortical (PFC)-striatal circuitry,
AAS may disrupt PFC-dependent processes such as behavioral flexibility. This was the focus
of one study. Adolescent male Long-Evans rats were treated chronically with high-dose
testosterone (7.5 mg/kg in water with 13 % cyclodextrin) or vehicle sc, and tested for set-
shifting and reversal-learning. For set-shifting, rats were trained on a visual cue task (VCT),
then were shifted to a direction cue task (DCT), or vice-versa. For reversal learning, rats
were first trained on VCT and were then required to press the opposite lever. Two-cue set-
shifting introduced a novel paradigm in which rats shifted from a 1-Light Visual Task (1LVT)
to a tone cue task (TCT). Testosterone-treated rats were significantly impaired on the set-
shift from DCT to VCT compared to vehicle-treated controls (trials to criterion: vehicle 240.9
± 29.9, testosterone 388.3 ± 59.3). However, on the set-shift from VCT to DCT, testosterone
did not affect performance. During reversal-learning, testosterone significantly increased
trials to criterion (vehicle: 495.9 ± 91.8 trials, testosterone: 793.7 ± 96.7 trials). In 2-cue set-
shifting, testosterone diminished performance and the difference showed borderline
significance (vehicle: 443.2 ± 84.4 trials, testosterone: 800.4 ± 178.2 trials). The results show
that testosterone impairs behavioral flexibility and have implications for understanding
cognitive and behavioral changes in human AAS users [150197].
Decision making: probability and effort discounting in male rats

Anabolic-androgenic steroid (AAS) abuse is implicated in maladaptive behaviors such as
increased aggression and risk taking. Impaired judgment due to changes in the
mesocorticolimbic dopamine system may contribute to these behavioral changes. While AAS
are known to influence dopamine function in mesocorticolimbic circuitry, the effects on
decision making are unknown. This was the focus of the present study. Adolescent male
Long-Evans rats were treated chronically with high-dose testosterone (7.5 mg/kg) or vehicle
(13 % cyclodextrin in water), and tested for cost/benefit decision making in two discounting
paradigms. Rats chose between a small reward (1 sugar pellet) and a large discounted
reward (3 or 4 pellets). Probability discounting (PD) measures sensitivity to reward
uncertainty by decreasing the probability (100, 75, 50, 25, 0 %) of receiving the large reward
in successive blocks of each daily session. Effort discounting (ED) measures sensitivity to a
work cost by increasing the lever presses required to earn the large reward (1, 2, 5, 10, 15
presses). In PD, testosterone-treated rats selected the large/uncertain reward significantly
less than vehicle-treated controls. However, during ED, testosterone-treated rats selected
the large/high effort reward significantly more than controls. These studies show that
testosterone has divergent effects on different aspects of decision making. Specifically,
testosterone increases aversion to uncertainty but decreases sensitivity to the output of effort
for reward. These results have implications for understanding maladaptive behavioral
changes in human AAS users [150198].
1168
Mood disorders
The knowledge concerning the long-term effect of former anabolic androgenic steroids
(AAS)-use on mental health is sparse. One study aimed to investigate whether previous
AAS-use affects mental health, present sociodemographic data, sport activity and substance
abuse in a retrospective 30-year follow-up study of former elite athletes. Swedish male-elite
power sport athletes (n=683) on the top 10 national ranking lists during any of the years
1960-1979 in wrestling, Olympic lifting, powerlifting and the throwing events in track and field
answered a questionnaire. At least 20 percent of the former athletes admitted previous AAS-
use. They had more often sought professional expertise for mental problems and had used
illicit drugs compared to those not having used AAS. The AAS-users also differed in former
sport activity pattern compared to non AAS-users. It is clear that a relationship exists
between use of AAS and mental-health problems. Further studies need to be done in order
to clarify this relationship [13146].
The true prevalence of these neuropsychiatric disorders is difficult to determine, but relatively
few individuals develop these adverse effects; they are found primarily in high-dose chronic
AAS abusers. In a study of 88 AAS-using athletes and 68 non-users, the incidence of major
mood disorders (mania, hypomania, and major depression) was significantly more frequent
in steroid users compared with non-users. Additionally, these mood disorders were more
frequent in current AAS users than in abstinent AAS users [13003].
Effects on the brain’s neurotrophy

Anabolic androgenic steroids (AAS) are synthetic androgen-like compounds that are abused
in sport communities despite their adverse effects. Nerve growth factor (NGF) influences
neuronal differentiation and survival, and it also mediates higher brain functions such as
learning and memory. Changes in NGF expression have been implicated in
neurodegenerative disorders, including Alzheimer disease. Hence, we decided to study the
effect of chronic AAS exposure on brain NGF profile, NGF-dependent cholinergic function,
and related behavioral performance. Male Wistar rats were injected for 4 weeks with either
nandrolone or stanozolol at daily doses (5.0 mg/kg, s.c.) that are considered equivalent to
those abused by humans. NGF levels and NGF receptor (TrkA and p75NTR) expression
were measured in the hippocampus and in the basal forebrain. Choline acetyltransferase
expression was evaluated in basal forebrain. Spatial learning and memory were assessed
using the Morris water maze. AAS treatment caused region-specific changes in the
expression of NGF and its receptors. Both nandrolone and stanozolol increased NGF levels
in the hippocampus and reduced NGF levels in the basal forebrain, reduced p75NTR
expression in the hippocampus, and failed to affect TrkA expression in the basal forebrain.
Finally, AAS treatment reduced the expression of choline acetyltransferase in the basal
forebrain and impaired the behavioral performance in the Morris water maze. It was
concluded that the evidence that supraphysiological doses of AAS cause neurotrophic
unbalance and related behavioral disturbances raises the concern that AAS abuse in
humans may affect mechanisms that lie at the core of neuronal plasticity [13147].
Influence of age on cognitive performance, impulsivity, and aggression in men
A growing translational literature suggests that adolescent exposure to anabolic-androgenic

steroids (AASs) leads to increased aggression and impulsivity. However, little is known about
the cognitive effects of AASs among AAS users or the differences between adolescent- and
adult-onset users. One study provides a test of the effects of acute naturalistic AAS use and
age of onset (adolescent vs adult) on measures of inhibitory control, planning and attention,
and decision making. Seventy-one active adult male AAS users completed self-report
measures of impulsivity and aggression, and a subsample (11 adolescent onset vs 11 adult
1169
onset) matched on current age were administered 4 computerized tests from the Cambridge
Neuropsychological Test Automated Battery (CANTAB) and the Iowa Gambling Task.
Multiple regression analyses and a series of 2 (adolescent vs adult) × 2 (on-cycle vs off-
cycle) analyses of variance (ANOVAs) were used to examine the differential effects of age of
onset and acute drug use on cognition and behavior. Regression analyses revealed larger
on-cycle effects for adolescent users than adult users. Subsample analyses indicated that
on-cycle users performed less well on cognitive measures of inhibitory control and attention,
but not on tests of planning or decision making. Adolescent onset was associated with
greater impulsivity and more acute sensitivity to AAS effects on attention. These preliminary
findings suggest the possibility that acute AAS use is associated with some differences in
inhibitory control and impulsivity and to a lesser degree, aggression. These effects may be
more potent for those initiating AAS use in adolescence [14255].
Spontaneous subdural haematoma
Spontaneous subdural haematoma is very rare in young patients. The complications of

anabolic steroid intake in weight lifters are numerous, yet subdural haematomas have not
been reported. It was reported on two cases of spontaneous subdural haematomas in young
weight lifters. Both patients underwent surgical evacuation and made a full recovery. A
review of the literature on the complications associated with valsalva manoeuvres is also
presented including hemodynamic and intracranial changes. It was proposed that patients on
chronic anabolic steroids may have vascular changes that predispose them to bleeding
during a Valsalva manoeuvre (VM) [05051].
Cerebral oedema
The usual side effects of anabolic steroid abuse are thromboembolic, hepatic, cardiac,
reproductive and psychiatric disorders. It was reported a case of lethal cerebral oedema
associated with massive abuse of anabolic steroids in a previously healthy 21 year old man
[00061].
Randomized trial on psychiatry of supraphysiological doses of anabolic steroids
Field studies of illicit anabolic-androgenic steroid users suggest that some develop manic or
aggressive reactions to these drugs-a potential public health problem. However, controlled
laboratory evaluations of these effects remain limited. In a randomized, placebo-controlled,
crossover trial, we administered testosterone cypionate for 6 weeks in doses rising to 600
mg/wk and placebo for 6 weeks, separated by 6 weeks of no treatment, to 56 men aged 20
to 50 years. Psychiatric outcome measures included the Young Mania Rating Scale (YMRS),
the Point Subtraction Aggression Paradigm (a computerized provocation test of aggression),
the Aggression Questionnaire of Buss and Perry, the Symptom Checklist-90-R, daily diaries
of manic and depressive symptoms, and similar weekly diaries completed by a "significant
other" who knew the participant well. Testosterone treatment significantly increased manic
scores on the YMRS, manic scores on daily diaries, visual analog ratings of liking the drug
effect, and aggressive responses on the Point Subtraction Aggression Paradigm. Drug
response was highly variable: of 50 participants who received 600 mg/wk of testosterone
cypionate, 42 (84 %) exhibited minimal psychiatric effects (maximum YMRS score, <10), 6
(12 %) became mildly hypomanic (YMRS score, 10-19), and 2 (4 %) became markedly
hypomanic (YMRS score, >20). The 8 "responders" and 42 "nonresponders" did not differ
significantly on baseline demographic, psychological, laboratory, or physiological measures.
It was concluded that testosterone administration, 600 mg/wk increased ratings of manic
symptoms in normal men. This effect, however, was not uniform across individuals; most
1170
showed little psychological change, whereas a few developed prominent effects. The
mechanism of these variable reactions remains unclear [00062].
Forensic experiences of use of anabolic steroids
Medicolegally investigated deaths among 34 male users of anabolic androgenic steroids

(AAS) are described. Nine persons were victims of homicide, 11 had committed suicide, 12
deaths were judged as accidental and 2 as indeterminate. In two cases of accidental
poisoning, the levels of pharmaceuticals and illicit drugs were considered too low to be the
sole cause of death and AAS was considered part of the lethal polypharmacia. Chronic
cardiac changes were observed in 12 cases. In two cases of accidental poisonous deaths,
these changes were regarded as contributory cause of death. Homicides, suicides, and
poisonings determined accidental or indeterminate in manner were related to impulsive,
disinhibited behavior characterized by violent rages, mood swings, and/or uncontrolled drug
intake. The observations in the present study indicate an increased risk of violent death from
impulsive, aggressive behavior, or depressive symptoms associated with use of AAS. There
are also data to support earlier reports of possible lethal cardiovascular complications from
use of AAS. Furthermore, a contributing role of AAS in lethal polypharmacia is suggested.
Finally, the observations indicate that use of AAS may be the gateway of approach to abuse
of other psychotropic drugs [00063].
Effects of alcohol intake, defensive behaviors and brain opioid peptides in the rat
One study investigated whether a relationship exists between nandrolone decanoate and
voluntary ethanol intake in laboratory rats. Animals were subjected to daily subcutaneous
injections with nandrolone decanoate (15 mg/kg) during 2 weeks. One group of animals was
tested for voluntary alcohol intake 1 week after the end of the 2-week treatment period and
another group received alcohol 3 weeks after the treatment. In addition, assessment of
defensive behaviors and immunoreactivity (ir) levels of the brain opioid peptides dynorphin B
and Met-enkephalin-Arg-Phe (MEAP) were performed. The nandrolone decanoate-treated
animals were significantly more aggressive and showed lower fleeing and freeezing reaction
than the oil-treated controls. Treatment with nandrolone decanoate enhanced voluntary
alcohol intake, regardless if it was presented 1 or 3 weeks after end of the treatment period.
These animals had a decreased activity of dynorphin B-ir in the nucleus accumbens,
decreased levels of MEAP-ir in the periaqueductal gray (PAG) and higher levels of MEAP-ir
in the hypothalamus compared to controls. In line with previous studies, this suggests that
the altered dynorphin B-ir activity may promote the rewarding effects of ethanol and thereby
increasing alcohol intake, whereas MEAP-ir may be associated with the ability to control the
aggressive reaction. Abuse of nandrolone decanoate may thus constitute a risk factor for
increased alcohol consumption and defensive aggression. In human, this constellation of
behavioral symptoms is closely related to acts of crimes and violence and is often observed
among those abusing anabolic androgenic steroids [00064].
Aggression after altering anterior hypothalamic-arginine vasopressin expression
One study examined the hypothesis that exposure to anabolic-androgenic steroids (AAS)
during adolescent development predisposes hamsters to heightened levels of aggressive
behavior by influencing the anterior hypothalamic-arginine vasopressin (AH-AVP) neural
system. To test this, adolescent male hamsters (Mesocricetus auratus) were treated with
high doses of AAS, tested for offensive aggression in the absence or presence of AH-AVP
receptor antagonists, and then examined for changes in AH-AVP expression and neural
organization. AAS exposure during adolescence significantly increased aggression intensity
1171
(number of attacks and bites) and initiation (latency to the first bite). Yet, only increases in
aggression intensity were inhibited by AH-AVP receptor antagonism. Adolescent AAS-
treated hamsters showed significant increases in AH-AVP fiber density and peptide content.
However, no alterations in AH-AVP neuronal organization or mRNA expression were found.
Together, these data suggest that adolescent AAS exposure increase aggression intensity
by altering AH-AVP expression and activity, providing direct evidence for a causal role of AH-
AVP expression and function in early onset AAS-stimulated aggression [00065].
Increased vasopressin V1A receptor binding
Repeated anabolic-androgenic steroid treatment during adolescence increases hypothalamic

vasopressin and facilitates offensive aggression in male Syrian hamsters (Mesocricetus
auratus). The current study investigated whether anabolic-androgenic steroid exposure
during this developmental period influenced vasopressin V1A receptor binding activity in the
hypothalamus and several other brain areas implicated in aggressive behavior in hamsters.
To test this, adolescent male hamsters were administered anabolic steroids or sesame oil
throughout adolescence, tested for offensive aggression, and examined for differences in
vasopressin V1A receptor binding using in situ autoradiography. When compared with control
animals, aggressive, adolescent anabolic steroid-treated hamsters showed significant
increases (20-200 %) in the intensity of vasopressin V1A receptor labeling in several
aggression areas, including the ventrolateral hypothalamus, bed nucleus of the stria
terminalis, and lateral septum. However, no significant differences in vasopressin V1A
receptor labeling were found in other brain regions implicated in aggressive responding, most
notably the lateral zone from the medial preoptic area to anterior hypothalamus and the
corticomedial amygdala. These data suggest that adolescent anabolic steroid exposure may
facilitate offensive aggression by increasing vasopressin V1A receptor binding in several key
areas of the hamster brain [02048].
Liver changes due to sex hormones (anabolic steroids and oral contraceptives)
Overview
To know the actual abuse of anabolic steroids by amateur athletes in our environment as
well as actions and secondary effects resulting from such abuse analytical observational
study from May 1997 to November 1998 were gathered. Forty-three therapy courses with
anabolic steroids among 39 male athletes were studied. Diet and training were standardized
for all participants. A verification was made that the test group started from a basal state.
Duration of therapy was 6 weeks and the mean total dose was 2,928 mg. Significant
differences were found in the test group regarding basal and post-therapy values for:
transaminases, cholesterol, HDL-cholesterol, LDL-cholesterol, LH, FSH, free testosterone,
17-beta-estradiol and arm muscular section. The inclusion of testosterone in therapy
introduced a significant difference with respect to the use of synthetic anabolic agents alone,
in total testosterone and 17-beta-oestradiol, but neither with respect to free testosterone nor
arm muscular section. An 85 percent of individuals in the problem group stated to complete
two therapy courses in a year. It was concluded that the use of anabolic steroids increases
the lean muscular mass. The most relevant secondary effects included: increased
transaminase serum levels, change in the lipid profile and suppression of the hypothalamus-
pituitary gland-gonad axis [00066].
The liver is a target tissue for androgens and, therefore, men develop hepatocellular
carcinoma more frequently than women. Both benign and malignant tumors have been
1172
reported in AS users [03002].
Drugs may cause several overlapping syndromes of cholestasis, the pathophysiological

syndrome resulting from impaired bile flow. These reactions comprise approximately 17
percent of all hepatic adverse drug reactions (ADRs) and they may be severe. Causes of
“pure” (bland) cholestasis include oestrogens and anabolic steroids; rarer associations are
with antimicrobials and NSAIDs. “Cholestatic hepatitis” is a common drug reaction in which
liver injury and inflammation cause significant elevation of serum alanine aminotransferase
(ALT) as well as cholestasis. Chlorpromazine and ketoconazole are classic examples, but it
is now exemplified by amoxycillin-clavulanate and other oxy-penicillins. Chronic cholestasis
results from small bile duct injury leading to the vanishing bile duct syndrome (VBDS), a
disorder mimicking primary biliary cirrhosis, or from injury to larger bile ducts causing
secondary sclerosing cholangitis. Whilst there is increasing evidence of a genetic
predisposition to cholestatic drug reactions, there are currently no pretreatment tests to
predict drug safety. Prevention of severe reactions therefore relies on early detection of liver
injury and prompt drug withdrawal. Symptomatic management includes relief of pruritus and
correction of fat-soluble vitamin deficiency. In small cohort studies, ursodeoxycholic acid
(UDCA) arrested progressive cholestasis in two-thirds of cases, but evidence for use of
corticosteroids is anecdotal. This review considers diagnosis, pathogenesis, prevention and
management of drug-induced cholestasis, with particular reference to frequently- and newly-
described causes [03059].
AASs may occasionally cause hepatotoxicity, with consequences including peliosis hepatis
(an accumulation of blood-filled cysts in the liver), and various types of hepatic tumors.
Virtually all AAS-associated hepatotoxic effects are associated with orally active 17alpha-
alkylated AASs. The frequency of AAS-induced hepatotoxicity is likely overestimated,
however, because rhabdomyolysis from heavy workouts can increase transaminases, and
this finding may be erroneously interpreted as evidence of abnormal liver function [14426].
Testosterone is metabolized rapidly in the body; however, esterification of the 17-hydroxyl

group renders the molecule more hydrophobic. When these esters of testosterone (such as
testosterone enanthate and cypionate) are administered in an oily suspension, they are
released very slowly into the aqueous plasma because of their hydrophobicity. This extends
their duration of action. These esters are readily de-esterified to testosterone in the body.
Investigations of the structure-activity relationships have established that removal of the 19-
methyl group increases the anabolic activity; thus, 19-nortestosterone (nandrolone) is a
potent AAS and a very popular training drug that accounts for a large number of positive
tests. 7-alkyl substitutions of 19-nortestosterone molecule may further increase the anabolic
to androgenic activity. 17-alkyl substitutions render the molecule resistant to degradation;
thus 17-alkylated androgens can be administered orally. Stanozolol is a 17-alkylated
androgen that can be taken orally or by injection. Orally administered 17-alkylated androgens
are hepatotoxic. Stanozolol is also nonaromatizable. Other substitutions in the steroid A-ring
may alter the susceptibility of the steroid molecule to aromatization. A number of nonsteroidal
selective androgen modulators, which display tissue-specific activation of androgen
signaling, are in development. Although the Food and Drug Administration has not approved
these novel nonsteroidal selective androgen receptor modulators for clinical use, some of
them are already being sold illicitly on the Internet [14017].
Hepatotoxicity includes peliosis hepatis (an accumulation of blood-filled cysts in the liver),
and various types of hepatic tumors. Virtually all AAS-associated hepatotoxic effects are
associated with orally active 17-alkylated AAS. The frequency of AAS-induced hepatotoxicity
is likely overestimated, however, because rhabdomyolysis from heavy workouts can increase
transaminases, and this finding maybe erroneously interpreted as evidence of abnormal liver
1173
function [14017].
The liver is a hormone-sensitive organ, and in fact both normal liver and hepatocellular
carcinoma (HCC) tissues from male and female mammals have been shown to express
specific estrogen receptors (ERs). Experimentally, estrogens may act as liver tumor inducers
or promoters in vivo, and are involved in stimulating hepatocyte proliferation in vitro.
Moreover, anti-estrogens like tamoxifen have been shown to reduce levels of ERs and to
inhibit hepatocyte proliferation following partial hepatectomy. As regards the role of
androgens, it has also been observed that androgen receptors (ARs), specifically activated
by testosterone, are present in normal liver tissue from both males and females and that their
expression is increased in tumor tissue and in the surrounding liver of individuals with HCC.
In addition, observations from clinical and epidemiological studies have highlighted that the
long-term use of OCs and anabolic androgenic steroids (AASs) can induce benign and
malignant hepatocellular tumors. One study provided definite and quantitative evidence that
OC use was significantly, although modestly associated with FNH. The time-risk relation
gave convincing support to the existence of a real association, given that there was a direct
trend in risk with duration and an inverse trend with age at first use. Benign tumors of the
liver are often discovered incidentally in asymptomatic individuals during diagnostic imaging
or exploratory laparotomy performed for other reasons. Hemangiomas are the most common
benign liver tumors, followed in prevalence by focal nodular hyperplasia (FNH) and the rarer
condition of adenoma; their growth and development have been linked to hormonal
stimulation. Long-term use of oral contraceptives (OCs) and anabolic androgenic steroids
(AASs) can induce both benign (hemangioma, adenoma, and focal nodular hyperplasia,
FNH) and malignant (hepatocellular carcinoma, HCC) hepatocellular tumors. Hepatic
adenomas (HAs) are rare, benign neoplasms usually occurring in young women, the
development and the complications of which have been related to the strength of OCs and
the duration of their use. HA incidence has fallen since the introduction of pills containing
smaller amounts of estrogens. In recent times AASs have also been proven to be involved in
the development of hepatic adenoma. Apparently, androgen-induced HAs are relatively rare.
However, the possibility that an oral AAS can induce liver cell proliferation must be taken into
account and sportsmen taking AASs over a long period should be considered a group at risk
for developing hepatic sex FNH is a benign lesion, most commonly seen in young women,
which is thought to represent a local hyperplastic response of hepatocytes to a vascular
abnormality. Because of the female predominance and the young age at onset, a role of
female hormones has been suggested. Furthermore, a large proportion of women with FNH
(50-75%) are OC users. Liver hemangiomas (LHs) are the most common benign liver tumors
and are seen more commonly in young adult females. The female predilection and clinical
observations of LH growth under conditions of estrogenic exposure suggest a possible role
for estrogen in the pathogenesis of LHs. HA has been strongly associated with the use of
OCs; in fact, it has been calculated that about 320 newcases are diagnosed each year,
mostly attributable to OC use. Consequently, in contrast with what happens for LH and FNH,
at least for HA there is an agreement among authors about the fact that the association
between OCs and HA is strong and depends on the duration of use. Furthermore,
unresected lesions may decrease in size in young women once they stop OC use. All these
data taken together suggest that the association between HA and OC use is one of cause
and effect. HCC has become one of the most widespread tumors in the world in recent
years, representing the sixth leading cancer and the third most common cause of death from
cancer. Apart from liver cirrhosis, numerous other factors responsible for its onset have been
proposed: hepatitis infections from virus B (HBV) and C (HCV), alcohol, smoking, and
aflatoxin. However, regardless of etiology, chronic liver diseases progress at unequal rates in
the two sexes, with the major sequelae, such as cirrhosis and HCC, being more frequent in
men than in women. These epidemiological data have prompted researchers to investigate
the relationship between sex hormones and liver tumors. The human liver expresses
1174
estrogen and androgen receptors and experimentally both androgens and estrogens have
been implicated in stimulating hepatocyte proliferation and may act as liver tumor inducers or
promoters. As regards the role of estrogens in HCC, it seems that in the physiological status
of premenopausal women, in the absence of other risk factors for liver disease, they have a
somewhat protective role against the development of HCC [06074].
Athletes and nonathletes have been using anabolic-androgenic steroids (AAS) for a long
time, in an inadequate and unsurveilled manner, with the aim of improving sports
performance or for cosmetic purposes. AAS consumption is becoming more widespread and
involving younger people, and there is a trend for self-administration of higher doses and for
combining AAS with other potentially harmful drugs. Almost any subject abusing AAS will
experience adverse effects. Therefore, adverse effects from these exposures, including liver
toxicity, are expected to increase in the years to come. It was described a representative
case of intrahepatic cholestasis with the intention to discuss AAS-related liver toxicity
(including the potential therapeutic role of ursodeoxycholic acid) and to comment on several
aspects of the clinical scenario the gastroenterologist should be aware of [07061].
Cholestatic jaundice with intrahepatic cholestasis and variable degrees of hepatocellular

necrosis on liver biopsy is themost commonly reported serious pathologic abnormality of the
liver associated with AASs abuse. Rarely, case reports associate the presence of multiple,
dilated liver cysts filled with blood in the liver (peliosis hepatitis) with chronic use of AASs.
The pathogenesis of peliosis hepatis is unknown. Other pathologic abnormalities of the liver
detected in the autopsy of AAS abusers include focal nodular hyperplasia and adenomas.
Rarely, the abuse of androgenic-anabolic steroids are associated with the development of
blood-filled cysts (peliosis) involving the liver, spleen, bone marrow,l ymph nodes, and lung.
A 9-year-old bodybuilder was found dead at home; an autopsy demonstrated peliosis of the
lung with the left pleural cavity filled with blood [13003].
AAS can induce elevations in liver enzymes (alanine- and aspartate-aminotransferases), but
this effect is typically seen with orally administered 17-alkylated AAS that exhibit high first-
pass effects in the liver [04018].
Liver function disturbances and diseases due to AAS treatment in patients, as well as in
AAS-abusing athletes, have been of great concern since animal studies have clearly shown
the hazardous effects of AAS on the liver. Taking into account the results of the trials, it is
plausible that AAS can induce serious liver disorders such as subcellular changes of
hepatocytes, impaired excretion function, cholestasis, peliosis hepatis and hepatocellular
hyperplasia, and carcinomas in humans.Additionally, several case reports have associated
the occurrence of aforementioned liver disorders with the abuse of AAS in young, healthy
athletes. These disease conditions are mainly attributed to the administration of 17-[alpha]-
alkylated steroids, that is, methyltestosterone, oxymetholone, fluoxymesterone, norethandro-
lone and metandienone. Injectable testosterone cipionate and enantate preparations do not
appear to affect liver function enzymes, whereas (nor-)testosterone esters may induce
parenchymal lesions of the liver. Some researchers have proposed that the occurrence of
AAS-induced liver disease may be dependent on the liver condition before starting drug
administration. Several studies have investigated the effects on serum liver enzyme activities
in athletes. In most studies, the common liver enzymes ASAT, alanine aminotransferase
(ALAT), [gamma]-glutamyl transpeptidase (GGT), lactate dehydrogenase (LDH) and alkaline
phosphatase (AP) were studied. Monitoring of liver function enzymes during AAS
administration provided equivocal results. However, the majority of longitudinal studies
reported no changes due to AAS, although elevations of ASAT or ALAT have been observed
within several weeks of taking steroids in some studies. These elevations were attributed to
the intake of oral steroids and tended to return to baseline levels within several weeks after
1175
cessation. On the other hand, serum levels of GGT, AP and LDH remained unaffected in all
studies. It has been stated that elevations of serum aminotransferase levels may represent
muscle damage rather than hepatic dysfunction because of the close relationship to
increments of serum CK levels. They demonstrated that GGT was the most distinctive
enzyme for the detection of hepatic dysfunction in exercising volunteers. Therefore, the
authors recommended evaluation of hepatic function in AAS users to determine CK and GGT
levels in addition to ASAT and ALAT 04[002].
AAS use associated with liver injuries among Brazilian young men
The recreational use of anabolic-androgenic steroids (AAS) has reached alarming levels
among healthy people. However, several complications have been related to consumption of
these drugs, including liver disorders. Between February 2007 and May 2012 asymptomatic
bodybuilders who were ≥18 years old and reported AAS use for ≥6 months were enrolled. All
had clinical evaluations, abdominal ultrasound (AUS), and blood tests. 182 individuals were
included in the study. The median age (interquartile range) was 26.0 years (22.0-30.0) and
all were male. Elevated liver enzyme levels were observed in 38.5 percent (n=70) of AAS
users, and creatine phosphokinase was normal in 27.1 percent (n=19) of them. Hepatic
steatosis was observed by AUS in 12.1 percent of the sample. One individual had focal
nodular hyperplasia and another had hepatocellular adenoma. One case each of hepatitis B
and C virus infection was found. A diagnosis of toxic liver injury was suggested in 23 (12.6
%) AAS users without a history of alcohol or other medications/drugs consumption, or
evidence of other liver diseases. It was concluded young Brazilian recreational AAS users
presented a wide spectrum of liver injuries that included hepatotoxicity, fatty liver, and liver
neoplasm. They also presented risk factors for liver diseases such as alcohol consumption
and hepatitis B and C virus infection. The results suggest that the risk of AAS use for the liver
may be greater than the esthetic benefits, and demonstrate the importance of screening AAS
users for liver injuries [150206].
Aminotransferase elevations: hepatitis or rhabdomyolysis?
The literature clearly suggests that unsupervised use of anabolic steroids may induce
hepatocellular carcinoma, cholestatic jaundice, peliosis hepatitis, and general liver
dysfunction. Currently, numerous reports suggest that physicians should diagnostically
evaluate anabolic steroid-related hepatic damage by monitoring alanine aminotransferase
(ALT) and aspartate aminotransferase (AST). Most anabolic steroid-related hepatitis cases
are thought to arise from the oral use of C-17 alkylated androgens. Today, the use of C-17
alkylated androgens is minimal because of two primary factors: the widely reported side
effects of such androgens and reduced production of these androgens by pharmaceutical
companies. Despite the decrease of C-17 alkylated steroid use, however, many physicians
continue to report hepatic dysfunction in athletes abusing other anabolic steroids, using
elevations in ALT and AST as their primary criteria for such diagnoses. This is problematic
because physicians are apparently neglecting to consider the skeletal muscle damage that
results from resistance training in which steroid-abusing athletes engage. This concomitant
resistance training releases various types of aminotransferases into the circulation. Based on
the literature, physicians also presumably fail to use gammaglutamyltransferase (GGT)
testing, which is a more sensitive diagnostic tool for hepatic dysfunction than ALT and AST
testing. Physicians also do not seem to be paying enough attention to creatine kinase (CK)
levels, which serve as a more sensitive and specific marker for muscle damage than either
ALT or AST levels. In a report, it was concluded that reports of anabolic steroid-induced
hepatitis based solely on ALT and AST levels may be unfounded [01066].
1176
The use of anabolic steroids among competitive athletes, particularly bodybuilders, is
widespread. Numerous reports have noted "hepatic" dysfunction secondary to anabolic
steroid use based on elevated serum aminotransferase levels. The authors' objective was to
assess whether primary care physicians accurately distinguish between anabolic steroid-
induced hepatotoxicity and serum aminotransferase elevations that are secondary to acute
rhabdomyolysis resulting from intense resistance training. Surveys were sent to physicians
listed as practicing family medicine or sports medicine in the yellow pages of seven
metropolitan areas. Physicians were asked to provide a differential diagnosis for a 28-year-
old, anabolic steroid-using male bodybuilder with an abnormal serum chemistry profile. The
blood chemistries showed elevated aspartate aminotransferase (AST), alanine
aminotransferase (ALT), and creatine kinase (CK) levels, and normal gamma-
glutamyltransferase (GGT) levels. In the physician survey (n=84 responses), 56 percent
failed to mention muscle damage or muscle disease as a potential diagnosis, despite the
markedly elevated CK level of the patient. Sixty-three percent indicated liver disease as their
primary diagnosis despite normal GGT levels. Prior reports of anabolic steroid-induced
hepatotoxicity that were based on aminotransferase elevations may have overstated the role
of anabolic steroids. Correspondingly, the medical community may have been led to
emphasize anabolic steroid-induced hepatotoxicity and disregard muscle damage when
interpreting elevated aminotransferase levels. Therefore, when evaluating enzyme elevations
in patients who use anabolic steroids, physicians should consider the CK and GGT levels as
essential elements in distinguishing muscle damage from liver damage [01060].
Metabolism of anabolic steroids in the liver
Anabolic androgenic steroids are the xenobiotic substrates that are metabolized in the body
by the protective enzyme systems. Mixed function oxygenase enzymes include a group of
enzymes which play an essential role in the metabolism of a broad range of xenobiotics
including endogenous and exogenous substrates. Cytochrome P-450, a member of mixed
function oxygenase enzymes, plays an important role in oxidative metabolism of drugs and
xenobiotics entering human body. Various anabolic steroids are found either to increase or
decrease the activity of cytochrome P-450. However, effect of nandrolone decanoate, most
commonly abused anabolic steroid, on cytochrome P-450 activity is still fragmentary. In one
study, albino mice were administered intramuscular 2.5 mg of nandrolone decanoate
injection at 15 days interval. Investigation shows a significant increase of cytochrome P-450
(nmol/mg) activity in liver tissue as compared to that of kidney tissues. A tissue specific and
dose specific increase of cytochrome P-450 activity is observed. Mean cytochrome P-450 is
found highest in liver tissue on 45th day whereas the activity in kidney tissue is noticed on 90th
day of treatment. From the above observation, nandrolone decanoate can be suggested as a
potent inducer of cytochrome P-450 activity like other anabolic steroids [09061].
Enzyme elevations
Various studies have demonstrated transient elevations of liver function tests (elevated
plasma alkaline phosphates, aminotransferases, conjugated bilirubin, and plasma proteins)
with and without significant hepatic injury. The orally 17-alpha alkylated steroids have a
higher incidence of hepatotoxicity than other preparations. The mechanism of action is most
likely from a direct toxic effect due to the brief period of time between exposure and liver
damage and a dose-related effect. The most common used measures of hepatotoxicity are
aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate
dehydrogenase (LDH). Values are usually in the range of two to three times normal. These
changes often mimic the effects seen with oral contraceptives. Elevations in AST, ALT, and
gamma-glutamyl-transferase (GGT) tend to peak within 2 to 3 weeks of consumption even at
1177
relatively low doses, and will usually return to baseline within several weeks upon
discontinuation. GGT was the most sensitive enzyme to detect hepatic dysfunction.
However, physicians must be careful in evaluating serum elevations of these enzymes,
because strenuous exercise alone can cause muscle breakdown, leading to transaminase
elevations. In addition, with the exception of LDH, the enzymes can be found in other body
tissues confounding the picture even more [06031].
Cholestatic liver disease
The spectrum of drug-induced cholestasis ranges from 'bland' reversible cholestasis to

chronic forms due to the vanishing bile duct syndrome. Agents known for many years to
cause cholestasis include estrogens and anabolic steroids, chlorpromazine, erythromycin,
and the oxypenicillins; structurally similar congeners of these drugs (tamoxifen, newer
macrolides) may also cause cholestasis. Contemporary drugs linked to cholestastic liver
injury include ticlopidine, terfenadine, terbinafine, nimesulide, irbesartan, fluoroquinolones,
cholesterol-lowering 'statins,' and some herbal remedies (greater celandine, glycyrrhizin,
chaparral). Amoxillin-clavulanate, ibuprofen, and pediatric cases of the vanishing bile duct
syndrome are recent additions to a long list of drugs associated with the vanishing bile duct
syndrome. Particular human leukocyte antigen profiles have recently been identified among
those who have developed cholestasis with specific drugs (tiopronin and amoxicillin-
clavulanate), and the mechanistic relevance of these genetic associations is being explored.
The treatment of drug-induced cholestasis is largely supportive. The offending drug should
be withdrawn immediately. Cholestyramine or ursodeoxycholic acid are used to alleviate
pruritus, with rifampicin and opioid antagonists being reserved for those who fail first line
therapy. Nutritional support is essential for those with prolonged cholestasis, a subgroup who
are at risk of developing biliary cirrhosis and liver failure. Timely referral for liver transplant
assessment is crucial in these patients [01059].
Elevated level of bile can cause bile cast nephropathy, which can be seen in patients with
severe cholestatic liver disease. Stanozolol is a C17alpha-alkylation steroid derived from
dihydrotestosterone and its major adverse effect is cholestatic jaundice. It was reported two
bodybuilders who received stanozolol for 6 weeks and developed jaundice. Serum total
bilirubin was around 50 mg/dL. Liver biopsy showed intrahepatic cholestasis. In spite of fluid
and albumin therapy, serum creatinine increased and the patients experienced oliguria. Urine
sediment showed granular cast and normal erythrocyte count. Protein excretion in 24-hour
urine was less than 1000 mg in both patients. Hemodialysis was started on and renal biopsy
revealed acute tubular epithelial cell damage along with bile pigment (cast) deposition,
compatible with bile cast-related nephropathy. Serum bilirubin decreased gradually and urine
output increased. Serum creatinine was around 1.5 mg/dL in both of the patients 2 months
after discharge [150203].
In the face of increasing societal pressure to achieve bodily perfection, young men in
particular sometimes turn to anabolic steroids to help them achieve the body they want. The
health consequences of this choice are often overlooked. It was described two cases of
severe cholestatic liver disease in young men who had taken anabolic steroids with the aim
of enhancing their body image. Both patients needed a prolonged stay in hospital for
treatment resistant pruritus. The second case was associated with considerable
psychological morbidity, so much so that the patient felt he had to leave school. The agent
implicated in both the cases of severe cholestatic liver injury was methandrostenolone. This
is a weak androgen receptor agonist and has long been recognised as a cause of liver
damage. This fact seems to be well known among users of anabolic steroids as many
internet steroid forums recommend taking the drugs for no more than four weeks to avoid
going “yellow” [12025].
1178
Many different formulations and types of anabolic steroids are available to users. However, it
is the 17alpha alkylated steroids, such as methandrostenolone and methyltestosterone that
have the most capacity to be hepatotoxic – 17alpha alkylation slows down metabolism of the
steroids in the liver, thereby exposing hepatocytes and cholangiocytes to the drug for longer.
Fewer of the injectable anabolic steroids are 17alpha alkylated, so use of oral anabolic
steroids is more commonly associated with abnormal liver function. Anabolic steroids vary in
their androgenic and anabolic properties, and body builders often use several steroids with
the intent of producing differing results. Most of these drugs are sourced either illegally or via
the internet. The actual anabolic steroids used, and the true dosage, are often unknown to
the user. Anabolic steroids are freely available online and there seems to be no regulation of
the quality or quantity of drugs dispensed in the various formulations. Several “dietary
supplements” have been found to contain substantial amounts of anabolic steroids. A report
from Portugal described a case in which cardiomyopathy induced by anabolic steroids had
caused fulminant liver failure in a bodybuilder who took large doses of anabolic steroids. A
further, Canadian case report has described the simultaneous occurrence of cholestatic
jaundice, acute kidney injury, and acute pancreatitis [12025].
Steroids induce a wide range of hepatic disorders ranging from impaired excretion, cellular
hepatocyte changes, cholestasis, peliosis hepatis, and hepatocellular hyperplasia to
carcinomas. Androgen-related cholestasis has been observed in varying frequency from a
few cases to 17 percent in some studies. The cholestasis results from the reduced bile
transport and disruption of intrahepatic microfilaments. This jaundice appears to be transient
in nature and is secondary to biliary stasis in the biliary canonicals without any structural
hepatic injury. This is in contrast to the associated inflammation and necrosis seen with other
forms of hepatitis. There may also be a relationship between cholestasis and
hypercholesteremia [06031].
Cholestasis is common, but often is asymptomatic or associated with subclinical elevation of

hepatic transaminases. Hepatocellular hyperplasia and elevations of transaminases,
conjugated bilirubin, alkaline phosphatase, and lactate dehydrogenase occur. Early elevation
of transaminases without increasing gamma-glutamyl transpeptidase may represent muscle
damage; serum creatine phosphokinase should be measured. Dose-dependent jaundice is
common after several months of AAS use and usually resolves after discontinuing AAS use.
Steroid “cycling” is believed to reduce cholestasis and jaundice. Benign hepatic adenomas
and rarer hepatocellular carcinoma have been reported in association with AAS use.
Regression of adenomas after avoidance of AAS has occurred; death from carcinoma also
has occurred. Peliosis hepatis is the presence of blood-filled cavities in the liver; this has
occurred with iatrogenic AAS use and with AAS abuse. Sometimes reversible, peliosis
hepatis can cause liver failure, and the rupture of these cysts can cause fatal internal
hemorrhage [07058].
Though possession of androgenic anabolic steroids (AAS) is illegal, non-prescription use of

AAS persists. It was described two Caucasian males (aged 25 and 45 years) with cholestatic
hepatitis following ingestion of the dietary supplement Mass-Drol (“Celtic Dragon”) containing
the AAS 2alpha-17alpha-dimethyl-etiocholan-3-one,17beta-ol. Despite substantial
hyperbilirubinaemia peak gamma-glutamyl transferase (GGT) remained normal. Besides
blan' intralobular cholestasis, liver biopsy in both found deficiency of canalicular expression
of ectoenzymes as seen in ATP8B1 disease. In the older patient, bile salt export pump
marking (encoded by ABCB11) was focally diminished. It was hypothesized that AAS had
either induced inhibition of normal ATP8B1/ABCB11 expression or triggered initial episodes
of benign recurrent intrahepatic cholestasis (BRIC) type 1/or 2. On sequencing, ATP8B1 was
normal in both patients although the younger was heterozygous for the c.2093G>A mutation
1179
in ABCB11, a polymorphism previously encountered in drug-induced liver injury. Thus, AAS
marketed as dietary supplements continue to cause hepatotoxicity in the UK; underlying
mechanisms may include unmasking of genetic cholestatic syndromes [13140].
It was describde two Caucasian males (aged 25 and 45 years) with cholestatic hepatitis
following ingestion of the dietary supplement Mass-Drol (“Celtic Dragon”) containing the AAS
2alpha-17alpha-dimethyl-etiocholan-3-one,17beta-ol. Despite substantial hyperbilirubinaemia
peak gamma-glutamyl transferase (GGT) remained normal. Besides bland intralobular
cholestasis, liver biopsy in both found deficiency of canalicular expression of ectoenzymes as
seen in ATP8B1 disease. In the older patient, bile salt export pump marking (encoded by
ABCB11) was focally diminished. We hypothesized that AAS had either induced inhibition of
normal ATP8B1/ABCB11 expression or triggered initial episodes of benign recurrent
intrahepatic cholestasis (BRIC) type 1/or 2. On sequencing, ATP8B1 was normal in both
patients although the younger was heterozygous for the c.2093G>A mutation in ABCB11, a
polymorphism previously encountered in drug-induced liver injury. It was concluded that AAS
marketed as dietary supplements continue to cause hepatotoxicity in the UK; underlying
mechanisms may include unmasking of genetic cholestatic syndromes [13142].
Drug-induced-liver-injury, DILI
It was observed an increase in hepatotoxicity (drug-induced-liver-injury, DILI) reporting
related to the use of anabolic androgenic steroids (AAS) for bodybuilding. Data on 25 cases
of AAS DILI reported to the Spanish (20) and Latin-American (5) DILI Registries were
collated and compared with previously published cases. AAS DILI increased from
representing less than 1 percent of the total cases in the Spanish DILI Registry in the period
2001-2009 to 8 percent in 2010-2013. Young men (mean age 32 years), requiring
hospitalisation, hepatocellular injury and jaundice were predominating features among the
AAS cases. AAS DILI caused significantly higher bilirubin values independent of type of
damage when compared to other drug classes. Furthermore, the cholestatic AAS cases
presented significantly higher mean peak bilirubin and serum creatinine values, compared to
the hepatocellular cases. In a logistic regression model, the interaction between peak
bilirubin values and cholestatic damage was associated with the development of AAS-
induced acute kidney impairment (AKI) (OR 1.26), with 21.5 ×ULN being the best bilirubin
cut-off point for predicting AKI risk (AUCROC 0.92). No fatalities occurred. It was concluded
that illicit recreational AAS use is a growing cause of reported DILI that can lead to severe
hepatic and renal injury. AAS DILI is associated with a distinct phenotype, characterised by
considerable bilirubin elevations independent of type of damage. Although hepatocellular
injury predominates, acute kidney injury develops in cholestatic cases with pronounced
jaundice [150201].
Combination herbal and designer drugs

It was reported a case of cholestatic jaundice as a result of combination herbal and designer
supplement use. A 50-year-old Hispanic male presented to the hospital with a 1-week history
of significant painless jaundice; total bilirubin on admission was 29.4 mg/dL. He reported use
of both herbal (creatine and whey protein) and designer (Incredible Bulk and Spartan 45)
supplements concurrently for approximately 2 months. Upon admission, all supplements
were discontinued and multiple laboratory and diagnostic tests were ordered. On day 6 of his
hospital admission, a liver biopsy was performed, the results of which indicated drug-induced
hepa to toxicity. On day 9 he was discharged with prescriptions for ursodeoxycholic acid and
hydroxyzine. Three months post hospital discharge, the patient continued to be supplement-
free and bilirubin had decreased substantially. Anabolic-androgenic steroids are capable of
causing hepatotoxicity, and multiple cases reported in the literature support this. A case
report described hepato toxicity secondary to both creatine and whey protein consumption,
and several reports have described liver damage secondary to designer supplement use. To
1180
our knowledge, this is the first case to describe hepatotoxicity as a result of combination
herbal and designer supplement use. The Roussel Uclaf Causality Assessment Method
(RUCAM) score for drug-induced hepatotoxicity indicated a highly probable correlation
between the use of combination supplements and cholestatic jaundice [13141].
Non-alcoholic fatty liver disease (NAFLD)
Industrial toxin and drugs have been associated with non-alcoholic fatty liver disease
(NAFLD); in these cases, the disease has been termed toxicant-associated steatohepatitis
(TASH). One study hypothesized that the use of anabolic-androgenic steroids (AAS) could
also be a risk factor to TASH or better toxicant-associated fatty liver disease (TAFLD)
development. A case-control study including 180 non-competitive recreational male
bodybuilders from 2007 to 2009 was performed. Ninety-five had a history of intramuscular
AAS use (cases; G1) and 85 were non-users (controls; G2). They underwent a clinical
evaluation and abdominal ultrasound, and their blood levels of aminotransferases, creatine
phosphokinase (CPK), lipids, glucose and insulin were measured. TAFLD criteria: history of
AAS use >2 years; presence of hepatic steatosis on ultrasound and/or aminotransferase
alterations with normal CPK levels; exclusion of ethanol intake ≥20 g/day or use of other
drugs; and exclusion of obesity, dyslipidaemia, diabetes and other liver diseases.
Homeostasis model assessment for insulin resistance ≥3 was considered insulin resistant.
All cases were asymptomatic. Clinical and laboratorial data were similar in G1 and G2.
TAFLD criteria were observed in 13 percent of the G1 cases and 2 percent of controls had
criteria compliant with non-alcoholic fatty liver related to metabolic conditions. Odds ratio was
6.0 (95 % confidence interval 1.3 to 27.6). These results suggest that AAS could be a
possible new risk factor for TAFLD. In this type of fatty liver disease, the individuals had a
low body fat mass and they did not present insulin resistance [11066].
Peliosis
Other rare hepatic lesions include some potentially life-threatening lesions. Peliosis hepatis is
a blood-filled cyst seen in many case reports in patients taking oral androgens, and is often
correlated with more prolonged use. In the majority of these cases, the lesions were
identified incidentally, most on autopsy, and the patients were completely symptom free.
Several case reports of patients show direct mortality from internal hemorrhage or hepatic
failure secondary to these lesions. Both cholestasis and peliosis hepatis are believed to be
explained by similar processes. Oral 17-alpha alkylated androgens produce hepatocyte
hyperplasia, with enlarged hepatocytes occluding both hepatic venous return and sinusoids.
Sinusoidal dilatation at the peripheral zone of the hepatic lobule is a common finding with
anabolic steroid use [06031].
Hepatocellular adenoma and carcinoma
One of the major risk factors of developing hepatocellular adenoma (HCA) is the use of oral
contraceptives, which stimulate liver expressed estrogen and androgen receptors,
predominantly in women between 15 and 45 years of age. Accordingly, 90 percent of HCA
are diagnosed in women. Other risk factors include glycogen storage disease I and III and
treatment with anabolic steroids in patients with Fanconi’s anemia. A few reports indicate that
anabolic androgenic steroids (AAS) may also lead to the formation of HCA. Relevant
complications of HCA include hemorrhage and malignant transformation into hepatocellular
carcinoma (HCC) depending on size and beta-catenin activation. In the literature are more
reports of patients with Fanconi’s anemia, which received medical treatment with AAS and
subsequently developed HCCs. Furthermore a few cases are described from bodybuilders
1181
with AAS abuse that subsequently developed HCC. These patients require close surveillance
to detect possible malignant transformation from HCA into HCC or lesions that are at risk of
bleeding or rupture. The morphological distinction of adenoma from well-differentiated
hepatocellular carcinoma is challenging and requires elaborated imaging techniques and
histology. It was reported a 29-year old male professional bodybuilder who presented with
mid-epigastric pain at the emergency unit. Ultrasound showed a severe hepatomegaly with
multiple lesions. Contrast-enhanced ultrasound revealed a heterogeneous pattern with signs
of hepatocellular carcinoma. CT scan of the abdomen confirmed multiple hypervascular
lesions and central areas of necrosis without contrast enhancement. Subsequent diagnostics
included fine needle aspiration (FNA) of suspicious lesions and mini-laparoscopy to establish
the diagnosis of a β-catenin and testosterone-receptor positive hepatocellular carcinoma
embedded in multiple adenomas. The patient was subsequently treated by liver
transplantation and remains tumor-free 27 month after surgery [150207].
Medical literature documents isolated cases of androgen- associated hepatic tumors. Many
of these reports were in patients with known hereditary anemias being treated with long-term
androgens. Most of these tumors are benign adenomas, but early detection may lead to
prevention of life-threatening complications associated with these tumors. Hepatic adenomas
are usually found in young women taking oral contraceptives, and are still relatively
uncommon. These adenomas are hypervascular tumors with relatively thin-walled capsules [.
They rarely transform to malignant tumors, but have risks of sudden rupture and bleeding,
leading to hemoperitoneum which is a life-threatening condition. A particular problem is
differentiating adenomas from hepatocellular carcinomas by ultrasound . If the differentiation
is not obvious by clinical and histologic findings, a surgical resection is recommended.
Treatment protocols for these lesions are difficult to formulate secondary to their varied
prognosis, their difficult resection, and their unknown potential for malignancy. However,
regression of the majority of these lesions after discontinuation of the steroids raises the
question if there is truly malignant potential attributable to AAS use. Most of these lesions are
linked to usage of the orally administered 17-alpha alkylated AAS. Sources recommend
repeat ultrasound every 6 months with consideration of excision if late diagnosis is made.
Nonsurgical options should be the optimal approach because many of the tumors will regress
after discontinuation of the AAS, especially if detected early. Prompt detection of these
lesions prevents important, potentially life-threatening sequelae and possible malignant
deterioration [06031].
In a case report a 35-year-old male bodybuilder was found to have a hepatocellular

carcinoma (HCC) arising in a pre-existing hepatic adenoma following recreational anabolic
steroid use. Malignant transformation to HCC from a pre-existing hepatic adenoma confirmed
by immunohistochemical study has previously not been reported in athletes taking anabolic
steroids [08146].
Hepatocellular adenomas
Several liver disorders have been reported to be associated with AAS nabolic androgenic
steroids (AAS) consumption, namely cholestasis, peliosis hepatis, and liver tumours.
Although most of these tumours are benign, early detection is important in order to avoid the
associated risks of life threatening haemorrhages and malignant degeneration.4 These
hepatic alterations are caused almost exclusively by 17a-alkylated AAS. AAS are used illicitly
at high doses by bodybuilders. The misuse of these drugs is associated with serious adverse
effects to the liver, including cellular adenomas and adenocarcinomas. It was reporedt two
very different cases of adult male bodybuilders who developed hepatocellular adenomas
following AAS abuse. The first patient was asymptomatic but had two large liver lesions
which were detected by ultrasound studies after routine medical examination. The second
patient was admitted to our hospital with acute renal failure and ultrasound (US) studies
1182
showed mild hepatomegaly with several very close hyperecogenic nodules in liver,
concordant with adenomas at first diagnosis. In both cases the patients have evolved
favourably and the tumours have shown a tendency to regress after the withdrawal of AAS.
The cases presented here are rare but may well be suggestive of the natural course of AAS
induced hepatocellular adenomas. In conclusion, sportsmen taking AAS should be
considered as a group at risk of developing hepatic sex hormone related tumours.
Consequently, they should be carefully and periodically monitored with US studies. In any
case, despite the size of the tumours detected in these two cases, the possibility of
spontaneous tumour regression must also be taken in account [05043].
Hepatocellular necrosis.
Athletes and bodybuilders often misuse androgenic/anabolic steroids. When used in

therapeutic doses, these drugs produce clinical jaundice in just a small number of recipients.
It was presented a 26-year-old male bodybuilder who self-administered high doses of
androgenic/anabolic steroids that induced liver damage. One month before admission to the
hospital, he used testosterone enanthate (500 mg intramuscularly, twice weekly), stanozolol
(40 mg/d), and methylandrostenediol (30 mg/d by mouth, for 5 weeks). On admission, his
bilirubin level was 470 micromol/L (direct, 360 micromol/L), his aspartate aminotransferase
(AST) level was 5,870 IU/L, his alanine aminotransferase (ALT) level was 10,580 IU/L, his
alkaline phosphatase (ALP) level was 152 IU/L, his gamma-glutamyl-transpeptidase level
was 140 IU/L, his albumin level was 27.6 g/L, and his prothrombin time was 29 percent.
During the patient's prolonged hospitalization, multiple tests and liver biopsy were performed,
showing only toxic hepatic lesions. The patient was provided with supportive medical
treatment. Clinical signs and laboratory findings improved substantially 12 weeks after the
patient discontinued androgenic/anabolic steroids. The reasons for presenting this case were
the much higher values of AST and ALT levels than reported in other studies, although the
values of bilirubin and ALP were similar to those found in the literature. To our knowledge, it
is the first case of toxic hepatitis induced by androgenic/anabolic steroids with predominantly
hepatocellular necrosis instead of intrahepatic cholestasis [02035].
Case reports associate AAS abuse with hepatic enlargement, peliosis hepatis, serious
cholestatic jaundice [2,17-dimethyldihydrotestosterone(methasterone)], and hepatorenal
failure, particularly following the use of oral 17alpha-alkylated AAS (e.g.methandrostenolone,
methyltestosterone, oxymetholone, oxandrolone, and stanozolol). Typically, non-17-alkylated
anabolic steroids are not associated with hepatocellular damage. Jaundice typically resolves
several months after cessation of AAS use, and liver transplantation is not usually necessary.
Case reports also associate the use of high doses of AAS with the development of
hepatocellular necrosis. A 26-year old malebody builder reportedly used testosterone
enanthate (500 mg intramuscularly twice weekly), stanozolol (40 mg orally/day), and
methylandrostenediol (30 mg/day orally for 5 weeks). He subsequently developed evidence
of severe hepatitis with hepatic dysfunction that required a prolonged hospitalization. Peliosis
hepatis is a rare illness characterized by blood-filled spaces within the hepatic parenchyma
that typically occurs in association with a variety of chronic wasting diseases (e.g.
tuberculosis and malignancy). This condition has been associated with both the chronic use
of pharmacologic doses of AAS and the chronic, intermittent abuse of AAS by bodybuilders.
The appearance of peliosis hepatis is not related to the dose of AAS or duration of use.
Some athletes using AAS take polyunsaturated phospholipids and vitamin complex to reduce
the elevation of hepatic aminotransferases often associated with the use of AAS; however,
there are inadequate data to determine the efficacy of this combination supplement [13003].
Spontaneous hepatic rupture
1183
Anabolic androgenic steroids are commonly used at high doses by bodybuilders and athletes
to enhance physique and improve performance levels. It was reported a case of
spontaneous hepatic rupture with life-threatening haemorrhage associated with a past history
of anabolic steroid use [07062].
Fatal liver cyst rupture due to anabolic steroid use

Liver cysts are commonly found incidentally from imaging scans or at autopsy. These benign
neoplasms vary in size and represent a heterogeneous group of disorders, for which the
demographics, risk factors, apparent inciting event, clinical presentation, and outcome are
varied. Complications that can develop from a liver cyst include development of spontaneous
hemorrhage, infection, and/or obstruction. Although the etiology of liver cysts varies, fatal
rupture of a hemorrhagic liver cyst due to anabolic steroid use is a rare occurrence. In fact,
there are few reported cases in journal literature. It was reported a case of a fatal liver cyst
rupture with resultant hemoperitoneum in the presence of anabolic steroid (stanozolol) use
[150207].
Hepatitis
The incidence of drug-induced hepatic injury has been increasing as a result of more
widespread use of workout supplements containing anabolic steroids to increase muscle
mass. Synthetic androgenic steroids are shown to cause cholestatic liver injury, but the exact
mechanism of injury is not completely understood. It was presented a case of a healthy,
young, active duty Army male soldier who developed pruritis and jaundice shortly after
starting to take a body-building supplement containing anabolic steroids, and was
subsequently found to have significant biopsy proven drug-induced liver injury [150202].
Hepatotoxicity
It was observed an increase in hepatotoxicity (DILI) reporting related to the use of anabolic
androgenic steroids (AAS) for bodybuilding. Data on 25 cases of AAS DILI reported to the
Spanish (20) and Latin-American (5) DILI Registries were collated and compared with
previously published cases. AAS DILI increased from representing less than 1 percent of the
total cases in the Spanish DILI Registry in the period 2001-2009 to 8% in 2010-2013. Young
men (mean age 32 years), requiring hospitalisation, hepatocellular injury and jaundice were
predominating features among the AAS cases. AAS DILI caused significantly higher bilirubin
values independent of type of damage when compared to other drug classes. Furthermore,
the cholestatic AAS cases presented significantly higher mean peak bilirubin and serum
creatinine values, compared to the hepatocellular cases. In a logistic regression model, the
interaction between peak bilirubin values and cholestatic damage was associated with the
development of AAS-induced acute kidney impairment (AKI) (odds ratio 1.26 ) with 21.5
×ULN being the best bilirubin cut-off point for predicting AKI risk (AUCROC 0.92). No
fatalities occurred. It was concluded that illicit recreational AAS use is a growing cause of
reported DILI that can lead to severe hepatic and renal injury. AAS DILI is associated with a
distinct phenotype, characterised by considerable bilirubin elevations independent of type of
damage. Although hepatocellular injury predominates, acute kidney injury develops in
cholestatic cases with pronounced jaundice [14750].
Hypertensive encephalopathy
Hypertensive encephalopathy (HE) is one of the possible causes of posterior reversible

encephalopathy syndrome (PRES). PRES is a clinicoradiological entity. It is characterized by
1184
severe headache, seizures, visual disturbances, alteration in consciousness, nausea and
vomiting. Since the initial report, PRES is now associated with many medical conditions.
Hypertension is nearly always present in patients with PRES. The clinical presentation of
HE/PRES may be nonspecific. Magnetic resonance imaging (MRI) is the preferred imaging
method used to detect PRES. In cases of drug-induced PRES, particularly in young persons,
the diagnosis can be missed. HE/PRES in the setting of anabolic-androgenic steroids (AAS)
use has not earlier been reported. I describe a 20-year-old man who developed HE/PRES
associated with AAS used for bodybuilding. A 20-year-old man presented with severe
headache. He had been using AAS (methandrostenolone 25 mg/day and methenolone
enanthate 300 mg/week) for about 3 months for the purpose of bodybuilding. Blood pressure
had been monitored earlier and he had been normotensive. There was no history of
headache or other diseases. He took no medication besides AAS. The headache was
followed by nausea, vomiting, visual disturbances and confusion. On presentation in the
emergency department he was sleepy, but arousable. General physical and neurologic
examination was normal. Fundoscopic examination was normal. He had a blood pressure of
190/100 mmHg. Plasma testosterone was very low 1.2 and 1.7 nmol/L (normal range 8-
30 nmol/L) indicating exogenous androgen use. Computer tomography/angiography of the
brain was normal. Repeated blood pressure monitoring displayed marked hypertension. The
blood pressure was aggressively treated with intravenous drugs. AAS use was stopped.
Extensive investigations were ordered to exclude cerebral infection/inflammation, ischemic
stroke, vasculitis and cerebral venous thrombosis. An electroencephalogram showed diffuse
slowing indicating an encephalopathy. Cerebrospinal fluid examination was normal apart
from a high pressure of 35 mmH2O. A brain MRI displayed an increased signal in the
cortex/white matter on images throughout the parietal, occipital, and frontal lobes. No
contrast enhancement was seen and diffusion weighted imaging showed no signs of
ischemia. The MRI findings were consistent with posterior reversible encephalopathy
syndrome due to severe acute hypertension. Repeated cerebral angiographies displayed no
signs of vasospasm. All serological tests were normal. Oral antihypertensive treatment
continued. Other secondary causes of hypertension were excluded. The patient was
discharged after 5 weeks. An MRI after 3 months displayed marked regression of the
abnormalities. At follow-up after 6 months and 1 year he felt well. His blood pressure
remained normal after discontinuation of AAS/antihypertensives. The case study highlights a
patient with HE/PRES in the setting of AAS use. The patient had no earlier known
hypertension and he recovered completely after discontinuation of AAS. Consequently,
HE/PRES induced by AAS was the only rational cause of HE/PRES in this case. Abuse of
AAS is estimated to be common both in athletes and in nonathletes. AAS abuse in
sportsmen has been associated with direct cardiovascular effects such as myocardial
changes and indirect cardiovascular effects such as abnormal plasma lipoprotein, alterations
in blood pressure, cardiac arrhythmia and myocardial infarction. The connection between
AAS abuse and blood pressure is not clear. Some studies have not observed any link. An
association between AAS abuse and hypertension has been found in other studies. When a
link is observed the underlying mechanism is likely to be the renal retention of sodium from
AAS. Blood pressure response to AAS abuse normally displays a dose-response relation.
Very high blood pressures (as high as 195/110 mmHg) have been noted in relation to the
use of AAS in fit sportspersons with no other identifiable cause. The effects of AAS abuse on
blood pressure can persist for long periods. Interestingly, in young and healthy persons
HE/PRES may occur at blood pressure levels as low as 160/100 mmHg, as in this case.
HE/PRES is usually reversible through control of blood pressure or removal of the underlying
cause. HE/PRES can occur in the setting of AAS use. Patients on AAS should have regular
monitoring of blood pressure. AAS use is probably common in certain groups. Consequently,
in young people presenting with hypertension and, as might be the case, a resulting PRES,
AAS should be considered a possible underlying cause [150204].
1185
There was one case reported of hypertensive encephalopathy associated with anabolic-
androgenic steroids used for bodybuilding [14751].
Nonobstructive sinusoidal dilatation of the liver
Sinusoidal dilatation found in the absence of an impaired sinusoidal blood outflow has been
so far of unclear significance. Sinusoidal dilatation may actually be a nonspecific feature of
impaired portal venous blood inflow, whatever the cause, or a feature of severe systemic
inflammatory reaction syndrome, whatever the cause. Sinusoidal dilatation is mainly located
in the centrilobular area even in the absence of an outflow block. A predominantly periportal
location is specifically found in oral contraceptive users, associated with an inflammatory
condition. There is strong evidence for the association of sinusoidal dilatation and oxaliplatin-
based chemotherapy but not for estroprogestative steroids or thiopurine derivatives.
Exposure to anabolic androgen steroids appears to cause sinusoidal changes different from
a mere sinusoidal dilatation. There is thus evidence of activation of the interleukin-6 and
vascular endothelial growth factor pathways in sinusoidal dilatation, but the mechanisms
linking the activation of these pathways with the microvascular changes must be identified
[150205].
Hepatoprotective effects of silymarin in androgenic-anabolic steroid-induced liver

damage
The use and abuse of anabolic-androgenic steroids (AAS) commonly induces liver damage.
The study included 40 male Wistar rats, divided into 4 groups of 10 rats each. Animals in the
first experimental group (M), were subjected to progressive systematic forced swimming test,
5 days a week, during 8 weeks. Animals in this group were treated with AAS methandienone,
2 mg/kg BW/day, per os, before swimming, 5 d/w for 8 weeks. After swimming, animals were
given three times more food than the laboratory animals of the same age and kind. Animals
in the second group (M+S), were subjected to progressive forced swimming test, 5 d/w 8
weeks. Animals in this group were treated with methandienone equally as the experimental
group M and received the same amount of food. Apart from that, they received silymarin 20
mg/kg BW/day. Animals in the third group (K), represented the control group, which was
neither subjected to swimming test, nor treated with methandienone or silymarin. Animals in
this group received the same amount of food as animals in groups M and M+S. Animals in
the fourth group (C), also represented a control. This group was not exercised nor treated,
and animals received a standard amount of food for laboratory animals of this kind and age.
Quantitative analysis of obtained hemataxylin-eosin, periodic acid shift and
enzymohistochemical preparations was processed using Digital Image Analysis System:
Microimage 3.0. It was established that processes in the nuclei of animals in groups M and K
were significantly more intensive in relation to groups M+S and C. The investigation of
glycogen showed significantly higher density in the cells of groups M and M+S in comparison
to groups K and C. Also, there was a significant difference between groups M+S and M.
Density of enzyme activity of glutamate dehydrogenase in hepatocytes of animals in the
group M+S was significantly higher in relation to the remaining three groups. A statistically
significant difference was not found in enzyme activity of succinate dehydrogenase and
lactate dehydrogenase. In cell nuclei of animals in the experimental group M, in the absence
of silymarin effect, methandienone causes damages which induce regenerative processes
and in this way increase high intensity activity. Silymarin significantly increases the glycogen
density in hepatocytes. Increased activities of GDH are attributed to cell vitality. The present
results thus show hepatoprotective effects of silymarin in androgenic-anabolic steroid
induced liver damage [03067].
1186
Anabolic steroids and reproductive system and male infertility
There is no doubt that lifestyle factors can be detrimental to fertility. The aim of one pilot
study was to identify initial prevalence rates for behaviour-related fertility disorders in a
clinical sample of couples wanting a child. Between February and August 2010, all patients
coming for the first time to Heidelberg University's Women's Hospital for consultation on
involuntary childlessness were asked to fill out a questionnaire designed by the authors of
this article. The questionnaire was based on a review of the relevant literature, with special
reference to the latest research findings on behaviour detrimental to fertility. Of the 156
couples addressed, 110 women and 100 men took part in the study. For behaviour-related
infertility, 9 percent of the women and 3 percent of the men in our sample were classified on
the basis of BMI <18.5, sexual disorders, or abuse of anabolic steroids. If it was included
smokers, these figures increase: 11 percent female smokers and 18 percent male smokers.
A further 19 percent of the women practised sport to an excessive degree; and 26 percent of
the women and 53 percent of the men had a BMI ≥25. The prevalence of behaviour-related
fertility disorders should not be underestimated. For the prevention of behaviour-related
fertility disorders, it is important to inform the population about lifestyle-mediated fertility risks
[12144].
It was reviewed the incidence of male infertility secondary to intake of anabolic products and
our experience and outcomes with treatment. There is a variety of such substances
(testosterone, nandrolone, stanozolol, etc.) in their intake may be unique or combinations,
both orally and parenterally. Comparisons between patients and case series are difficult
because of the hiding of this practice and various consumption practices and doses
employed. Most of the patients recover normal spermatogenesis does by stopping intake of
anabolic substances. The period of time until recovery is 6 months. Patients not recovering
after six months were given tamoxifen 20 mg/24-hour, if having a normal or inhibited
hypothalamus-hypophysis axis. Duration of abuse, doses, and anarchical consumption made
response to treatment with antiestrogen drugs or gonadotropins unpredictable in patients not
responding to conservative treatment [05059].
One-third of infertile couples may have a male factor present. Illicit drug use can be an
important cause of male factor infertility and includes use of anabolic-androgenic steroids,
marijuana, opioid narcotics, cocaine, and methamphetamines. The use of these illicit drugs is
common in the United States, with a yearly prevalence rate for any drug consistently higher
in males compared with females. The aim of one study was to provide a review of recent
literature on the prevalence and effects of illicit drug use on male fertility and to aid health
professionals when counseling infertile men whose social history suggests illicit drug use.
Anabolic-androgenic steroids, marijuana, cocaine, methamphetamines, and opioid narcotics
all negatively impact male fertility, and adverse effects have been reported on the
hypothalamic-pituitary-testicular axis, sperm function, and testicular structure. The use of
illicit drugs is prevalent in our society and likely adversely impacting the fertility of men who
abuse drugs [12145].
AAS abuse inhibits gonadotropins secretion, endogenous testosterone production and

spermatogenesis. AAS-induced sperm alterations include oligozoospermia, azoospermia,
decreased sperm motility, and abnormal sperm morphology. These often result in decreased
fertility in males. After AAS withdrawal the inhibited HPT axis functions will restore, but not
always, within several months. In bodybuilders with a history of long-term AAS abuse, at
least 6/12 months are needed for a full recovery of testicular functions. Clomiphene may
1187
successfully restore AAS-induced male HPG dysfunction and gonadotropins have been used
to recover from AAS-linked azoospermia [12094].
The negative impact of AAS abuse on male fertility is well known by urologists. The
secondary hypogonadotropic hypogonadism is often highlighted when AAS and fertility are
being discussed. On the other hand, the patterns of use, mechanisms of action and direct
effects over the testicle are usually overseen. One study reviews the vast formal and
"underground" culture of AAS, as well as their overall implications. Specific considerations
about their impact on the male reproductive system are made, with special attention to the
recent data on direct damage to the testicle. This kind of overview is absolutely unique,
offering a distinguished set of information to the day-by-day urologists. For several decades,
testosterone and its synthetic derivatives have been used with anabolic and androgenic
purposes. Initially, these substances were restricted to professional bodybuilders, becoming
gradually more popular among recreational power athletes. Currently, as many as 3 million
anabolic-androgenic steroids (AAS) users have been reported in the United States, and
considering its increasing prevalence, it has become an issue of major concern. Infertility is
defined as the failure to achieve a successful pregnancy after 12 months or more of regular
unprotected intercourse, with male factor being present in up to 50 percent of all infertile
couples. Several conditions may be related to male infertility. Substance abuse, including
AAS, is commonly associated to transient or persistent impairment on male reproductive
function, through different pathways. Herein, a brief overview on AAS, specially oriented to
urologists, is offered. Steroids biochemistry, patterns of use, physiological and clinical issues
are enlightened. A further review about fertility outcomes among male AAS abusers was also
presented, including the classic reports on transient axial inhibition, and the more recent
experimental reports on structural and genetic sperm damage [11571].
Infertility is generally defined as the inability to conceive a pregnancy or the failure to do so

within a reasonable period (typically 12 months). Approximately 85 percent of couples
conceive a first pregnancy within 12 months. The prevalence of infertility has increased over
the past 10 years, with approximately 10 million affected couples in the United States.
Roughly 40-50 percent of infertility is either due to, or is contributed by, a male factor. Given
how common the condition is, men and their partners are understandably concerned and
interested in identifying and eliminating risk factors for male infertility. One article reviewed
the available literature on various aspects of male infertility related to athletic pursuits. These
include the effects of exercise on semen parameters, hormonal axes, and testicular health.
Due to the prevalence and particular relevance of anabolic steroid use by athletes and the
impact of steroid use on fertility, this topic was also reviewed [10448].
In a case report of primary gonadal failure due to the chronic abuse of anabolic steroids used
for bodybuilding the clinical symptoms, levels of serum T, FSH, and LH were given. It was a
case of initially secondary gonadal failure resulting from anabolic steroid use with
subsequent primary gonadal failure and infertility. It adds to the current literature and
illustrates that the side effects of anabolic steroids can be prolonged and irreversible [11324].
Long-term side effects of high doses of anabolic androgenic steroids self-administration were
evaluated in this study. Twenty male bodybuilders, voluntarily starting steroid self-
administration, were followed every 6 months over 2 years. Physical examination,
haematological, metabolic and endocrine variables, semen analysis, hepatic and prostate
ultrasound and echocardiographic evaluations were performed. LH values were suppressed
at 18 and 24 months and FSH at 6, 12, 18, and 24 months and SHBG values significantly
lowered at 12, 18, and 24 months. A significant decrease in spermatozoa count and fertility
index occurred. HDL-cholesterol was reduced at 18 and 24 months and Apo A-1 at 12, 18,
and 24 months. The most important long-term adverse effects were lower fertility and the
1188
impairment of lipid profile associated with an increased cardiovascular risk [07071].
Although the same androgen receptor mediates both the anabolic and androgenic effects of
steroids, the specific structural characteristics of individual AASs determine the balance
between the anabolic and androgenic effects. Masculinization results from the presence of
high doses of AASs in women. The aromatase cytochrome P450 (CYP19) enzyme complex
converts testosterone to estradiol, which binds to the estrogen receptor. Although this
conversion usually accounts for a small percentage of testosterone biotransformation at
physiologic doses, excess amounts of testosterone increase the formation of estradiol.
Gynecomastia is a consequence of excessive estradiol concentrations in chronic male AAS
abusers that result from the peripheral conversion of excess testosterone to estradiol. Drug
sused by male AAS abusers to prevent the feminizing effects of estradiol include the use of
aromatase inhibitors (e.g. anastrozole, and aminoglutethimide) and drugs that block estrogen
receptor (clomiphene and tamoxifen) [13003].
All AASs suppress gonadotropin secretion and therefore suppress endogenous testicular
function. Adverse effects of AASs on the male reproductive system include reduced hormone
levels (gonadotrophic hormones, endogenous testosterone, and sex-hormone-binding
globulin), impotence (erectile dysfunction), alterations in sperm morphology, and reductions
in sperm count, spermmotility, and the size of the testicles. These changes are not
responsive to the administration of human chorionic gonadotropin, and resolution of the
alterations in sperm count occurs several months after cessation of AAS use with some
individuals requiring up to 30 weeks of abstinence. Although chronic AAS abuse produces
hypogonadism, decreased serum testosterone, and impaired spermatogenesis, these
changes are reversible within a few months to 1 year following cessation of AAS use.
Abnormalities of the female reproductive system following chronic AAS abuse include
menstrual irregularity, deep voice, increased libido, and increased clitoris size along with
elevated sex-hormone-binding globulin. Anabolic-androgenic steroids are contraindicated in
pregnant women with potential effects including masculinization of the female fetus, clitoral
hypertrophy of the female fetus, decreased birth weight, and premature bone maturation
[13003].
Data from a recent retrospective study found that 21 percent of 382 hypogonadal patients
seeking testosterone replacement therapy (TRT) had earlier AAS exposure [14427].
For men who have previously used AAS, a unique condition known as anabolic steroid-
induced hypogonadism (ASIH) becomes a real concern. Clearly described in 1990 by Jarow
and Lipshultz, ASIH has recently been identified as a potentially underrecognized cause of
hypogonadism in young men. Symptomatic hypogonadism is a potential consequence of
AAS use and may depend on dose, duration, and type of AAS used. Complete endocrine
and metabolic assessment should be conducted. Management strategies for anabolic
steroid-associated hypogonadism (ASIH) include judicious use of testosterone replacement
therapy, hCG, and selective estrogen receptor modulators. Thus, although complications of
AAS use are variable and patient specific, they can be successfully managed. Treatment of
ASIH depends on the type and duration of AAS use. Specific details regarding a patient's
AAS cycle are important in medical management [14427].
Recently, a retrospective database review of 6,033 hypogonadal men found ASIH to be a

common cause of profound hypogonadism (T < 50 ng/dL). Even more surprising, it was
found that as many as one out of five men who were being treated for symptomatic
hypogonadism had previously used AAS. These important new data identify ASIH as a
concerning and preventable cause of hypogonadism, especially in younger hypogonadal
men. Despite being the only class of US Drug Enforcement Administration scheduled drugs
1189
for which the Diagnostic and Statistical Manual of Mental Disorders does not recognize a
dependence syndrome, expert psychiatrists now appreciate AAS dependence as a valid
diagnostic entity. The primary goal of counseling patients with ASIH should be to deter future
and potentially harmful use of nonprescribed AAS. Understanding the patient's motivations
for use can help to deliver the most effective counseling and may identify treatable
pathologies such as primary hypogonadism, delayed puberty, or psychopathology, all of
which could be safely addressed through medically supervised treatment strategies.
Symptomatic hypogonadism is common after completion of an AAS cycle. After a complete
endocrine and metabolic assessment, management strategies for hypogonadism include use
of transient TRT, SERMs, and hCG. Responses may be quite variable, depending on
specific characteristics of AAS use. Recognition of the specific details of the user's AAS cycle
is important for their subsequent medical management. Use of AIs may be more problematic
in light of recent evidence suggesting that their use may lead to potential sexual side effects
[14427].
The adverse endocrine effects are gender specific. In males, this endocrine suppression can
lead to hypogonadotrophic hypogonadism, testicular atrophy, reduced sperm count,
decreased sperm motility, abnormal sperm morphology, infertility, and changes in libido.
These effects generally worsen with larger doses of AAS taken for longer periods of time.
This AAS-induced hypogonadal state is transient and reversible after discontinuation of AAS.
However, restoration of hypothalamic-pituitary homeostasis, endogenous testosterone, and
spermatogenesis takes between 3 and 12 months, and AAS-induced hypogonadism may
require treatment with human chorionic gonadotrophin [04018].
Abuse of anabolic androgenic steroids (AASs) may be an aetiological factor in male infertility
among recreational power athletes. They try to avoid AAS-induced deterioration in
spermatogenesis by combining doses of human chorionic gonadotrophin (HCG) and/or
antiestrogens with their AAS abuse. Eighteen healthy male power athletes using massive
doses of AASs were recruited for the study. Semen samples were collected during AAS
abuse and 1.5 and 6 months after cessation of the abuse. They were also asked about their
reproductive activity six years after the study. At the end of the AAS cycle, the sperm count
was 33 + 49 x 106/mL, and only one subject had azoospermia. At 1.5 months after cessation
of the AAS cycles, the mean sperm concentration was 30 + 42 x 106/mL, and after six
months 77 + 70 x 106/ml. There were significant differences between the sample drawn six
months after cessation of AAS abuse and both samples drawn during and 1.5 months after
the abuse. There was a significant positive correlation between HCG dose during the cycle
and the relative amount of morphologically abnormal spermatozoa. The concomitant abuse
of HCG and supraphysiological AAS thus dose causea a transient impairment on semen
quality in males, although spermatogenesis is maintained with this regimen despite
prolonged abuse of massive doses of AAS [04061].
Since AAS are derived from testosterone they exert important effects on the sex hormones
and the reproductive system. They will suppress the hypothalamic-pituitary-gonadal axis,
which acts as a feedback system. Consequently, exogenous administration of AAS will
disturb the endogenous production of testosterone and gonadotrophins (luteinising hormone
[LH] and follicle-stimulating hormone [FSH]). In males, suppression of gonadotropin
production induces testicular atrophy and reduces semen production and quality. Studies
have shown that the use of AAS may dramatically lower serum gonadotrophin
concentrations; a decline can be observed within 24 hours. Serum testosterone levels will
also decrease, except when exogenous testosterone is administered in amounts usually
practised by strength athletes. However, it was proven that a close dose-response
relationship exists between the administered dose of testosterone and serum levels of
testosterone. Administration of weekly doses of intramuscular testosterone > 300 mg
1190
increase serum levels of testosterone and free testosterone, whereas weekly doses of 25 or
50 mg resulted in lower serum levels of testosterone and free testosterone. Previously, it has
been demonstrated that the administration of high doses of testosterone in polydrug abusers
will induce supraphysiological levels of serum total and free testosterone and estradiol.
Serum concentrations of androstenedione and dihydrotestosterone closely follow the same
pattern. The high serum levels of estradiol, androstenedione and dihydrotestosterone can be
explained by peripheral conversion of AAS [04002].
The administration of supratherapeutic doses of AAS will reduce the quantity and quality of
semen production in male athletes and may lead to infertility within months. This is in
agreement with the results of research into the use of androgens for contraceptive purposes
in males, although this method of contraception is still experimental and not yet reliable.
Once the steroid intake is stopped, the exact time needed for full recovery of reproductive
function is not known and may vary depending on the doses taken and duration of AAS
abuse. After long-term (6 months) polydrug administration, full recovery may take at least 4-5
months. However, in some individuals complete restoration of normal reproductive function
may take more than a year. Long-term administration of high doses of AAS may provoke
hypogonadotrophic hypogonadism, characterised by testicular atrophy, oligo- or
azoospermia, low serum concentrations of LH and FSH, and of endogenous testosterone
and precursors. A number of athletes try to prevent or reverse this disturbance of the
reproductive system by using human chorionic gonadotrophin or clomifene together with the
AAS or immediately after the end of the AAS course, although scientific rationale for such
regimens is not available. Treatment of hypogonadotrophic hypogonadism with human
chorionic gonadotrophin resulted in a testicular responsiveness comparable with that in
prepubertal boys. On the other hand, in recent case studies it has been reported that
clomifene may successfully restore AAS-induced pituitary-gonadal dysfunction and treatment
with both human chorionic gonadotrophin (hCG) and human menopausal gonadotrophins
(hMG) reversed persistent azoospermia due to the misuse of AAS. Moreover, Karila and
coworkers demonstrated that in AAS abusers spermatogenesis can be maintained by using
hCG during an AAS course, although sperm quality may be impaired [04002].
In men, a dose dependent decrease of LH and FSH is documented that may lead to reduced
sperm count and abnormal sperm morphology. These findings are reversible in about 4
months after cessation of AS. In children and adolescents, AS use may lead to premature
closure of epiphyses and therefore stunted growth [03002].
Androgens play a crucial role in the development of male reproductive organs such as the
epididymis, vas deferens, seminal vesicle, prostate and the penis. Furthermore, androgens
are needed for puberty, male fertility and male sexual function. High levels of intratesticular
testosterone, secreted by the leydig cells, are necessary for spermatogenesis. Intratesticular
testosterone is mainly bound to androgen binding protein and secreted into the seminiferous
tubules. Inside the sertoli cells, testosterone is selectively bound to the androgen receptor
and activation of the receptor will result in initiation and maintenance of the spermatogenic
process and inhibition of germ cell apoptosis. The androgen receptor is found in all male
reproductive organs and can be stimulated by either testosterone or its more potential
metabolite dihydrotestosterone. Severe defects of the androgen receptor may result in
abnormal male sexual development. More subtle modulations can be a potential cause of
male infertility. Treatment of an infertile man with testosterone does improve
spermatogenesis, since exogenous administrated testosterone and its metabolite estrogen
will suppress both GnRH production by the hypothalamus and Luteinising hormone
production by the pituitary gland and subsequently suppress testicular testosterone
production. Also, high levels of testosterone are needed inside the testis and this can never
be accomplished by oral or parenteral administration of androgens. Suppression of
1191
testosterone production by the leydig cells will result in a deficient spermatogenesis, as can
be seen in men taking anabolic-androgenic steroids. Suppression of spermatogenesis by
testosterone administration is also the basis for the development of a male contraceptive.
During cytotoxic treatment or irradiation suppression of intratesticular testosterone production
cells may prevent irreversible damage to the spermotogonial stem cells [03055].
It was reported a case of symptomatic hypogonadism induced by the abuse of multiple

steroid preparations that was subsequently reversed by clomiphene in a case report.
Clomiphene citrate, 100-mg challenge for 5 days, was followed by treatment at same dose
for 2 months. Clinical symptoms, and androgen declined in aging male questionnaire, total T,
FSH, LH with reversal of symptoms, normalization of T levels with LH surge, restoration of
pituitary-gonadal axis. It was concluded that clomiphene citrate is used typically in helping to
restore fertility in females. This represents the first case report of the successful use of
clomiphene to restore T levels and the pituitary-gonadal axis in a male patient. The axis was
previously shut off with multiple anabolic steroid abuse [03056].
To document for the first time the successful treatment using human chorionic gonadotropin
(hCG) and human menopausal gonadotropins (hMG) of anabolic steroid-induced
azoospermia that was persistent despite 1 year of cessation from steroid use it was reported
a married couple with primary subfertility secondary to azoospermia and male
hypogonadotropic hypogonadism. The husband was a bodybuilder who admitted to have
used the anabolic steroids testosterone cypionate, methandrostenolone, oxandrolone,
testosterone propionate, oxymetholone, nandrolone decanoate, and methenolone enanthate.
Twice-weekly injections of 10,000 IU of hCG (Profasi; Serono) and daily injections of 75 IU of
hMG (Humegon; Organon) was given for 3 months. Semen analyses returned to normal after
3 months of treatment. The couple conceived spontaneously 7 months later. It was
concluded that steroid-induced azoospermia that is persistent after cessation of steroid use
can be treated successfully with hCG and hMG [03057].
Since in men androgen levels decrease with age and result in symptoms of hypogonadism,
the use of testosterone supplementation to treat symptoms resulting from hypogonadism is
increasing. One potential complication of this treatment is the possibility of an increased risk
of prostate cancer. Although most authorities agree that androgen is involved in the
exacerbation of existing carcinoma of the prostate, the action of androgens on the
carcinogenic process is not well understood. Attempts to demonstrate a correlation between
hormone levels and prostate cancer have yielded inconsistent results. No clear evidence
exists that androgen supplementation to restore physiologic levels produces any deleterious
effects on the prostate. It is highly doubtful that when testosterone therapy is administered to
middle-aged or older men, any potential prostate cancer promotion effect will be clinically
manifested in the absence of already established cancer. It is, however, imperative that
existing or developing prostate cancer be ruled out before initiation and during androgen
replacement therapy. As with any therapeutic regimen, careful monitoring of the patient
receiving treatment is recommended and constitutes good medical care.Testosterone levels
decrease as men age. It is estimated that roughly one out of two men more than 50 years of
age has free testosterone levels that are below those considered normal for younger men.
Testicular atrophy and decline in Leydig cell number lead to this decrease in serum
testosterone. Decline in androgen levels and resulting symptoms have been referred to as
the male climacteric, andropause, and PADAM (partial androgen deficiency of the aging
male). The symptoms of decreasing testosterone in the aging male are certainly less
dramatic and occur over a longer time period than those of menopause in the female. Men
experience a continuous, slow decline in testosterone beginning in approximately the fourth
decade of life. The indication for testosterone therapy is hypogonadism resulting from
deficiency of testosterone. Hypogonadism can be primary (testicular failure or resulting from
1192
testicular trauma), secondary (pituitary or hypothalamic failure), or sometimes both. Common
causes of hypogonadism are aging, obesity; severe systemic illness such as AIDS, uremia,
and hepatic cirrhosis; and medications such as ketoconazole, glucocorticoids,
spironolactone, lactone, cimetidine, phenytoin, and flutamide. Potential liabilities of androgen
replacement in the hypogonadal male include polycythemia, gynecomastia, azoospermia,
exacerbation of sleep apnea, and prostatic disease. Possible prostatic complications include
increase in lower urinary tract symptomatology caused by benign prostatic hyperplasia (BPH)
or initiation or promotion of prostate carcinoma. The effectiveness of testosterone therapy in
ameliorating the signs and symptoms of hypogonadism in the aging male will lead to
increased implementation of this therapy. There have been multiple attempts to correlate the
administration of testosterone to prostate carcinogenesis, but the studies have failed to
produce consistent results. Similarly, the studies which attempt to correlate increased
testosterone with increased PSA levels have been unconvincing. Nor have the studies been
able to link DHT, the more active metabolite of testosterone, to the development of
carcinoma. The prevailing opinion is that restoring testosterone levels to physiologic levels
offers no increased risk of carcinoma. However, there is little doubt that the studies show a
deleterious effect on existing clinical carcinoma of the prostate. With the elimination of the
presence of an existing carcinoma of the prostate, through physical examination and
laboratory studies, before initiation of testosterone therapy, and the continuous monitoring of
the patient throughout therapy, testosterone therapy will prove safe with regard to prostate
health [03058].
Dose-dependent effects of testosterone on sexual function
The dose-response relationships are central to the issue of testosterone replacement

therapy, within the context of both hypogonadal men and older men with declining
testosterone levels. Dosages are also important when considering anabolic applications of
testosterone for treating sarcopenia associated with chronic illness. The critical issue is
whether increases in muscle mass and function can be achieved with dosages that will not
adversely affect lipid profile, cardiovascular risk, and the prostate. The dose-response
relationship and mechanisms of androgen action on the muscle for controlling it are being
investigated [00069].
Pituitary side effects of anabolic steroids
Pituitary function is influenced by several drugs, including anti-depressant, opioids,

glucocorticoids, chemotherapeutic agents, immunomodulators and the newly developed
tyrosine kinase inhibitors. In most instances, treatment with these drugs negatively affects
pituitary function, but in rare cases an activation of specific hypothalamic-pituitary axes may
be observed. Several of the observed pituitary side effects are reversible after drug
withdrawal, but pituitary function deficiency may persist long-term. In addition to the well
known drugs, recent evidence shows that also non-steroidal anti-inflammatory drugs impair
gonadal axis at pituitary level, while antipsychotic phenothiazines alter TSH response to TRH
and TSH levels. Atypical antipsychotics may decrease TRH-stimulated TSH. Tricyclic
antidepressant drugs interfere with the hypothalamo-pituitary-thyroid axis by decreasing TSH
response to TRH. Anabolic-androgenic steroids, marijuana, cocaine, methamphetamines,
and opioid narcotics negatively impact fertility, also acting at hypothalamic-pituitary level.
Many of the drugs administered routinely in the intensive care unit significantly impact the
hypothalamic-pituitary axis. Therefore, an increased awareness on pituitary side effects of
drugs commonly used in clinical practice is necessary in order to rule out possible
pharmacological interference when assessing patients with pituitary deficiencies [14754].
1193
AAS abuse has been proven to have the potential to dramatically disturb the hypothalamic-
pituitary-gonadal axis, although other endocrine systems have also been shown to be
susceptible for these substances. Deregulation of glucose metabolism secondary to insulin
resistance and impairment of thyroid function have been observed [04002].
Anabolic androgenic steroids (AAS) are the favoured appearance and performance
enhancing drugs (APED) used in competitive athletics, by body-builders and in recreational
sports. Many AAS, often obtained from the internet and dubious sources, have not
undergone proper testing and are consumed at extremely high doses and in irrational
combinations, also with other drugs. Controlled clinical trials investigating undesired side-
effects of AAS are lacking since ethical restrictions prevent exposing volunteers to potentially
toxic regimens, thus making it difficult to establish a causal relationship between AAS abuse
and possible sequelae. Because of the negative feedback in the regulation of the
hypothalamic-pituitary-gonadal axis, in men AAS cause reversible suppression of
spermatogenesis, testicular atrophy, infertility and erectile dysfunction (anabolic steroid
induced hypogonadism). Should spermatogenesis not recover after AAS abuse, a pre-
existing fertility disorder may have resurfaced. AAS frequently cause gynecomastia and
acne. In women, AAS may disrupt ovarian function. But as chronic strenuous physical activity
leads to menstrual irregularities and, in severe cases, to the female athlete triad (low energy
intake, menstrual disorders and low bone mass), it is difficult to disentangle effects of sports
and AAS. Acne, hirsutism and (irreversible) deepening of the voice are further consequences
of AAS misuse. There is no evidence that AAS cause breast carcinoma. Detecting AAS
misuse through the control network of the World Anti-Doping Agency (WADA) not only aims
to guarantee fair conditions for the athletes, but also to protect them from medical sequelae
of AAS abuse [150001].
Pituitary function is influenced by several drugs, including anti-depressant, opioids,

glucocorticoids, chemotherapeutic agents, immunomodulators and the newly developed
tyrosine kinase inhibitors. In most instances, treatment with these drugs negatively affects
pituitary function, but in rare cases an activation of specific hypothalamic-pituitary axes may
be observed. Several of the observed pituitary side effects are reversible after drug
withdrawal, but pituitary function deficiency may persist long-term. In addition to the well
known drugs, recent evidence shows that also non-steroidal anti-inflammatory drugs impair
gonadal axis at pituitary level, while antipsychotic phenothiazines alter TSH response to TRH
and TSH levels. Atypical antipsychotics may decrease TRH-stimulated TSH. Tricyclic
antidepressant drugs interfere with the hypothalamo-pituitary-thyroid axis by decreasing TSH
response to TRH. Anabolic-androgenic steroids, marijuana, cocaine, methamphetamines,
and opioid narcotics negatively impact fertility, also acting at hypothalamic-pituitary level.
Many of the drugs administered routinely in the intensive care unit significantly impact the
hypothalamic-pituitary axis. Therefore, an increased awareness on pituitary side effects of
drugs commonly used in clinical practice is necessary in order to rule out possible
pharmacological interference when assessing patients with pituitary deficiencies [14649].
The data of the present case demonstrate that the abuse of androgenic anabolic steroids
(AAS) may lead to serious health effects. Although most clinical attention is usually directed
towards peripheral side effects, the most serious central side effect, hypothalamic-pituitary-
dysfunction, is often overlooked in severe cases. Although this latter central side-effect
usually recovers spontaneously when AAS intake is discontinued, the present case shows
that spontaneous recovery does not always take place. It was suggest that hypothalamic-
pituitary dysfunction should always be considered in the differential diagnosis in athletes
seen with typical presentation of anabolic steroid use. In order to regain normal
hypothalamic-pituitary function, supraphysiological doses of 200 microg LH-RH should be
1194
considered when the physiological challenge test with LH-RH (50 microg) fails to show an
acceptable response [03066].
Hypogonadism
Testosterone (T) deficiency syndrome (TDS) is a prevalent condition, commonly managed
with exogenous T. Despite an abundance of T formulations, alternative treatments are often
sought for various reasons. To evaluate outcomes of alternative therapies, a PubMed search
was performed of all publications that included men with TDS from 1990 through October
2013, with results summarized. Proposed mechanisms of action were also reviewed to
provide a pathophysiologic basis for reported outcomes. Nonpharmacologic therapies that
increase endogenous T are weight loss, exercise, and varicocelectomy, while medications
used off-label include aromatase inhibitors, human chorionic gonadotropin, and selective
estrogen receptor modulators. All reported therapies increase T, while changes in estradiol
and adverse events vary by therapeutic class. Although limited data preclude direct
comparisons between therapies, exercise and weight loss alone or in combination with
medications may be considered first line. The role for surgical therapy in TDS remains
undefined and requires further study [14650].
Androgen- or anabolic steroid-induced hypogonadism (ASIH) is no longer confined to

professional athletes; its prevalence amongst young men and teenagers using androgens
and/or anabolic steroids (AASs) is rising fast, and those affected can experience significant
symptoms. Clinicians are increasingly encountering demanding, well-informed men affected
by ASIH, yet lacking authoritative information on the subject may struggle to project a
credible message. In one overview the methods and drugs that men use in an attempt to
counteract ASIH (with a view to either preventing its onset, or reversing it once it has
developed) and summarize the scientific evidence underpinning these. The main channel for
obtaining these drugs is the Internet, where they can be readily sourced without a valid
prescription. An Internet search using relevant terms revealed a huge number of websites
providing advice on how to buy and use products to counteract ASIH. Drugs arising
repeatedly in our search included human chorionic gonadotrophin (hCG), selective oestrogen
receptor modulators (SERMs) and aromatase inhibitors (AIs). The quality and accuracy of
the online information was variable, but review of medical literature also highlighted a lack of
scientific data to guide clinical practice. It is important for clinicians to be aware of the AAS
user's self-treatment strategies with regard to ASIH side-effect mitigation. By ensuring that
they are well-informed, clinicians are more likely to retain the credibility and trust of AAS
users, who will in turn likely be more open to engage with appropriate management [14752].
Growth hormone and IGF-1 diminish the catabolic effects of hypogonadism in man
Severe gonadal androgen deficiency can have profound catabolic effects in man.
Hypogonadal men develop a loss of lean body mass, increased adiposity, and decreased
muscle strength despite normal GH and insulin-like growth factor I (IGF-I) concentrations.
We designed these studies to investigate whether GH or IGF-I administration to male
subjects with profound hypogonadism can diminish or abolish the catabolic effects of
testosterone deficiency. Moreover, we also examined the nature of the interactions among
GH, IGF-I, and androgens in specific genes of the im system. A group of 13 healthy subjects
(mean age, 22 + 1 yr) was studied at baseline (D1) and 10 weeks after being made
hypogonadal using a GnRH analog (GnRHa; D2). At 6 weeks from baseline they were
started on either recombinant human (rh) IGF-I (60 microg/kg, sc, twice daily) or rhGH (12.5
microg/kg, sc, daily) for 4 weeks. On each study day subjects had infusions of L-[13C]leucine;
indirect calorimetry; isokinetic dynamometry of the knee extensors; determination of body
composition (dual energy x-ray absortiometry) and hormone and growth factor
concentrations, as well as percutaneous muscle biopsies. Their data were compared with
those of previously studied male subjects who received only GNRHA: Administration of
1195
rhIGF-I and rhGH to the hypogonadal men had similar effects on whole body metabolism,
with maintenance of protein synthesis rates, fat oxidation rates, and fat-free mass compared
with the eugonadal state, preventing the decline observed with hypogonadism alone. This
was further amplified by the molecular assessment of important genes in muscle function.
During rhIGF-I treatment, im expression of IGF-I declined, and IGF-binding protein-4
increased, similar to the changes during GnRHa alone. However, rhGH administration was
associated with a marked increase in IGF-I and androgen receptor messenger ribonucleic
acid concentrations in skeletal muscle with a reciprocal decline in IGF-binding protein-4
expression in the hypogonadal men. The gene expression for myostatin did not change.
These effects were accompanied by a much greater increase in plasma IGF-I concentrations
after rhIGF-I compared with the concentrations achieved during rhGH. It was concluded that
rhGH and rhIGF-I both may be beneficial in preserving lean body mass and sustaining rates
of protein synthesis during states of severe androgen deficiency in man; GH may affect the
im IGF system via an a paracrine, local production of IGF-I; and androgens may be
necessary for the full anabolic effect of GH/IGF-I in man. These hormones, particularly GH,
may play a role in the treatment of hypogonadal men rendered hypogonadal
pharmacologically or those unable to take full testosterone replacement. The latter requires
Pathophysiology of infertility: Feedback suppression
Use of AAS results in hypogonadotropic hypogonadism by feedback suppression of the

hypothalamic-pituitary-gonadal (HPG) axis via inhibition of pulsatile GnRH release and a
subsequent decrease in LH and FSH. The duration of suppression and the resultant
symptomatic ASIH is highly variable and due to multiple factors, including differences in the
choices of drugs, amounts used, and durations of use. There may be differences between
individual users regarding the response kinetics of the HPG axis. It is suggested that younger
men may have a more “elastic axis” capable of recovering GnRH pulsation and gonadotropin
secretion faster and more completely than older AAS users. It is possible that shorter
durations, lower doses, younger ages, and higher testosterone levels at baseline are
associated with a quicker recovery of HPG axis function after AAS use. Considerable
variation exists regarding drug combinations, dosing, and duration of use. Up to 90 percent
of AAS users combine or “stack” multiple androgens, a practice that users believe provides
the greatest results while minimizing unwanted side effects. Traditionally, a typical
bodybuilding cycle includes a stack of multiple AAS at a combined dose of 500-1,500
mg/week and lasts on average 4-12 weeks. The most commonly used androgens reported
by multiple surveys are single-ester testosterone, nandrolone, stanazolol, metandienone, and
trenbolone. In contrast, medical TRT is given at fixed replacement doses and may not be a
good model to describe pharmacodynamics in the AAS user. Although some evidence exists
describing the recovery period after exogenous testosterone used as a hormone
replacement, it is likely that a more complex and global endocrine disruption exists for the
AAS user because of the stacking and cycling of multiple high-dose synthetic androgens and
other ancillary drugs, culminating in a unique pharmacologic milieu. Beyond the systemic
consequences of hypogonadotropic hypogonadism, non-human animal studies suggest
direct testicular toxicity results from synthetic androgen use [14427].
Infertility and the molecular biology of the androgen receptor
Mutations that totally disrupt androgen receptor function cause the well known testicular
feminizing syndrome or complete androgen insensitivity syndrome, wherein a 46 XY
individual is completely feminized at birth. Recently it has been increasingly obvious that
androgen receptor mutations not only result in the complete androgen insensitivity syndrome,
1196
but can cause a wide spectrum of milder insensitivity syndromes including ambiguous
genitalia in newborn infants, and “idiopathic” male infertility in otherwise normal males.
Characterization of the molecular and structural mechanisms of androgen receptor
dysfunction in these cases has led to directed hormonal therapy. Thus the differential
response of a Met807Thr mutant androgen receptor to dihydrotestosterone but not
testosterone, have been used to restore male genital development in an infant with partial
AIS. Of greater significance, because they affect larger numbers of patients, are the
mutations and polymorphisms that result in depressed spermatogenesis and male infertility
in phenotypic males. Studies involving Singaporean, Australian, North American and
Japanese subjects have established that increases in length of a trinucleotide repeat (CAG)
tract, encoding a polyglutamine stretch in the transactivation domain of the androgen
receptor, are associated with increased risk of defective spermatogenesis and
undermasculinization. Independent of the CAG repeats, missense amino-acid substitutions in
the ligand-binding domain, involving residues 727, 798 and 886 cause infertility through a
novel mechanism. Pathogenicity is transmitted, not through defective ligand binding, but
through defective protein-protein interactions between receptor domains and coactivator
proteins that are essential for gene transcription. Elucidation of the molecular and structural
basis of androgen receptor dysfunction in these cases allows precise genetic counseling and
can lead to the design of rational hormonal therapy [01063].
Idiopathic male hypogonadotropic hypogonadism (IMHH)
Adult-onset idiopathic male hypogonadotropic hypogonadism (IMHH) is a very rare but

treatable disease. This study was conducted to examine the efficacy and safety of a
combination of human chorionic gonadotropin (hCG) and recombinant human follicle-
stimulating hormone (rhFSH) for inducing spermatogenesis in men with adult-onset IMHH.
Seven men (34-45 years of age) with azoospermia and/or sexual dysfunction, with a low
serum testosterone concentration, and apulsatile secretion of luteinizing hormone, were
referred to our hospital for infertility. All had normal secondary sexual characteristics.
Thorough endocrinologic examination and magnetic resonance imaging revealed no
identifiable cause of hypogonadotropic hypogonadism. Adult-onset IMHH was diagnosed in
all cases and treatment was started with 150 IU rhFSH and 5,000 IU hCG, both administered
two times per week. Spermatogenesis was restored in five of the seven patients. During
treatment one patient achieved spontaneous pregnancy with his wife, and spermatozoa
recovered from the other four patients were frozen for future use in intracytoplasmic sperm
injection [14753].
Physiology of reproduction-endocrinology
Exogenous steroids exert their affect on adrenal hormones by negative feedback on the
hypothalamicpituitary-gonadal axis. Large doses of exogenous AAS lead to a decrease in
both follicle-stimulating hormone and luteinizing hormone serum concentrations. There is
some controversial evidence to show that high doses of exogenous steroids lead to
spontaneous increases in growth hormone (GH) production. In one study of men with
hypogonadotropic hypogonadism, testosterone enanthate administration resulted in marked
increases in spontaneous GH secretion. This elevation of GH provides the negative feedback
that leads to decreased endogenous testosterone and gonadotropin release. Suppression of
gonadotropin release leads to oligospermia and hypogonadism. This is reflected by
decreased levels of testicular precursors of testosterone, namely serum pregnenolone,
progesterone, 17-hydroxyprogesterone, and 17-hydroxypregnenolone. Supraphysiologic
doses of AAS may lead to infertility due to decrease in both quantity and quality of sperm
after several months of use. The return of normal levels of FSH and LH concentrations after
1197
discontinuation of AAS can take 6 to 12 weeks, whereas normalization of endogenous serum
testosterone levels requires several additional weeks even after the return of normal
gonadotropin levels. The amount of time needed for full recovery of normal reproductive
function varies both on dose and duration of steroid use and can range from 4 to 5 months to
several years. Many athletes turn to human chorionic gonadotrophin or clomiphene to
reverse or even maintain spermatogenesis following or during courses of AAS [06031].
Since its isolation and characterization in 1935, there have been further studies on
testosterone which have led to the synthesis of numerous derivatives with properties different
from the original molecule. It is in 2011 estimated that there are as many as three million
AAS users in the USA. Interestingly, two thirds of US users are non-competitive
bodybuilders, or even non-athletes, who use these substances for aesthetic purposes only.
The negative impact of AAS abuse on male fertility is well known by urologists. Infertility is
defined as the failure to achieve a successful pregnancy after 12 months or more of regular
unprotected intercourse, and male-factor infertility is the cause in about 50 percent of all
infertile couples. Several conditions may explain male-factor infertility. Some are identifiable,
but not reversible; others may be identified and also reversed. Hypogonadotrophic
hypogonadism is a typical example of a reversible condition, whereas primary testicular
impairment is often related to a less reversible one. The secondary hypogonadotropic
hypogonadism is often highlighted when AAS and fertility are being discussed. On the other
hand, the patterns of use, mechanisms of action and direct effects over the testicle are
usually overseen. One review studied the vast formal and "underground" culture of AAS, as
well as their overall implications. Specific considerations about their impact on the male
reproductive system are made, with special attention to the recent data on direct damage to
the testicle. For several decades, testosterone and its synthetic derivatives have been used
with anabolic and androgenic purposes. Initially, these substances were restricted to
professional bodybuilders, becoming gradually more popular among recreational power
athletes. Currently, as many as 3 million anabolic-androgenic steroids (AAS) users have
been reported in the United States, and considering its increasing prevalence, it has become
an issue of major concern. Infertility is defined as the failure to achieve a successful
pregnancy after 12 months or more of regular unprotected intercourse, with male factor being
present in up to 50 percent of all infertile couples. Several conditions may be related to male
infertility. Substance abuse, including AAS, is commonly associated to transient or persistent
impairment on male reproductive function, through different pathways. Infertility after AAS
abuse commonly presents as oligozoospermia or azoospermia, associated with
abnormalities in sperm motility and morphology. According to most reports, sperm quality
tends to recover spontaneously within 4-12 months after discontinuation. However, the
negative effect on semen quality may persist for longer periods. A hypogonadotrophic
hypogonadism state is induced, characterized by decreased serum testosterone
concentrations, testicular atrophy and impaired spermatogenesis. These effects result from
the negative feedback of androgens on the hypothalamic-pituitary axis, and possibly from
local suppressive effects of exogenous androgens on the testis. FSH and luteinizing
hormone (LH) concentrations are typically low. In addition, during AAS use, serum androgen
concentrations may be supraphysiologically high, but the hypogonadotrophic state lowers the
intratesticular testosterone concentrations required to maintain normal spermatogenesis. The
management of AAS-induced male infertility has also been extensively reported. Simple
discontinuation of AAS use may lead to fertility recovery in a certain proportion of male users,
but there is little literature and considerable disagreement regarding the management of such
cases. Patients may also be actively treated, in a manner similar to that used for other forms
of hypogonadotropic hypogonadism infertility, requiring the induction of spermatogenesis
with gonadotropins or gonadotropin analogues, including i.m. injections of hCG, human
menopausal gonadotropin (hMG) or even recombinant FSH. The use of hCG alone, or in
combination with hMG, has been reported to be a successful treatment for this group of
1198
patients. Fertility restoration has been reported, even in situations of persistent azoospermia
up to 5 years after AAS discontinuation so AAS-associated male infertility may be treatable
because of its endocrine nature. Considering the prevalence of AAS abuse and the
favourable results after treatment, it is reasonable to consider it during the infertility
consultation [11028].
In men, chronic AAS use can lead to decreased endogenous testosterone production and
hypogonadotropic hypogonadism associated with testicular atrophy. Chronic AAS abuse
causes a decrease in gonadotropins, luteinizing hormone (LH), and follicle-stimulating
hormone (FSH) as part of the negative feedback system of the hypothalamic-pituitary-
gonadal axis. LH and FSH are needed for spermatogenesis so when these hormones are
decreased, there is a decrease in sperm count and mobility as well as an increase in the
number of morphologically abnormal sperm. One study found a 73 percent overall decrease
in sperm count; three individuals had azoospermia with chronic use of high-dose AASs. In
individuals who did not experience azoospermia, there was a 10 percent increase in the
number of immotile sperm and a 30 percent decrease in the number of motile sperm.
Overall, fertility was severely reduced. Decreases in gonadotropins can be seen within 24
hours of beginning AASs. Infertility may result within months. After cessation of use,
gonadotropin and testosterone secretion are suppressed for months to years. Usually, the
infertility is spontaneously reversible, typically within 1 year of cessation of AAS abuse, but it
may take longer in long-term users. At least one user of multiple AASs did not recover fertility
spontaneously and required treatment with LH-releasing hormone to regain normal levels of
testosterone and fertility. Men also may experience priapism, impotence, prostatic
hypertrophy, difficulty/pain with urination, and a possible increased risk for prostate cancer.
The risk for these consequences increases with dose and duration of use [07058].
Exogenous steroid administration thus provides feedback inhibition of luteinizing and follicle-
stimulating hormones, which leads to testicular atrophy and decreased spermatogenesis.
This testicular impairment is reversed upon cessation of AAS use. Excess steroids undergo
peripheral aromatization to estrogens, which results in feminizing changes of high voice pitch
and male gynecomastia. In long-term AAS abuse, this gynecomastia is irreversible, leaving
surgical correction as the only solution. In addition to the female side effects of decreased
menstruation and breast tissue atrophy, virilizing effects also occur and include deepened
voice, clitoromegaly, and hirsutism. Sometimes these effects are irreversible, even after
discontinuation of AAS use [07008].
To develop an understanding of hypogonadal men with a history of anabolic-androgenic

steroid (AAS) use and to outline recommendations for management a review of published
literature and expert opinions was made, but no quality studies met the inclusion criteria for a
meta-analysis. Symptomatic hypogonadism is a potential consequence of AAS use and may
depend on dose, duration, and type of AAS used. Complete endocrine and metabolic
assessment should be conducted. Management strategies for anabolic steroid-associated
hypogonadism (ASIH) include judicious use of testosterone replacement therapy, hCG, and
selective estrogen receptor modulators. It was concluded that although complications of AAS
use are variable and patient specific, they can be successfully managed. Treatment of ASIH
depends on the type and duration of AAS use. Specific details regarding a patient's AAS
cycle are important in medical management [14051].
High doses of anabolic-androgenic steroids (AAS) are used by some athletes to increase
muscle mass, that is often associated with male infertility. The aim of one study was to
investigate the possible cause/s of male infertility using a rat model by analysing sperm
quality, including its protamine content and DNA integrity, as well as pregnancy rate. Five
groups of male Wistar rats were treated for 10 weeks as follows: nandrolone decanoate (10
1199
mg/kg per week) (ND); running exercise (50 min per day, 5 days a week) (EX); Combination
of ND and exercise (ND-EX); nandrolone decanoate solvent (Sham); and control without any
injection or exercise (CO). Deterioration in sperm quantity was observed in all test groups.
The frequency of fertile rats was decreased in the ND-EX and ND groups. Chromomycin-A3
staining showed a protamine deficiency in the epididymal spermatozoa in the ND-EX rats.
Chromatin analysis indicated an abnormal maturation of the sperm nuclei in all test groups
compared with the controls. TUNEL analyses showed a highly significant increase in
apoptosis in the EX, ND, and ND-EX groups. The data show that a combination of exercise
and high doses of nandrolone decanoate negatively influences the DNA integrity and
protamine content resulting in lower sperm quality and reduced pregnancy rate [14052].
Anabolic steroid-induced hypogonadism in young men
It was examined whether men with anabolic-steroid-induced hypogonadism (ASIH) seeking

testosterone supplementation therapy (TST) regretted their decision to use anabolic-
androgenic steroids (AAS) and what their reasons were for this regret. An anonymous,
prospective survey was distributed to 382 men seeking follow-up treatment for
hypogonadism. Prior AAS use was confirmed by self-report, and men were categorised
based upon whether they regretted (R) or did not regret (NR) their use of AAS. The average
patient age was 40 ± 1 years (n=79) and 15 percent expressed regret over AAS use. No
demographic differences were identified between those who regretted AAS use (n=12) and
those who did not (n=67). Regret was not related to ASIH diagnosis or to AAS-related side
effects like increased aggression, mood disorders, erectile dysfunction, acne, fluid retention
or dyslipidemia. Those who regretted AAS use were significantly more likely to have not
comprehended the negative impact on future fertility. Actual fertility issues were comparable
in men who regretted AAS use (17 %) and those who did not (13 %). A total of 15 percent of
men regretted using AAS. A lack of awareness regarding the negative long-term effects on
fertility was the primary factor related to regret of AAS use in men with ASIH [14651].
Use of anabolic androgenic steroids (AAS) has not been traditionally discussed in
mainstream medicine. With the increased diagnosis of hypogonadism, a very heterogeneous
population of men is now being evaluated. Within this larger population of patients, the
existence of anabolic steroid-induced hypogonadism (ASIH), whether transient or
permanent, should now be considered. An initial retrospective database analysis of all
patients (2005-2010, n=6033) seeking treatment for hypogonadism was conducted.
Subsequently, an anonymous survey was distributed in 2012 to established patients
undergoing testosterone replacement therapy (TRT). Profound hypogonadism, defined as a
testosterone =50 ng/dL, was identified in 1.6 percent (n=97) of the large retrospective cohort
initially reviewed. The most common etiology was prior AAS exposure, identified in 43
percent (42/97) of men. Because of this surprising data, a follow-up anonymous survey of
our current hypogonadal patient population (n=382; mean age 49 ± 13 years) was then
performed which identified 21 percent of patients (n=80; mean age 40 ± 8 years) with prior
AAS exposure. Hypogonadal men <50 years old were greater than 10 times more likely to
have prior AAS exposure than men >50 (OR 10). Prior AAS use was significantly negatively
correlated with education level and number of children. Prior AAS use is common in young
men seeking treatment for symptomatic hypogonadism, and ASIH is the most common
etiology of profound hypogonadism. These findings suggest a necessary refocused approach
in the evaluation and treatment paradigms of young hypogonadal men [13152].
The use of anabolic androgenic steroids has not been traditionally discussed in mainstream
medicine. With the increased diagnosis of hypogonadism a heterogeneous population of
men is now being evaluated. In this larger patient population the existence of anabolic steroid
induced hypogonadism, whether transient or permanent, should now be considered. It was
1200
performed an initial retrospective database analysis of all 6,033 patients who sought
treatment for hypogonadism from 2005 to 2010. An anonymous survey was subsequently
distributed in 2012 to established patients undergoing testosterone replacement therapy.
Profound hypogonadism, defined as testosterone 50 ng/dL or less, was identified in 97 men
(1.6 %) in the large retrospective cohort initially reviewed. The most common etiology was
prior anabolic androgenic steroid exposure, which was identified in 42 men (43 %). Because
of this surprising data, it was performed an anonymous follow-up survey of our current
hypogonadal population of 382 men with a mean ± SD age of 49 ± 13 years. This identified
80 patients (21 %) with a mean age of 40 ± 8 years who had prior anabolic androgenic
steroid exposure. Hypogonadal men younger than 50 years were greater than 10 times more
likely to have prior anabolic androgenic steroid exposure than men older than 50 years (OR
10). Prior anabolic androgenic steroid use significantly correlated negatively with education
level and number of children. It was concluded that prior anabolic androgenic steroid use is
common in young men who seek treatment for symptomatic hypogonadism and anabolic
steroid induced hypogonadism is the most common etiology of profound hypogonadism.
These findings suggest that it is necessary to refocus the approach to evaluation and
treatment paradigms in young hypogonadal men [13153].
In addition to decreased sperm counts and testes volumes, some AASs abusers experience
lack of libido and erectile function as well as other signs of hypogonadism. This occurs
especially in those men abusing aromatisable AASs, resulting in high oestrogen levels.
Although physiological levels of oestrogens are necessary for normal sexual function, the
extremely high doses and the imbalance between testosterone and estradiol appear to be
the cause of sexual dysfunction in these cases. This may also occur during the recovery
phase after termination of exogenous AASs supply, when endogenous production has not
yet resumed full activity. This type of hypogonadism (31) has been referred to as “anabolic
steroid-induced hypogonadotrophic hypogonadism” and more recently, simply as “anabolic
steroid-induced hypogonadism” (ASIH). In a large US urology department, 96 of 6033 (1.6
%) patients consulting for hypogonadism suffered from ASIH. One-quarter of these patients
presented with infertility and three quarters with sequelae of hypogonadism. Many of these
AASs abusers were first overlooked and only diagnosed after renewed interviewing following
inconclusive investigations. This reflects the fact that patients seeking medical care only
reluctantly admit to AASs abuse, and targeting AASs abuse directly should be part of the
routine work-up of hypogonadal men. Cessation of AASs abuse is the prime measure to treat
ASIH. In addition, various therapeutic attempts have been undertaken to overcome ASIH and
to hasten recovery. Human chorionic gonadotropin (hCG) has been given in individual cases,
and has also been combined with tamoxifen or clomiphene to counteract the increased
oestrogen levels under hCG, inducing or worsening gynecomastia. However, hCG
administration simultaneously with AASs in order to maintain fertility in power athletes
showed an increase in morphologically abnormal sperm in comparison with a control group
receiving only AASs. This may be due to the lack of FSH under stimulation by hCG alone.
Therefore, although sperm counts were maintained under this treatment, fertility may still be
compromised due to deteriorated sperm morphology. In cases with erectile dysfunction,
PDE5 inhibitors have been prescribed, but again no systematic studies exist. The continuing
lack of conclusive studies prevents clear recommendations on how to treat ASIH except to
stop AASs and other drug intake immediately and to await recovery in patience. It also
remains ethically questionable whether the consequences of hormone abuse should be
counteracted by additional hormone treatment, when normal conditions can be reconstituted
by strict termination of abuse and waiting for spontaneous recovery. Gonadotropin treatment
may be justified only if no improvement has been observed within 24 months [150001].
Androgen- or anabolic steroid-induced hypogonadism (ASIH) is no longer confined to

professional athletes; its prevalence amongst young men and teenagers using androgens
1201
and/or anabolic steroids (AASs) is rising fast, and those affected can experience significant
symptoms. Clinicians are increasingly encountering demanding, well-informed men affected
by ASIH, yet lacking authoritative information on the subject may struggle to project a
credible message. In one article, it was overviewed the methods and drugs that men use in
an attempt to counteract ASIH (with a view to either preventing its onset, or reversing it once
it has developed) and summarize the scientific evidence underpinning these. The main
channel for obtaining these drugs is the Internet, where they can be readily sourced without a
valid prescription. An Internet search using relevant terms revealed a huge number of
websites providing advice on how to buy and use products to counteract ASIH. Drugs arising
repeatedly in our search included human chorionic gonadotrophin (hCG), selective oestrogen
receptor modulators (SERMs) and aromatase inhibitors (AIs). The quality and accuracy of
the online information was variable, but review of medical literature also highlighted a lack of
scientific data to guide clinical practice. It is important for clinicians to be aware of the AAS
user's self-treatment strategies with regard to ASIH side-effect mitigation. By ensuring that
they are well-informed, clinicians are more likely to retain the credibility and trust of AAS
users, who will in turn likely be more open to engage with appropriate management [150211].
Prolonged hypogonadism in males following withdrawal AASs

To assess the frequency and severity of hypogonadal symptoms in male long-term anabolic-
androgenic steroid (AAS) misusers who have discontinued AAS use 24 male former long-
term AAS users and 36 non-AAS-using weightlifters, recruited by advertisement in
Massachusetts, USA were studied. Five of the former users were currently receiving
treatment with physiological testosterone replacement, leaving 19 untreated users for the
numerical comparisons below. The Structured Clinical Interview for DSM-IV, questions
regarding history of AAS use, physical examination, serum hormone determinations and the
International Index of Erectile Function (IIEF) were used. Compared with the 36 non-AAS-
using weightlifters, the 19 untreated former AAS users displayed significantly smaller
testicular volumes and lower serum testosterone with five users showing testosterone levels
below 200 ng/dl despite abstinence from AAS for 3-26 months. Untreated former users also
displayed significantly lower scores on the IIEF sexual desire subscale. In the overall group
of 24 treated plus untreated former users, seven (29 %) had experienced major depressive
episodes during AAS withdrawal; four of these had not experienced major depressive
episodes at any other time. Two men (8 %) had failed to regain normal libidinal or erectile
function despite adequate replacement testosterone treatment. Among long-term anabolic-
androgenic steroid misusers, anabolic-androgenic steroid-withdrawal hypogonadism appears
to be common, frequently prolonged and associated with substantial morbidity [150212].
Secondary hypogonadism
It was presented the case of a 16-year-old male who presented reporting a 6-month history
of lowered mood, fatigue, anhedonia, disturbed sleep and heightened anxiety. On further
questioning he reported restricted eating and weightlifting for at least 1 h on a daily basis.
Investigations revealed findings compatible with secondary hypogonadism. The potential
causes of secondary hypogonadism including structural lesions, muscle dysmorphia and use
of illicit anabolic steroids are discussed [14652].
Azospermi
Male infertility is commonly seen at urology clinics and 10 to 20 percent of infertile males are
found to be azoospermic. Azoospermia is classically categorized as nonobstructive or
obstructive. This classification tailors the evaluation, diagnosis and proper treatment. We
performed a retrospective study to provide an updated etiology of azoospermia in patients in
the United States in a universal health care model. It was retrospectively reviewed the
records of men with azoospermia who presented to our institution between 2004 and 2012.
1202
Laboratory data were analyzed, included semen analysis, follicle-stimulating hormone,
luteinizing hormone, testosterone, semen fructose and genetic studies. Patients underwent
scrotal exploration as indicated for testis biopsy and sperm extraction. It was r eviewed 139
outpatient records. Nonobstructive azoospermia was diagnosed in 99 men (71%), including
33 (34 %) identified with Sertoli-cell only syndrome. Other etiologies included an idiopathic
cause in 25 cases (26 %), Klinefelter syndrome in 9 (9 %), maturation arrest in 9 (9 %), Y
chromosome microdeletion in 5 (5 %), cryptorchidism in 4 (4 %), trauma in 4 (4 %),
exogenous testosterone supplementation in 4 (4 %) and other genetic disorders in 6 (6 %).
Obstructive azoospermia was identified in 40 men (29 %), of whom 16 (40 %) had congenital
bilateral absence of the vas deferens. Other etiologies included an idiopathic cause in 11
cases (28 %), an iatrogenic condition due to a surgical cause in 5 (13 %), ejaculatory duct
obstruction in 3 (8 %), trauma in 1 (3 %), retrograde ejaculation in 1 (3 %), vas deferens
occlusion in 2 (5 %) and unilateral absence of the vas deferens in 1 (3 %). The study
delineates the etiology of azoospermia in men with universal access to care [150210].
Azoospermia may sometimes be related to the use of androgenic anabolic steroids. It was
reported a case of an azoospermic man who had abused androgenic anabolic steroids and
who recovered spermatogenesis six months after cessation of abuse and the administration
of hormonal therapy. An azoospermic 34-year-old man came to Regional Referral Center for
Male Infertility. The recovery of spermatogenesis was observed after the cessation of abuse
of steroids and the administration of hormonal therapy. Ultrastructural analysis of sperm was
carried out by transmission electron microscopy, and the meiotic segregation of
chromosomes 1, 9, 18, X, Y was investigated. Mathematically elaborated transmission
electron microscopy data highlighted seminal features close to normal fertility. Fluorescence
in situ hybridisation showed a high frequency of XY disomy in sperm. The findings confirm
the recovery of spermatogenesis but suggest a possible relationship between altered meiotic
segregation and the abuse of androgenic anabolic steroids [07072].
Combined testosterone and progestogen preparations are a promising approach to male

hormonal contraception. It was investigated the effect of s.c. etonogestrel with depot
testosterone on spermatogenesis in normal men over a period of 48 weeks. Fifteen healthy
men received three s.c. 68 mg etonogestrel implants. Testosterone pellets (400 mg) were
administered at 12 weekly intervals. Nine men completed 48 weeks of treatment. Four
subjects chose to discontinue after 6 months, one man withdrew from the study early for
personal reasons and one was withdrawn due to illness. Sperm concentrations of <1 x 106/ml
were achieved in all men by 16 weeks of treatment. All men became azoospermic, although
the time to achieve this varied from 8 to 28 weeks. Azoospermia was maintained in eight of
the nine men treated for 48 weeks, one subject showing partial recovery from 40 weeks.
Testosterone levels remained in the physiological range throughout. Treatment did not result
in weight gain, change in body composition or decline in high-density lipoprotein cholesterol
concentrations. It was concluded that the combination of three etonogestrel implants with
depot testosterone results in rapid and consistent suppression of spermatogenesis. This can
be maintained for up to 1 year and may therefore be a suitable approach for a long-acting
male hormonal contraceptive [04062].
Reversible hypogonadism and azoospermia

It was reported a case of reversible hypogonadism and azoospermia resulting from anabolic-
androgenic steroid abuse in a body-builder with primary personality disorder. A keen body
builder, a 20-year-old man, developed acute aggressive and destructive behavior after 10-
month use of Bionabol (mean total dose of 1,120 mg per month), and Retabolil (mean total
dose of 150 mg per month). He was found to meet the Diagnostic and Statistical Manual of
Mental Disorders-IV ed. (DSM-IV) criteria for Borderline personality disorder. On admission
to the hospital the clinical profile of the patient showed extremely low levels of serum
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testosterone. Values increased to normal levels 10 months after withdrawal of steroids. The
semen was azoospermic at the beginning of the study period, oligospermic five months later,
and reached 20 x 106 sperm per mL ten months after the steroid discontinuation. Anabolic
steroids can greatly affect the male pituitary-gonadal axis. A hypogonadal state,
characterized by decreased serum testosterone and impaired spermatogenesis, was induced
in the patient. This condition was reversible after the steroid withdrawal, but the process took
more than ten months. His personal imbalance could be considered a personality trait rather
than a result of the anabolic-androgenic steroid use. There were probably dispositional
personality characteristics that contributed to anabolic steroid abuse in our patient. The
hypogonadal changes which occurred after his long-term steroid abuse were for the most
part reversible [00070].
Suppression of spermatogenesis
As endogenous testosterone is the major regulator of the hypothalamo-pituitary-testicular

axis, it is not surprising that exogenous testosterone and AASs exert a suppressive effect on
the hypothalamo-pituitary system. The resulting suppression of luteinizing hormone (LH) and
follicle-stimulating hormone (FSH) leads to a decrease in intratesticular testosterone and
secreted testosterone, as well as to a decrease in spermatogenesis and sperm production.
This effect forms the basis for clinical trials in hormonal male contraception. As there are no
systematic investigations of the effects of doping with high-dose AASs on testicular function,
contraceptive trials may serve as a model for what happens under AASs suppression. Male
hormonal contraceptive trials use testosterone alone in therapeutic doses or in combination
with gestagens to induce azoospermia or severe oligozoospermia compatible with
contraceptive protection. Testosterone derivatives used for doping, such as 19-
nortestosterone and MENT, have also been applied in contraceptive trials. The kinetics of
sperm suppression and recovery are quite well known from these carefully conducted trials
using therapeutic doses. Of 1549 healthy eugonadal men participating in 30 different clinical
trials, after cessation of medication, 67 percent showed a return to sperm concentrations
above 20 million/mL within 6 months, 90 percent within 12 months, 96 percent within 16
months and 100 percent within 24 months. While the different regimens used in the 30 trials
were not identical, they used similar steroid doses; the differences in recovery time appear to
be determined more by the characteristics of the individual than by the therapeutic regimen.
Nevertheless, all men returned to fertile levels of sperm counts. Thus a 6 to 24-month span
provides a time frame for recovery in AASs abusers, although it has to be kept in mind that
doses used for doping far exceed those used for male contraception, and therefore even
longer periods may be anticipated [150001].
These results may help to interpret case reports and small retrospective studies in AASs
abusers. These show a wide spectrum of sperm counts in AASs users under treatment, as
well as after cessation of abuse, ranging from normal levels to azoospermia. LH and FSH
correlate grossly with sperm counts, i.e. the lower the gonadotropins, the lower the sperm
counts tend to be. Parallel to the decline in spermatogenesis, testicular volumes decrease
significantly because the tubules occupy about 95 percent of the testes volume and their
atrophy causes shrinkage of the testis. Varying doses, preparations, and combinations of
AASs and other APED make it difficult to draw general conclusions from individual
observations, but it is clear that the recovery of sperm counts correlates positively with
duration of time since last intake of AASs, as do sperm morphology and motility. As AASs
may be abused for many years at high doses and in varying combinations, it is impossible to
predict accurately their impact on spermatogenesis without proper investigations. In some
AASs abusers, recovery may take irritatingly long. This, in addition to individual
predisposition, is most likely due to the depot effects of the huge steroid concentrations
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consumed. For example, after termination of nandrolone abuse metabolites could still be
detected in urine after more than a year in some men. In other candidates, sperm counts
may not reach the normal range after cessation of doping, possibly due to preexisting fertility
problems. When infertile men with subnormal sperm counts were included in a contraceptive
trial using testosterone undecanoate alone, all returned to their (subnormal) baseline levels
after cessation of testosterone undecanoate administration, but did not become better or
worse than before the trial. In analogy, those AASs users who apparently do not return to
normal sperm counts may never have had normal values before initiation of AASs abuse. In
conclusion, to date there is no indication that AASs abuse causes permanent damage to
spermatogenesis, although suppression may cause transient azoospermia and recovery may
take up to 2 years. However, no systematic investigations exist to provide a definitive
answer. Considering the great number of teenage boys using AASs, the question arises
whether their use by boys around puberty may be harmful to spermatogenesis. Although
systematic investigations in pubertal AASs users are lacking, treatment of over-tall boys with
high doses of testosterone – close to doping doses – for reduction of final height provides an
analogy. Initially it was suspected that this treatment would be harmful to the testes and
leave permanent damage. However, when the proper control groups were co-investigated,
the incidence of subnormal semen parameters was the same in both groups, indicating that
at this age the testes do not differ from adult men in their capacity to recover from
suppression [150001].
Oligospermia responds to low dose estrogen-testosterone combination therapy
A man having severe oligospermia, due to partial maturation arrest at spermatid stage, was
given low dose estrogen-testosterone combination therapy for three months. His sperm
count increased enormously, following which his wife conceived and delivered a healthy
baby at term. A 34-year-old man, married for four years without having any child, attended
the low-resource private clinic on August 28, 2000. His semen analysis (SA), done on July 7,
1998, showed a sperm count (C) of 0.025106 per mL, of which only few sperm were motile
(M) and 15 percent of them were of normal morphology (N). He received mesterolone 25 mg
orally daily for three months and his SA, done on October 24, 1998, showed: C- 0.075106 per
mL, M- 40 percent and N-15 percent. Subsequently, he received clomiphene citrate 25 mg
orally daily for few months. His SA was done on June 1, 2000 and again on August 1, 2000,
and on both occasions it showed few weakly motile sperms only. The volume of the ejaculate
was always normal (1.5 to 4.0 mL). On August 11, 2000 testicular biopsy was done and it
showed similar histological pattern in both the testes: number and diameter of seminiferous
tubules were normal, majority of them showed maturation arrest at spermatid stage, while
few tubules showed maturation up to spermatozoa stage; there was no thickening of tubular
basement membrane and interstitium was normal. The man did not have past history of any
significant illness or sexual dysfunction, nor was he exposed to heat or chemicals. On
physical examination: he was normotensive and androgenized; his testes were of normal
consistency but the left one was a bit small in size, there was no evidence of any varicocele
or any epididymitis. Since no assurance of a cure could be given, the man refused to spend
on costly hormonal assay and therapy. So, after taking informed consent, he was given low
dose estrogen-testosterone combination therapy (ETCT). He received one tablet of
combined low dose estrogen and testosterone orally daily (ethinyl estradiol 0.0044 mg and
methyl testosterone 3.6 mg) for three months. On November 6, 2000, seventy days after
starting of the treatment, his SA showed: C- 35106 per mL, M- 25 percent and N-25 percent.
He took the ETCT for three months till November 28, 2000 and on December 12, 2000, his
SA showed: C- 40106 per mL, M- 28 percent and N-32 percent. His SA was repeated on
February 9, 2001, seventy-three days after stopping of the treatment, and it showed: C-
20106 per mL, M- 60 percent and N-70 percent. He did not report of any adverse effects and
none were detected on physical examination. This man had received mesterolone and
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clomiphene, but the sperm count remained less than 0.1106 per mL on four occasions
between July 1998 and August 2000; his sperm count increased after taking ETCT and was
more than 20106 per mL on three occasions between November 2000 and February 2001.
Though combination of testosterone and estrogen, at higher doses, has been shown to have
contraceptive effects in men, yet this case study is being reported so that it may stimulate
interest in the conduct of further controlled studies; until then this low dose estrogen
testosterone combination therapy, for the treatment of oligospermia, remains unproven
[02038].
Spermatogenesis
It was reviewed the current literature for the effect of hormones used in rejuvenation clinics
on the maintenance of spermatogenesis. Exogenous testosterone and anabolic androgenic
steroids suppress intratesticular testosterone production, which may lead to azoospermia or
severe oligozoospermia. Therapies that protect spermatogenesis involve human chorionic
gonadotropin (hCG) therapy and selective estrogen receptor modulators (SERMs). The
studies examining the effect of human growth hormone (HGH) on infertile men are
uncontrolled and unconvincing, but they do not appear to negatively impact
spermatogenesis. At present, routine use of aromatase inhibitors is not recommended based
on a lack of long-term data. The use of hormones for rejuvenation is increasing with the
aging of the Baby Boomer population. Men desiring children at a later age may be unaware
of the side-effect profile of hormones used at rejuvenation centers. Testosterone and
anabolic androgenic steroids have well-established detrimental effects on spermatogenesis,
but recovery may be possible with cessation. Clomiphene citrate, human growth hormone
(HGH)/insulin-like growth factor-1 (IGF-1), human chorionic gonadotropin (hCG), and
aromatase inhibitors do not appear to have significant negative effects on sperm production,
but quality data are lacking [13151].
Histopathlogy
Experiments in animal models mainly report AAS-induced Leydig cell alterations, but cellular
morphology anomalies have also been reported. The decrease in this population of cells is
accompanied by low testosterone and LH levels in all papers reviewed, especially in those
papers reporting on adult animal models. Immunohistochemical findings have suggested
decreased steroidogenesis in testicular tissue, hence spermatogenesis was considered
unchanged by some other authors. Nethertheless, specific end-stage spermatogenesis
impairment, with a lack of advanced forms of spermatids, has been described. After AAS
discontinuation, Leydig cells tend to proliferate but remain below the regular counts, even
after longer periods. Clearly, long-lasting, or possibly persistent effects of AAS use cannot be
ruled out [11028].
Impact on semen quality
The use of a combination of hCG and steroids is a common practice among AAS users. The
goal is to avoid the impact of LH suppression after long-term AAS administration, which may
lead to a persistent state of hypogonadism and low-quality semen. Restoration of
spermatogenesis has been described; however more abnormal and hypokinetic
spermatozoa are found, even after hCG “post-cycle” use, showing a potential for persistent
alterations after the discontinuation of AAS use [11028].
The purpose of one study was to assess the influence of the administration of high doses of
androgenic anabolic steroids (AAS) on endocrine and semen parameters. Thirty volunteering
bodybuilders were studied (ages ranging between 27 + 4 years). A history of anabolic steroid
1206
administration was recorded for fifteen subjects, and results of semen analysis and
endocrine parameters were compared with data from fifteen bodybuilders not using steroids.
In those subjects using AAS, eight had sperm counts under the lower normal limit (20 x 106
sperm/mL), three had azoospermia, two polyzoospermia, and two had normal sperm counts.
The percentage of morphologically normal sperm was significantly reduced, only 18 percent
had normal spermatozoa. In the control group, only one subject had oligozoospermia. The
hormonal parameters revealed reduced FSH and PRL levels. LH, T, E2 and DHEA levels did
not vary [01064].
Aneuploidies and ultrastructural changes in spermatozoa
The innovative use of both transmission electron microscopy and fluorescence in situ
hybridization (FISH) has recently been reported in an AAS user sperm sample, searching for
genetic and ultrastructural consequences of steroid abuse. Immaturity, necrosis and
apoptosis were assessed, and a high percentage of structurally normal spermatozoa were
found, which showed the absence of a correlation between AAS and ultrastructural sperm
changes. In contrast to these findings, FISH sperm analysis revealed XY and chromosomes
1 and 9 disomies, suggesting anomalies in the meiotic process and genetic damage among
AAS users [01128].
Sexual orientation and anabolic-androgenic steroids in adolescent boys
It was compared the lifetime prevalence of anabolic-androgenic steroid (AAS) misuse among
sexual minority versus heterosexual US adolescent boys, and secondarily, sought to explore
possible intermediate variables that may explain prevalence differences. Participants were
17,250 adolescent boys taken from a pooled data set of the 14 jurisdictions from the 2005
and 2007 Youth Risk Behavior Surveys that assessed sexual orientation. Data were
analyzed for overall prevalence of AAS misuse and possible intermediary risk factors. Sexual
minority adolescent boys were at an increased odds of 5.8 (95 % confidence interval 4.1 to
8.2) to report a lifetime prevalence of AAS (21 % vs 4 %) compared with their heterosexual
counterparts. Exploratory analyses suggested that increased depressive symptoms/
suicidality, victimization, and substance use contributed to this disparity. This is the first
known study to test and find substantial health disparities in the prevalence of AAS misuse
as a function of sexual orientation. Prevention and intervention efforts are needed for sexual
minority adolescent boys [14053].
Spontaneous corpus cavernosum abscess
Abscess of the corpus cavernosum is an unusual infection and can develop after trauma, as
a complication of cavernosography; after intracavernous injection of vasoactive agents or
perineal abscess drainage; with intermittent self-catheterization, seeding from periodontal
infection or tuberculosis; as a result of neglected penile fracture; or spontaneously from
undetermined causes. Androgenic anabolic steroids (AASs) are structurally related to
testosterone, bind to androgen receptors, and exert masculinizing and anabolic effects to
varying degrees. The adverse effects of long-term use of AAS remain unknown. Abscess
formation of the corpus cavernosum is very rare. Here, it was reported a case of long-term
anabolic androgenic steroid (AAS) abuse that is suspected to have facilitated the
development of a corpus cavernosum abscess in a healthy bodybuilder. Cultures obtained
from the abscess contained Staphylococcus epidermidis, a microorganism that almost
exclusively affects immunocompromised patients. Therefore, prompt drainage of pus from
cavernosal bodies should be the primary aim of the treatment. This case illustrates the
1207
potential danger of AAS suppressing the immune system and causing a serious infection
[150213].
Testicular tumours
In connection with AASs abuse, testicular germ cell tumours have not been reported in the
literature. A single case of a leiomyosarcoma in a former GDR weightlifter has been reported.
He used oral turinabol at high doses (up to 20 tablets/day) from 18 to 23 years of age. He
developed gynaecomastia under the treatment and was, at the age of 32, operated for a
unilateral intratesticular leiomyosarcoma. As these tumours are extremely rare and have
been described in hamsters after treatment with testosterone propionate and
diethylstilbestrol, the authors suspected a causal relationship between AASs abuse and the
sarcoma. As this remains the only reported case, possible involvement of AASs in the
pathogenesis of the tumour remains unclear [150001].
Prostate
Prostate development and growth are dependent on androgens. The prostate grows during
puberty and the small prostates of hypogonadal patients attain normal adult dimensions
under testosterone treatment. Furthermore, androgens are often considered promoters or
even initiators of prostate carcinoma so that one would expect a high rate of benign prostatic
hypertrophy (BPH) and prostate carcinoma in AASs abusers, often exposed to high doses of
various AASs. Despite these considerations, only one case has been reported of a
bodybuilder who used combinations of different oral and injectable AASs at high doses over
a time span of 18 years, occasionally augmented by growth hormone injections, who
developed an adenocarcinoma of the prostate at age 40. Considering this drug anamnesis, it
is tempting to suspect a causal relationship, but the lack of further case reports or systematic
investigations do not support this suspicion. The observation that the type and duration of
sport activities may influence the occurrence of prostate carcinoma (as well as erectile
dysfunction and infertility) makes interpretation of AASs abuse even more difficult. In cyclists
over 50 years of age, a clear positive correlation between the incidence of prostate cancer
and hours of weekly cycling time (<3.75 vs >8.5 h/week) was found. Furthermore, the
observation that hypogonadal men treated with therapeutic doses of testosterone do not
suffer from a higher incidence of prostate carcinoma than patients not treated with
testosterone supports the hypothesis that prostate carcinoma develops independently of
possible androgen treatment. Similarly, there are no clear indications, case reports or
systematic investigations demonstrating that AASs abuse causes BPH. As shown in the
preclinical model of the cynomolgus monkey, co-administration of testosterone and
norethisterone prevents the testosterone-induced prostate growth and hypertrophy. As
several AASs also have gestagenic activity in addition to androgenic effects, some AASs
may prevent testosterone-dependent prostate growth when given in combination, and this
may explain the low incidence of BPH in AASs abusers [150001].
Experimental
Regarding the penis, it is well known that adequate levels of testosterone are necessary to
maintain its normal morphology and the proper functioning of erectile bodies. Therefore, it
was hypothesized that AAS may affect penile morphology. There is no information on the
effects of supra-physiological doses of AAS on the corpus cavernosum tissue. Thus, one
study aimed to assess the penile morphological modifications of pubertal and adult rats
chronically treated with supra-physiological doses of AAS. To evaluate the penile
morphological modifications of pubertal and adult rats chronically treated with supra-
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physiological doses of anabolic androgenic steroids. Forty-eight male Wistar rats were
distributed into four groups: two control groups, 105- and 65-day-old (C105 and C65,
respectively) injected with peanut oil (vehicle); and two treated groups, 105- and 65-day-old
(T105 and T65, respectively) injected with nandrolone decanoate at a dose of 10 mg/kgg of
body weight. The rats were injected once a week for eight weeks. The rats were then killed
and their penises were processed for histomorphometric analyses. The mean of each
parameter was statistically compared. A corpus cavernosum reduction of 13 percent and 11
percent was observed in the T105 and T65 groups, respectively, when compared with their
respective control groups. The cavernosum smooth muscle surface density diminished by 6
percent and 13 percent in the T65 and T105 groups, respectively, when compared with their
controls. In contrast, the sinusoidal space increased by 17 percent in the T105 group and
decreased by 10 percent in the T65 group. It was concluded that the use of supra-
physiological doses of AAS promotes structural changes in the rat penis, by altering the
proportions of corpus cavernosum tissues, in both pubertal and adult treated animals
[150214].
One study was designed to systematically analyze and define the effects of 1-day, 2-weeks,
10-weeks intramuscular administration of testosterone-enanthate, widely used and abused
anabolic androgenic steroid (AAS), on main regulators of steroidogenesis and steroidogenic
genes expression in testosterone-producing Leydig cells of adult rats. The results showed
that prolonged (10-weeks) intramuscular administration of testosterone-enanthate, in
clinically relevant dose, significantly increased prolactin, but decreased Prlr2 and Gnrhr in
pituitary of adult rat. The levels of testosterone, Insl3, cAMP and mitochondrial membrane
potential of Leydig cells were significantly reduced. This was followed by decreased
expression of some steroidogenic enzymes and regulatory proteins such as Lhcgr, Prlr1/2,
Tspo, Star, Cyp11a1, Cyp17a1, Dax1. Oppositely, Hsd3b1/2, Hsd3b5, Hsd17b4, Ar, Arr19
increased. In the same cells, transcriptional milieu of cAMP signaling elements was disturbed
with remarkable up-regulation of PRKA (the main regulator of steroidogenesis). Increased
prolactin together with stimulated transcription of Jak2/Jak3 could account for increased
Hsd3b1/2 and Hsd3b5 in Leydig cells following 10-weeks in vivo treatment with testosterone-
enanthate. In vitro studies revealed that testosterone is capable to increase level of Prlr1,
Prlr2, Hsd3b1/2, Hsd3b5 in Leydig cells. Accordingly, testosterone-induced changes in
prolactin receptor signaling together with up-regulation of PRKA, Hsd3b1/2, Hsd3b5, Ar in
Leydig cells, could be the possible mechanism that contribute to the establishment of a new
adaptive response to maintain homeostasis and prevent loss of steroidogenic function.
Presented data provide new molecular insights into the relationship between disturbed
testosterone homeostasis and mammalian reproduction and are important in terms of wide
use and abuse of AASs and human reproductive health [150215].
Few data are available on adolescent users because most behavioral studies on anabolic-
androgenic steroids (AAS) abuse have been performed in adults. Studies evaluating the
impact of long-term effects of AAS abuse on the prepubertal phase are even more
uncommon. Accordingly, this study was developed to test the hypothesis that changes
induced by the use of AAS during the adolescent phase may be noted in the adult phase
even when the AAS treatment cycle is discontinued. Therefore, not only behavioral changes
but also possible autonomic and electrolyte disorders were evaluated. For this purpose, we
used male prepubertal, 26-day-old (P26) Wistar rats that were treated with vehicle (control,
n=10) or testosterone propionate (TP; 5 mg/kg intramuscular (IM) injection, AAS, n=10) five
times per week for 5 weeks, totaling 25 applications during the treatment. Aggression tests
were performed at the end of the cycle (P54-56), whereas open-field tests (OFTs), elevated
plus maze (EPM) behavioral tests and measurements of heart rate variability (HRV), fluid
intake and pathology were conducted in the adult phase (P87-92). The AAS group showed
greater aggressiveness in the pubertal phase and higher levels of horizontal and vertical
1209
exploration and anxiety-related behavior in the adult phase than the control group. HRV tests
showed an increase in sympathetic autonomic modulation, and hydroelectrolytic assessment
showed lower basal intake levels of hypertonic saline than the control group, without
statistically significant changes in the basal intake of water. These data together suggest that
the use of AAS during the prepubertal phase induces behavioral, autonomic and
hydroelectrolytic changes that manifest in the adult phase even when treatment is
discontinued in late adolescence in rats [14054].
Sexual behavior in male rats

Anabolic-androgenic steroids (AAS) increase libido and sexual behavior, but the underlying
behavioral mechanisms are unclear. One way AAS may enhance expression of sexual
behavior is by increasing the willingness to work for sex. In the present study, sexually-
experienced male rats received daily injections of testosterone at supraphysiologic doses
(7.5 mg/kg in water with 13 % cyclodextrin) or vehicle and were tested for appetitive sexual
behavior measured by operant responding for access to an estrous female. Initially, rats
were trained in their home cage to respond on a nose-poke under a 10-min fixed-interval
schedule for food reward. Once rats achieved stable response rates, the food was replaced
by a female, followed by mating for 10 min. There was no effect of testosterone on operant
responding for food (28.1 ± 4.4 responses/10min for testosterone, 30.6 ± 4.3 for vehicle) or
sex (35.0 ± 4.0 responses/10min for testosterone, 37.3 ± 5.2 for vehicle). However, rats
made significantly more responses for sex than for food, and responses for food and sex
were positively correlated among individuals. Additional groups of rats were trained to
respond on a lever for the female under a 2nd-order schedule of reinforcement, where 5
responses opened a door to show the female for 5s. After 15 door openings, the male gained
access to the female. There was no effect of testosterone on time to complete 75 responses:
38.4 ± 7.8min for vehicle controls versus 43.3 ± .6min for testosterone-treated rats. These
findings suggest that chronic high-dose testosterone does not enhance appetitive drive for
sexual behavior [14653].
Case report
A 32-year-old man complained about a reduction of testicular volume and loss of libido. He
had been abusing androgenic anabolic steroids for 7 years. Genital examination revealed
that both testicular volumes were reduced to 13 ml. Endocrinological investigations showed
luteinizing hormone, follicle-stimulating hormone and total testosterone levels to be low. The
level of free testosterone was documented to be high. Later, sex hormone-binding globulin
(SHBG) and calculated bioavailable testosterone (cBAT) levels were found to be low. Based
on these features, it was diagnosed hypogonadotrophic hypogonadism caused by AAS
abuse. Treatment with injections of human chorionic gonadotropin (hCG) was started. About
one month after treatment with hCG started, his symptoms and endocrinological features
were not improved. It is also reported that normal hormonal function usually recovers after
AAS are discontinued, but sometimes the condition is not reversible [08144].
Adverse effects and treatment recommendations
AAS users commonly report side effects that they consider to be esthetically unpleasing,
such as testicular atrophy, fluid retention, acne, gynecomastia, and alopecia. Sexual
dysfunction was reported by 25 percent of users, and symptoms of androgen deficiency,
including fatigue and depression, are common complaints, especially during a post-cycle
period. Polycythemia also is a common adverse event, occurring in about 40 percent of
patients. Long-term AAS users may have serious underlying hepatic, renal, and cardio-
vascular disease, with hypertension and dyslipidemia common among chronic users. ASIH
1210
arises from the combination of hyperandrogenism, resulting from the supraphysiologic
supplementation of AAS, and subsequent hypogonadism. This testosterone deficiency
occurs because typical AAS users alternate between “on-cycle” supraphysiologic plasma
androgen levels and periods of androgen deficiency where ancillary drugs such as SERMs,
AIs, and hCG are used in attempts to recover the HPG axis Via suppression of estrogen and
thus its negative feedback, the hypothalamus can restart the HPG axis [14427].
Commonly reported user-reported side effects and concerns, user strategies for manage-
ment, and physician recommendations [14427]:
What should physicians
Side effect User strategies for management
recommend?
Low Discontinue AAS

endogenous
SERMs to restart axis
testosterone Start recovery protocol with TRT,
SERMs, or hCG
Chronic gynecomastia likely

unresponsive to medical
management
Surgical management is best option
Tamoxifen
for chronic gynecomastia
Aromatase inhibitors
Gynecomastia Acute gynecomastia may be treated
with tamoxifen per SERM recovery
Cabergoline and bromocriptine
protocol
for galactorrhea
Avoid hCG use if possible. Use of
aromatase inhibitors is discouraged
because of possible sexual side
effects
Testicular atrophy will resolve

discontinuation of AAS and recovery
Testicular
of HPG axis function
atrophy hCG injections
hCG should be reserved for cases
unresponsive to first line SERM
treatment
PDE5 inhibitors should be first-line

PDE5 inhibitors
treatment
Herbal aphrodisiacs
Sexual Herbal aphrodisiacs should be
Cabergoline
dysfunction discouraged owing to contamination
Mesterolone for added
concerns
androgenic effects
Dapoxetine not yet approved for
Dapoxetine
sexual dysfunction
Users of oral AAS concerned Encourage discontinuation of oral

Hepatic
with hepatic function may take AAS and herbal supplementation
dysfunction
herbal supplements such as milk Perform complete metabolic panel to
thistle extract for liver protection assess liver function
Alopecia Users often prophylactically take Although AAS use may worsen
finasteride to prevent hair loss existing alopecia, 5-alpha-reductase
1211
What should physicians
Side effect User strategies for management
recommend?
inhibitor use should be discouraged
because it may worsen symptoms of
ASIH
Diagnostic and treatment recommendations
Initial testing typically consists of a hormonal panel (LH, FSH, E2, T, free T, SHBG, and
PRL), complete blood cell count, lipid profile, prostate-specific antigen, and a comprehensive
metabolic profile. Common post-cycle complaints include depressive mood alterations,
fatigue, lethargy, insomnia, and decreased libido, and any such symptoms should be
addressed. Physical examination should include height, weight, blood pressure, and body
mass index, and common signs consistent with AAS use, such as acne, gynecomastia,
testicular atrophy, skin striations, and alopecia should be noted if present. For AAS users
seeking treatment and assistance in permanently discontinuing AAS, certain steps should be
taken. Following establishment of a nonjudgmental, healthy, and trusting physician-patient
relationship, the patient should be counseled to discontinue all AAS as well as any self-
administered ancillary drugs and supplements. For the severely symptomatic patients, a 4-
week tapered course of transdermal or injectable TRT may provide immediate symptom
improvement. Simultaneous administration of a SERM (such as clomiphene citrate, 25 mg
every other day) will interact at the hypothalamus causing stimulation of LH and ultimately
increase intratesticular testosterone. For patients with ASIH-induced gynecomastia, 20 mg
tamoxifen daily will block the breast estrogen receptors and stimulate HPG axis recovery
[14427].
After 4 weeks of treatment with TRT and/or a SERM, repeated hormone panels should be
obtained. If the patient has had either a poor gonadotropin response or a poor T response,
the authors commence a 4-week course of hCG (1,000–3,000 IU, 3 times per week) while
continuing daily treatment with a SERM at the initial starting dose. If a patient develops
gynecomastia while on hCG, tamoxifen (10 mg b.i.d.) or anastrazole may be commenced.
After 8 weeks of hCG and adjunctive treatment, hormone levels should once again be
assessed. At this point, if the total serum testosterone remains low and the patient continues
to be symptomatic, primary testicular failure is likely. These patients will require a longer
duration of TRT to avoid permanent ASIH. If appropriately increased serum T and
gonadotropin levels are observed, the SERM may be reduced to 50 percent of its starting
dose at 10 weeks of treatment and continued through weeks 12-16 or until target serum T
level is achieved. Recovery of hormonal function may be limited in men with testicular failure,
and close monitoring is recommended [14427].
Management of sexual dysfunction

Erectile dysfunction and decreased libido are common complaints of AAS users, especially
during the post-cycle period when endogenous T levels are lowest. Adding to the complexity
of evaluating these patients, the types of AAS used may contribute uniquely to the
pathophysiology of AAS-induced sexual dysfunction. Certain synthetic AASs, such as
nandrolone, have a reputation for causing erectile dysfunction when used alone. This effect
is likely due to an unopposed progestin-like action of the steroid along with the relatively
lower androgenic activity of its 5-alpha metabolite dihydronandrolone (compared with
dihydrotestosterone). By concurrently administering injectable T, AAS users attempt to
mitigate the sexual side effect profile of synthetic AASs such as nandrolone [14427].
More than 25 percent of users report using PDE5i either prophylactically or as treatment for
erectile dysfunction. Several popular Internet AAS suppliers offer drugs such as dapoxetine,
bromocriptine, and cabergoline as well as PDE5i. In addition, AAS users commonly purchase
1212
over the counter herbal “aphrodisiacs” that have been previously found to contain designer
analogues of licensed PDE5i. Indeed, for AAS users, initial therapy for erectile dysfunction
consists of PDE5i. Although controversial, the restoration of a normal hormonal milieu may
be important for optimal response to oral PDE5i therapies [14427].
Management of inferility and testicular atrophy

AAS use can be an important cause of male-factor infertility. Although several recent reviews
have addressed the effects of androgen consumption on male fertility, some clinicians
remain unaware of the fact that the use of exogenous androgens suppresses the HPG axis
and, by decreasing intratesticular testosterone (ITT), results in infertility. Because an
adequate ITT concentration is necessary for spermatogenesis, it is not surprising that AAS
users have presented to fertility clinics with azoospermia or oligospermia as well as sperm
dysmorphia and dysmotility. A return of ITT is the most important factor for restoration of
spermatogenesis, and therefore initial management of AAS-induced infertility should parallel
strategies for the correction of the underlying hypogonadotropic hypogonadism. A review of
the literature suggests that most cases of AAS-induced oligospermia or azoospermia are
likely to resolve spontaneously within 4-12 months after AAS discontinuation. Although some
authors have argued for reserving medical treatment for cases of azoospermia lasting >24
months, SERMs and/or gonadotropins have been successfully used after much shorter
intervals of AAS cessation. Spermatogenesis recovery time, however, with or without
medical treatment, appears to be highly variable and is difficult or impossible to predict for an
individual patient. In a case series of four azoospermic men, it was reported on the
spontaneous return of sperm concentration to normal levels over a variable period of 5-18
months after AAS cessation [14427].
As far as we know, cases of persistent azoospermia despite exhaustive medical treatment

have not been described in the literature. Clearly, the management of AAS-induced male
infertility should begin with conservative or medical management. Histopathologic
abnormalities, such as sperm maturation arrest, have been described in AAS users and
animal models, and although there are no published data confirming the success of sperm
retrieval techniques with subsequent IVF-ICSI for cases of AAS-induced azoospermia,
invasive procedures such as microdissection testicular sperm extraction (micro-TESE) may
be used for the exceedingly rare case of unrelenting azoospermia that does not resolve after
a thorough attempt at medical treatment has been made. Regarding testicular atrophy, hCG
preserves testicular function and prevents testicular atrophy. Treatment with hCG is known to
increase testicular volume, based on studies in patients with hypogonadotropic
hypogonadism. Furthermore, AAS users typically self-administer hCG at low doses, such as
250-500 IU subcutaneously or intramuscularly daily or every other day for several weeks
toward the end of long cycles and through the first few weeks of their post-cycle regimen.
SERMs may be equally efficacious for the prevention of AAS-induced testicular atrophy,
although quality comparative studies are not available. hCG may be added to the protocol if
response to primary SERM treatment is inadequate [14427].
Reproductive-endocrine effects in women
The sexual and reproductive effects of AAS are more dramatic in women. Although AASs
have been developed to try to minimize androgen effects, all AASs exert some degree of
virilizing effects if given for long enough and in sufficiently large doses. Virilization occurs
with AAS use by women, regardless of the type used. Early effects include acne, deepening
of the voice, and changes in libido. Deepening of the voice occurs as a result of laryngeal
hypertrophy. Long-term use can lead to clitoral enlargement, male-pattern baldness, and
1213
alterations in pubic hair. Other virilizing effects include decreased body fat, breast atrophy,
amenorrhea or oligomenorrhea, uterine atrophy, and hirsutism. The changes in menses are
due to suppression of the hypothalamic-pituitary-gonadal axis. Some of these effects may be
irreversible with chronic use. AASs may act as a teratogen [07058].
In females, delayed menarche, dysmenorrhoea, oligomenorrhoea, secondary amenorrhoea,

anovulation and, as their consequence, infertility are the changes most often attributed to
AASs abuse. However, physical and athletic activity often result in reproductive
disregularities due to disruption of the GNRH pulse generator at the hypothalamic level. This
leads to a decrease in LH and FSH and thus to decreased oestrogen production. A
population-based survey among 3887 Norwegian women revealed that those who were
physically active on most days were 3.2 times more likely to have fertility problems than
inactive women. Exercising to exhaustion caused a further increase in fertility problems.
However, after terminating the active sport, the number of nulliparous women was the same
in the inactive and formerly active women. When the influence of physical activity in 2232
women undergoing IVF treatment was investigated, those women who exercised 4 or more
hours per week for 1 to 9 years were 40 percent less likely to experience a live birth in the
first IVF cycle than those who did not exercise at all. Among 717 of 849 elite female athletes
participating in the 2011 IAAF Championship using neither hormonal contraceptives nor
AASs, 168 were oligo- or amenorrhoic. Only five of the 849 women were identified as AASs
abusers. This indicates that ovulation and menstrual disorders leading to infertility are
common among physically active women and especially among competitive athletes, even
without AASs abuse [150001].
Furthermore, the type of sport and the body composition required influence reproductive
functions. Ballet dancers and competitive gymnasts start strenuous training at an early age
and retain a lean physique with extremely low fat mass. Consequently, their menarche
occurs 2 years later than in less active girls. In runners, menstrual disorders occur in 25
percent on average, with frequency correlating positively with distances covered per week.
Swimmers have fewer irregularities than other athletes, probably due to their higher
oestrogen-generating fat mass than other sportswomen. The lack of oestrogens may
become so severe that the syndrome of the “female athlete triad” (disturbed energy balance
due to disturbed eating behaviour, menstrual irregularities and low bone mineral density) has
been identified as a severe consequence of intensive sport activity. The high frequency of
reproductive anomalies among female athletes highlights the difficulty in disentangling the
effects of exhausting sport activities and of AASs abuse in the absence of controlled studies
and based on only few case reports. In cases of the female athlete triad, it has been
speculated that (moderate) AASs intake could prevent some of the symptoms. To approach
the possible influence of androgens on the female organism, investigations on the
therapeutic use of testosterone in women may be consulted. A large investigation with the
aim of evaluating the side effects of testosterone administration in therapeutic doses in
women showed no significant differences concerning the frequency of cerebrovascular
diseases, coronary heart disease, breast carcinoma, deep venous thrombosis/lung
embolism, diabetes mellitus, or acute hepatitis between women receiving testosterone
therapy and the control group. If changes in the reproductive system due to suppression of
the hypothalamic-pituitary-gonadal axis, such as dysmenorrhoea, secondary amenorrhoea
with anovulation or reduction of breast size, are attributed to AASs abuse, they should also
be reversible if caused by AASs. It can take weeks or months up to complete recovery of the
axis. In some cases, it has been reported that after cessation of AASs administration in
women it took up to 20 months until testosterone concentrations in serum dropped to normal
levels, correlating with observations on spermatogenesis in male AASs abusers (see above).
Concerning possibly irreversible side effects of AASs use in women, such as clitoris
hypertrophy, no well-documented case reports or studies are available [150001].
1214
In women users of AS, menstrual irregularities or cessation of menstruation are common
[03002].
Research conducted in adult laboratory rats and mice has demonstrated that AASs disrupt
estrous cyclicity and gonadotropin secretion. Of particular relevance to the present study are
clinical findings that use of AASs by adolescent girls has increased recently. The
physiological effects of AASs on the developing neuroendocrine axis have largely been
unexplored, and there is concern about the nature and duration of potential neuroendocrine
disruptions. Analyses of the effects of AASs in prepubertal laboratory animals can provide
insights into the consequences of AASs on the maturing female neuroendocrine system.
The female rat has been used as a model system for the examination of the neural and
hormonal events underlying puberty. Various aspects of female physiology and
neuroendocrine function have been used as markers of puberty. Specifically, vaginal opening
(VO) is one early and visible indicator of the increasing estrogen titers that accompany the
onset of puberty. The culmination of the maturation of the adult pattern of functioning of the
hypothalamic-pituitary-gonadal (HPG) axis is signaled by the first vaginal estrus (VE), itself
precipitated by ovulation. Last, the adult pattern of estrous cyclicity is established. These
three measures were used in the present study to assess the effects of long-term
administration of AASs on puberty in female rats. In a previous study, it was reported the
effects of a single injection of one AAS, stanozolol, on the onset of puberty. Administration of
stanozolol on Postnatal Day 21 (PN21) advanced the day of VO without affecting the day of
VE. These results suggest that a brief exposure to stanozolol does not produce the hormonal
conditions necessary to elicit true precocious puberty and maturation of the HPG axis. Users
of AASs typically take these compounds for prolonged periods of time (weeks or months) as
opposed to a single exposure. Use of anabolic-androgenic steroids (AASs) is becoming
increasingly popular among adolescent girls, yet the effects of AASs on female physiology
and development are not well understood. The present study compared the effects of chronic
exposure to three individual AASs, stanozolol (0.05-5 mg/kg), 17alpha-methyltestosterone
(0.5-5 mg/kg), and methandrostenolone (0.5-5 mg/kg) on the onset of puberty and estrous
cyclicity in the rat. Female rats received daily injections of AASs for 30 days (Postnatal Day,
PN, 21-51). Rats receiving the highest dose of each of the AASs (5 mg/kg) displayed vaginal
opening at a younger age than rats receiving the oil vehicle. The day of first vaginal estrus
was delayed in rats receiving stanozolol (5 mg/kg) or 17alpha-methyltestosterone (0.5-5
mg/kg) but not in rats receiving methandrostenolone. At the highest dose (5 mg/kg), each of
the AASs reduced the incidence of regular estrous cyclicity during the treatment period.
Concurrent administration (on PN21-51) of the androgen receptor antagonist, flutamide (10
mg/kg, twice daily), reversed the effects of 17alpha-methyltestosterone (5 mg/kg) on vaginal
opening. Flutamide administration also eliminated the effects of stanozolol (5 mg/kg) and
17alpha-methyltestosterone (5 mg/kg) on the day of first vaginal estrus. In contrast, rats
receiving flutamide and methandrostenolone (5 mg/kg) exhibited first vaginal estrus earlier
than controls. The present results indicate that chronic exposure to AASs during
development has deleterious effects on the female neuroendocrine axis and that these
effects appear be mediated via multiple mechanisms [03054].
Other female-specific side effects
The psychological effects of illicit AAS use in women have seldom been studied. In two
studies, female athletes reported an increase in aggressiveness when on steroids. It was
assessed the psychiatric status of female athletes attending gyms. Of these women, one-
third had a history of AAS abuse. The researchers observed a number of mental
1215
abnormalities among AAS users, including polysubstance dependence, hypomanic
symptoms, depressive symptoms during withdrawal, rigid dietary practices, non-traditional
sex roles and chronic dissatisfaction and preoccupation with their physiques (“muscle
dysmorphia”) [04002].
Female-specific side effects of AAS include hirsutism, increased facial hair, voice deepening,
clitoral hypertrophy, oligomenorrhea, amenorrhea, reduced breast tissue, and male-pattern
baldness. Even after the discontinuation of AAS, some of these changes, such as a deeper
voice, facial hair growth, and loss of scalp hair, may be permanent and devastating [04018].
Research exploring the effects of AAS in females is scarce; the administration of AAS will
induce masculinisation in women. Female bodybuilders reported the development of acne
vulgaris, changes in libido and alterations of the voice as the most pronounced adverse
effects in the first weeks of AAS use.Long-term AAS administration may induce loss of hair of
the head, alterations of pubic hair growth and enlargement of the clitoris. Furthermore,
menstrual irregularities and a reduction of the breasts usually occur. Finally, adolescents
may be prone to early closure of growth plates resulting in premature stop of length growth
[04002].
Hirsutism and alopecia
Hirsutism and alopecia are frequent, but in most instances reversible side effects of
androgen and AASs use in women. Assessment of body hair and hirsutism has to take
ethnic dispositions into account. The degree of increased facial or body hair growth depends
on dose and duration of AASs abuse and can be described according to the hirsutism score
by Ferriman-Gallwey, established in 1961. Based on the intensity of hair growth in nine
face/body areas, hirsutism can be diagnosed as mild, moderate, or severe. However, proper
analysis of the grade of hirsutism and alopecia in AASs abusers has not been undertaken
[150001].
Deepening of the voice
Lowering of the voice is caused by growth of the larynx in girls and by thickening of the vocal
chords in women after puberty and can be monitored objectively. As laryngeal tissue has
androgen receptors, the voice is part of the virilisation that androgenic substances and AASs
can cause in women. The voice is an important phenotypic characteristic of a person's
identity and changes are easily recognised during social contacts. The voice change can be
so pronounced that on the telephone women may be mistaken for men. It is accompanied by
hoarseness which may intensify upon longer use of the voice. This dysphonia may become a
problem for teachers, actors, and singers who are professionally dependent on their voices.
Such voice alterations are observed with endogenous elevation of testosterone levels e.g.
congenital adrenal hyperplasia or in women sensitive to the androgenic action of some oral
contraceptives. Effects of androgens prescribed for other than doping purposes in women
(endometriosis, climacteric complaints, low libido, cellulitis etc.) have been described in some
detail. In low-dose transdermal testosterone trials, 12/545 postmenopausal women receiving
placebo and 15/549 on testosterone reported voice changes, emphasising the importance of
controlled studies when evaluating subjective parameters. In contrast to acne, hirsutism,
alopecia and mammary atrophy, deepening of the voice due to AASs tends to be irreversible.
However, although deepening of the female voice is mentioned in all pertinent reviews
dealing with AASs abuse, it is surprising that no systematic investigations exist, and even
case reports are very rare. Most information is anecdotal and some comes from telephone
interviews or hotlines. For example, 11 percent of 217 women consulting the anti-doping
1216
hotline of a Swedish university hospital complained of hoarseness or lowering of the voice;
however, as only those with complaints use such hotlines, the figures are not representative.
As changes in the voice are mostly irreversible, androgen application must be suspended at
the earliest sign of symptoms, if they are to be avoided [150001].
Risk of breast cancer
Breast cancer is the most frequent carcinoma in women with 96 new cases per 100 000
women and year in Western Europe. This high prevalence has to be kept in mind when
considering any additional changes in AASs abusers. The effect of exogenous androgens on
the development of breast cancer has been discussed controversially in the scientific
literature. The lack of controlled studies and epidemiologic investigations contributes to the
uncertainties so that indirect evidence from other clinical situations has to be referred to. In
premenopausal women – the group to which most AASs abusers belong – most studies do
not demonstrate an association between serum testosterone levels and breast cancer risk. In
postmenopausal women, however, a small increase in the risk for breast cancer in
correlation with testosterone and androstenedione serum levels was found, but only in E+/P+
cancers [150001].
In recent years, low-dose testosterone – mainly transdermal – has been used for the
treatment of female sexual dysfunction, in particular of hypoactive sexual desire syndrome
(HSDD). In this context, the risk of breast cancer has become a concern. Recent reviews and
practice guidelines find no evidence for an increased risk, but also conclude that no
randomised controlled trials (RCTs) have been of sufficient size or duration to provide a
definitive answer concerning the impact of testosterone on breast cancer risk. Experience
with long-term hormonal therapy in transsexuals (female-to-male) aiming at virilisation
(standard therapy: testosterone enanthate 250 mg i.m. every second week or testosteone
undecanoate 1000 mg every 10-12 weeks for 2-3 years before surgical therapy, e.g.
mastectomy, ovarectomy and hysterectomy, and for years after that) shows no increased risk
for breast cancer. Since the 1970s, when hormonal therapy of transsexuals was first
documented, only one clinical case has been reported; in this case a mamma carcinoma of
the residual breast tissue developed 10 years after bilateral mastectomy and continuous
testosterone therapy. The polycystic ovary syndrome (PCOS) is characterised by a
significant increase in the testosterone concentration in blood and often serves as a model
for long-term testosterone exposure in women. Studies showed that the risk for breast
cancer in these women does not increase [150001].
Exogenous androgens are partially metabolised to oestrogens in breast tissue. However, not
all synthetic androgens are subject to aromatisation, e.g. tibolone and its metabolites cannot
be aromatised. This also applies to the metabolism of oralturinabol (chlordehydromethyl-
testosterone) predominantly used in the former GDR. Unless taken at extremely high doses,
the molecule is not aromatised, so that oestrogenic side effects become clinically irrelevant.
A large randomised study showed that postmenopausal women receiving estrogens
exclusively did not have an increased risk of mammary carcinoma, in contrast to women who
received an oestrogen/gestagen combination. The age of the patient and the duration of
oestrogen therapy are considered as risk factors for the development of breast cancer in
women. Comparable results have also been shown in other studies. However, women who
received hormone replacement therapy (estrogen or estrogen/gestagen preparations) at the
time point of the evaluation, in comparison with those who had never taken hormonal drugs,
had a higher risk for development of breast cancer. Women who in the past had received
hormonal therapy did not have a higher risk for mamma carcinoma. It has also been shown
that additional administration of testosterone during hormonal replacement therapy in
postmenopausal women (estrogen-gestagen preparations) inhibited the proliferation of
1217
breast cells and thereby decreased the risk of mammary carcinoma. A recent 5-year interim
analysis of a 10-year prospective study has demonstrated that in women treated with
testosterone implants the incidence of breast cancer was significantly reduced compared
with untreated women [150001].
In animals and also in postmenopausal patients androgens (e.g. testosterone, dihydro-

testosterone (DHT)) blocked proliferation of breast cells in vitro, caused by oestrogens as
well as expression of oestrogen receptor genes. The antiproliferative and proapoptotic
actions of androgens are probably mediated through the androgen receptor, despite the
potential of testosterone to metabolise to oestrogens. Before these interrelations were
known, advanced stages of mammary gland carcinoma had even been treated with
testosterone from the 1940s until the 1970s. The underlying clinical experience was that
testosterone inhibits rather than proliferates a breast carcinoma. A genetic disposition
concerning mutations in BRCA1 and BRCA2 genes (breast cancer gene) can exhibit a higher
risk for the development of a breast carcinoma. In conclusion, there are no appropriate
epidemiologic studies which clearly document or negate a causal connection between the
administration of AASs in young female athletes and the development of breast carcinoma
later in life. Nor is there an accumulation of case reports which would argue for such a
connection. Indirectly, it can be assumed that the use of AASs at young ages cannot be
causal for breast cancer. However, as in the case of clinical low-dose testosterone treatment,
sufficiently powered epidemiologic studies are required to provide a definitive answer
concerning the breast cancer risk in AASs abusers [150001].
Skin
The use of AASs can very rapidly lead to cutaneous changes in previously unaffected
athletes so that the dermatologist may be among the first physicians to be confronted with
AASs abuse. AASs act through the androgen receptor, presenting in epidermal and follicular
keratinocytes, sebocytes, sweat gland cells, dermal papilla cells, dermal fibroblasts,
endothelial cells, and genital melanocytes. The effects are mediated through affecting the
sebaceous gland growth and differentiation, hair growth, epidermal barrier homeostasis and
wound healing. The AR polymorphism appears to play a role in the severity of symptoms.
The most frequent skin manifestations are acne vulgaris, oily skin, seborrhoea, striae,
hirsutism and male pattern alopecia. The incidence of acne in AASs abusers ranges from 17
percent in persons consulting a Swedish anti-doping hotline to over 50 percent of athletes
taking part in a questionnaire aiming to identify unsupervised AASs regimens and side
effects of AASs. After elimination of the causal agent, these changes are mostly reversible.
To speed up recovery, anti-androgen therapy with cyproterone acetate or spironolactone
may be tried. However, severe forms of AASs-induced acne conglobata will leave extensive
scarring on the affected skin areas. After acne, striae distensae as a result of rapid muscular
hypertrophy, supported by AASs intake, is the most prevalent skin side effect in athletes,
especially in bodybuilders. Over 40 percent of athletes complained about stretch marks of
the skin with typical localisation in the musculus pectoralis or upper arm region. After
discontinuation of drug misuse, striae can persist as white streaks [150001].
Anabolic-androgenic steroids (AAS) abuse by the athletes has dramatically increased during
the recent decades. Consumption of high doses of AAS may cause skin diseases like acne
vulgaris and folliculitis are more common in AAS users. These AAS increase the activation of
sebaceous glands and consequently cholesterol and free fatty acids of the skin surface lipids
which in turn may provide a better condition for colonization of some lipophilic bacteria such
as Propionibacterium acnes and Staphylococcus aureus. Moreover, secretion of lipase by
these bacteria, known to be resistant to antimicrobial activities of the fatty acids, provides a
1218
suitable environment for colonization in sebaceous follicles which in turn may present as
sebaceous follicles comedones and inflamed lesions such as papules, pustules, and cysts.
One study aimed to investigate the potential side effects of AAS on the bacterial microflora
colonization of the bodybuilders` skin. The skin samples of 94 male bodybuilders (71 AAS
users, 23 non-AAS users) and 46 subjects of the control group, with similar gender and age,
were cultured and incubated in both aerobic condition to isolate Staphylococcus aureus and
anaerobic condition for Propionibacterium acnes. The isolated bacteria were identified by
standard microbiological techniques. The skin lesions were more frequent in the body
builders than the controls. Moreover, statistically significant differences were also observed
in skin lesions among the AAS users and the non-AAS user athletes. The prevalence of S.
aureus and P. acnes in the athletes was higher than that of the control group. In addition,
there was a significant difference in distribution of P. acnes between the bodybuilders who
used AAS and those who did not. It was concluded that a higher number of bacterial flora
was found in the bodybuilders particularly those using AAS in comparison to the controls,
which might be due to the influence of these AAS on the skin microflora and transmission of
the bacteria through the direct contact of the naked skin with the exercise instruments
[150223].
Cutaneous manifestations develop early in the use of anabolic-androgenic steroids, placing

dermatologists in a unique position to make an early diagnosis of AAS abuse in patients who
engage in competitive sports. One review of the literature focuses on dermatologic
presentations of anabolic-androgenic steroids use [09055].
The most common dermatologic effects of AAS abuse are alopecia, male pattern baldness,
and hirsutism, particularly in women. Other adverse dermatologic effects include keloid
formation, sebaceous cysts, comedones, seborrheic furunculosis, folliculitis, striae, and acne.
A common triad of chronic AAS abuse is the combination of acne, striae, and gynecomastia.
Hyperplasia of the pilosebaceous glands and increased sebum production cause a high
incidence of acne in chronic AAS abusers. These effects become prominent about 1 month
after initiation of AAS abuse, depending on the dose and frequency of use [13003].
Dermatologic changes such as acne, striae, alopecia, and hirsutism are induced by the
action of dihydrotestosterone on ARs in skin and sebaceous glands. High doses of AAS
cause acne by increasing skin surface lipids and the cutaneous population of
propionibacteria acnes. Cutaneous striae are the result of rapid gains in body mass, in which
the skin is unable to accommodate the rate of stretch, and a secondary effect that AAS may
have on collagen reducing skin elasticity [04018].
Moreover, the effects on skin may be profound. It has been found enlargement of sebaceous
glands, increase of sebaceous production and elevation of skin surface lipid cholesterol
levels due to AAS. In several case studies the development of dermatological adverse
effects have been described, including occurrence of acne vulgaris, acne fulminans,
hereditary coproporphyria, linear keloid formation and exacerbations of psoriasis. However,
whether these reported conditions are adverse effects of AAS or not has yet to be
established since, in contrast, AAS are used in experimental clinical trials in the treatment of
several dermatological disease conditions [04002].
Acne
A 22-year-old male amateur bodybuilder presented with a 3-month history of severe acne
lesions on his upper trunk and face, accompanied by arthralgia of several joints. He reported
the use of anabolic androgenic steroids (AAS) (testosterone enanthate, trenbolone acetate,
1219
drostanolone propionate, and methandrostenolone) for 3 months to increase his muscle
mass. Shortly after he discontinued AAS intake, he developed severe inflammatory acne with
painful rupturing and draining inflammatory nodules, pustules, and hemorrhagic ulcerations
on his upper trunk and face. Moreover, he described an immobilizing arthralgia of his right
ankle and both shoulder joints, as well as general symptoms including fatigue and a 15-kg
weight loss over the 6 weeks prior to presentation. As derivatives of the hormone
testosterone, AAS lead to hypertrophy of the sebaceous glands, increased sebum
production, and increased density of the Propionibacterium acnes population. The patient
developed AAS-induced acne fulminans with the typical unresponsiveness to systemic
antibiotics. After initial therapy with oral prednisolone, 0.5 mg/kg, and debridements, a clinical
response was achieved with isotretinoin, 0.75 mg/kg. In conclusion, it is important for health
care providers to keep in mind that androgen-induced acne is one of the most frequent
symptoms of AAS abuse. The most important measure is the immediate termination of AAS
administration [12141].
Severe cases of acne, especially on the face and back of AAS users, are common
dermatologic findings. Premature baldness is noted as well. It has been reported multiple
cases of serious muscular abscesses resulting from the common practice of shared needles
and shared steroid vials among adolescent AAS users. A limited knowledge of sterile
injection technique, as well as limited access to sterile needles and syringes are likely
additional causative factors in these infections [07008].
Abuse of anabolic-androgenic steroids (AAS) by members of fitness centers and others in

Germany has reached alarming dimensions. Besides health-threatening cardiovascular,
hepatotoxic and psychiatric long-term side effects of AAS, acne occurs in about 50 percent of
AAS abusers and is an important clinical indicator of AAS abuse, especially in young men
18-26 years of age. Both acne conglobata and acne fulminans can be induced by AAS
abuse. The dermatologist should recognize bodybuilding acne, address the AAS abuse, and
warn the patient about other potential hazards [07074].
Men experience male-pattern baldness, acne (mostly on the trunk), which likely is due to the
effects of DHT. Acne is the result of androgenic stimulation of sebaceous glands [07058].
Hirshutism
Hirsutism is a symptom or sign, which may have more serious associations than cosmetic
and psychological concern alone, such as adrenal hyperplasia and ovarian tumor,
particularly if it develops well after puberty. Some medicines having androgenic activity may
also cause this problem. It was presented a case of a young unmarried girl who was given
anabolic steroid for the treatment of dysmenorrhoea which resulted in hirsutism [06092].
Androgenic alopecia (baldness)
Adolescent androgenic alopecia is pattern hair loss occurring in boys and girls younger than
18 years, whereas early-onset androgenic alopecia refers to pattern hair loss before 35 years
of age. A number of studies published in the last decade have helped to elucidate the
prevalence of adolescent androgenic alopecia, have clarified the genetic as well as
physiologic mechanisms underlying hair loss, and have revealed the associated psychologic
and systemic morbidities. One article provides an overview of the pathophysiology,
diagnosis, and treatment of adolescent androgenic alopecia [11448].
In establishing a theory to predict male-pattern baldness, it was investigated the correlation
1220
of testosterone, epitestosterone, and dihydrotestosterone with 5alpha-reductase in hair using
gas chromatography-mass spectrometry. One hundred milligram hair samples were obtained
from a group of balding subjects and their sons, as well as from a corresponding aged-
matched, nonbalding group. The ratio of testosterone to epitestosterone was significantly
greater in the hair of balding fathers (n=19, age 28-50 years) and their sons (n=16, age 8-16
years) than in the hair of the nonbalding control subjects. These findings demonstrate that
analysis of terminal hair may not only provide a basis for predicting baldness when the
subject is still young, but also for preventing and treating male-pattern baldness by
controlling the steroid hormone balance [01069].
Effects on pancreas
One patient in the Intensive Care Unit was a case of acute pancreatitis. It was diagnosed on
the basis of raised amylase level with ultrasonographic findings of swollen pancreas with
inflammatory changes in patient of epigastric pain, which was later confirmed on computed
tomography scan. Androgenic anabolic steroids have grown in popularity amongst athletics
and bodybuilders due to their ability to enhance performance, muscle mass, and aesthetic
reasons. They are easily available and perceived to be safe. Recent estimates place AAS
use in the USA at 1 percent of the population. These agents have numerous side effects.
Pancreatitis as a complication of AAS is not much reported but of corticosteroids is well
documented. The usual causes of pancreatitis were excluded in our patient on the basis of
history, lab reports, and ultrasonography. Researchers have recently discovered evidences
that suggest anabolic steroids may demonstrate potentially new and serious adverse
consequences. Evidence obtained from a clinical trial suggests that acute pancreatitis and
acute kidney injury can be caused by the use of anabolic steroids like methandrostenolone. It
was reported a case of 50-year- old man who develop acute pancreatitis and acute kidney
injury which was attributed to AAS and described a case report of multi-organ damage after
the use of anabolic steroids. They suspected anabolic steroid causes hypercalcemia. They
also suspected that besides hypercalcemia, acute pancreatitis have resulted from overuse of
amino acid supplements [150216].
Effects on the immune system
Anabolic-androgenic steroids are potentially immunosuppressive based on a study

suggesting that chronic AAS abuse reduces immunoglobulins (i.e. IgA), increases natural
killer cell activity, and increases mitogen response to staphylococca lantigens. There were no
significant differences in T-cell subsets among steroid users and non-users in this study.
However, the clinical relevance of these potential immunosuppressive effects is unclear.
Sporadic case reports associated infectious complications from the parenteral use of AASs
including local abscess at the site of injection, septic arthritis, HIV, and hepatitis, usually as a
result of sharing contaminated needles [13003].
Infection risks after injections of anabolic steroids
In addition to the pharmacologic side effects of AAS, complications also result from the
injection technique used in self-administration. Infective complications usually result from
nonsterile injection technique, reusing needles, sharing needles, sharing multidose vials, and
contaminated drugs. Infections reported with AAS injection include bacterial abscesses,
septic arthritis, septic shock, and cross infection with blood-borne pathogens HIV, hepatitis B,
1221
and hepatitis C. Other injection complications arise from chronic needle stick injury or poor
injection technique. Frequent repeated injection into the same site can result in inflammation,
intramuscular fibrosis, dystrophic calcification, and oil-induced granuloma. Misplaced
injections have resulted in needle infections [04018].
To describe drug use, sexual risks and the prevalence of blood-borne viral infections among
men who inject image and performance enhancing drugs (IPEDs), 19 needle and syringe
programmes with 395 men who had injected PEDs were investigated Of the participants
(median age 28 years), 36 percent had used IPEDs for <5 years. Anabolic steroids (86 %),
growth hormone (32 %) and human chorionic gonadotropin (16 %) were most frequently
injected, with 88 percent injecting intramuscularly and 39 percent subcutaneously. Two-thirds
also used IPEDs orally. Recent psychoactive drug use was common (46 % cocaine, 12 %
amphetamine), 5 percent had ever injected a psychoactive drug and 9 percent had shared
injecting equipment. Viagra/Cialis was used by 7 percent, with 89 percent reporting
anal/vaginal sex in the preceding year (20 % had 5+ female-partners, 3 % male-partners)
and 13 percent always using condoms. Overall, 1.5 percent had HIV, 9 percent had
antibodies to the hepatitis B core antigen (anti-HBc) and 5 percent to hepatitis C (anti-HCV).
In multivariate analysis, having HIV was associated with: seeking advice from a sexual health
clinic; having had an injection site abscess/wound; and having male partners. After excluding
those reporting male partners or injecting psychoactive drugs, 0.8 percent had HIV, 8 percent
anti-HBc and 5 percent anti-HCV. Only 23 percent reported uptake of the hepatitis B vaccine,
and diagnostic testing uptake was poor (31 % for HIV, 22 % for hepatitis C) [13026].
The practice of self-injection of anabolic steroids (AS) in bodybuilders is common. AS are not
the only materials used by bodybuilders for muscle augmentation or image enhancement.
Other materials, for example, plant oils, silicon, Vaseline, and paraffin are also injected either
in a pure form or mixed with AS. Muscle bulking is the main aim. However, bodybuilders
undergo illicit injections for cosmetic, therapeutic, and sexual purposes. Even though the
practice of unsupervised injection is probably common in the sports community, site-specific
complications are underreported in the medical literature and mostly limited to case reports.
Complications can be clinically and pathologically challenging because some can be
confused with nonneoplastic and, more important, with neoplastic lesions. Bodybuilders are
reluctant to disclose information because of stigma and legal issues. This study attempts to
correlate the clinical manifestations and histomorphological features of different injected
materials used for different purposes by bodybuilders in our region. A series of 11 cases out
of 9 male bodybuilders was studied. A variety of clinical presentations and histological tissue
reactions was identified, with some overlapping features between some cases. We identified
5 basic tissue reaction patterns depending on the injected materials, site, and duration of
injection. Certain histological features provide useful hints in the absence of prior knowledge
of injection history. However, in other cases, a retrospective enquiry by clinicians is
warranted to avoid pitfalls. The medical and sports community should be aware of these
injection-site complications. Bodybuilders should be discouraged from this practice by
implementing appropriate educational and legislative measures [14758].
Despite intensive information on possible side effects and complications of performance-

enhancing substances in sports, the use of AAS (anabolic androgen steroids) is far common.
Particularly in sports like bodybuilding or weight lifting AAS are used for setting up muscle
mass and increasing muscle power. It was presented a case of a 27 year old bodybuilder,
who was transferred due to suspected malignant expansion of the upper limb to a
department of orthopaedic surgery, not knowing that the patient had injected AAS. At biopsy
the tumor was found to be an abscess formation, that had to be treated surgically with
curettage. The microbiological analysis detected an infection with Pseudomonas fluorescens
and Erwinia species. Erwinia species are associated with plants, Pseudomonas fluorescens
1222
is found in feces, sewage and soil. It is obvious, that the infection is caused by an
inappropriate injection of AAS or by the contamination of the injected substances [07077].
Detailed data on performance- and image-enhancing drug injections are difficult to obtain
because of the illicit and unsupervised way in which many these drugs are used, and the
hidden nature of the group. One study examined the patterns of use, risk behaviours and
related harm associated with injections. Data were obtained via a structured questionnaire
administered in face-to-face interviews with 60 men who used performance- and image-
enhancing drugs (primarily anabolic androgenic steroids) for non-medical purposes. Although
the rates of needle sharing were low (5 %), the men more frequently reported re-use of
needles/equipment, injecting from a shared container (bladders, vials, etc.), injecting other
illicit drugs, injecting insulin and targeting small muscle groups. Self-reports of being hepatitis
C antibody positive were associated with lifetime use of heroin and injection of other illicit
drugs. All HIV positive participants were gay or bisexual men. Participants reported a range
of other injection-related injuries and diseases such as fevers, scarring and abscesses.
“Risky injectors” (38 % of participants) were more likely to initiate performance- and image-
enhancing drugs use at a younger age, use these drugs in a larger number of cycles per
year and report involvement in a violent/aggressive incident than “low risk” injectors and
report involvement in a violent/aggressive incident than “low risk” injectors. Participants
mainly reported seeking information about performance- and image-enhancing drugs from
internet sites (62 %) and friends (55 %). It was concluded that over-reliance on personal
networks and internet forums limits this groups' access to objective harm reduction advice
and primary care services [08148].
In addition to the direct adverse effects of AAS, illicit users are vulnerable to infectious
complications associated with use of contaminated needles, contaminated products obtained
on the black market, or other risks associated with weightlifting and AAS use. Although
needle-sharing appears uncommon in modern American AAS users, one Internet survey
found that 65 out of 500 AAS users (13 %) reported unsafe needle practices, including
needle sharing, needle reuse, and sharing of multiple-dose vials. Moreover, respondents to
Internet surveys are likely better educated and more affluent than the population of AAS
users as a whole, so that Internet surveys likely underestimate the prevalence of unsafe
practices in the global population of AAS users. Thus it is not surprising that the literature has
documented various infectious complications of AAS use, including the blood-borne
pathogens, HIV, hepatitis B, and hepatitis C, as well as skin and soft tissue infections, most
notably due to community-acquired methicillin-resistant Staphylococcus aureus (MRSA). The
first report of HIV infection in an AAS user surfaced nearly 30 years ago and subsequent
reports in both the US, and Europe, have documented additional cases. AAS users have
also contracted hepatitis B and C. The greatest risk for transmission of HIV and other
diseases in AAS users appears to arise from needle sharing and other unsafe needle
practices. This is likely because the frequent use of injectable preparations, such as
testosterone and nandrolone, among long-term illicit AAS users. However, unsafe needle
practices represent only one possible risk factor for HIV and other infections in AAS users.
For example, a study of homosexual men in London gyms found that current AAS users
were significantly more likely than never-users to report unprotected anal intercourse with
partners of unknown serostatus, even in analyses adjusting for potential confounders. Given
that AAS are widely used by homosexual men, both illicitly, and as prescribed treatments for
the wasting syndrome associated with HIV infection, there is a clear opportunity for the
spread of HIV both through needles and sexual practices. AAS users are also likely to have
spent time in prison, and prisoners, in turn, are well-documented to display an elevated risk
for hepatitis and HIV. Studies have also reported soft tissue abscesses related to anabolic-
steroid injections [14017].
1223
In addition to the direct adverse effects of AAS, illicit users are vulnerable to infectious
complications associated with use of contaminated needles, contaminated products obtained
on the black market, or other risks associated with weightlifting and AAS use. Although
needle-sharing appears uncommon in modern American AAS users, one recent Internet
survey found that 65 of 500 AAS users (13 %) reported unsafe needle practices, including
needle sharing, needle reuse, and sharing of multiple-dose vials. Moreover, respondents to
Internet surveys are likely better educated and more affluent than the population of AAS
users as a whole, so that Internet surveys likely underestimate the prevalence of unsafe
practices in the global population of AAS users. Thus, it is not surprising that the literature
has documented various infectious complications of AAS use, including the blood-borne
pathogens, HIV, hepatitis B, and hepatitis C, as well as skin and soft tissue infections, most
notably due to community-acquired methicillin-resistant Staphylococcus aureus (MRSA). The
first report of HIV infection in an AAS user surfaced nearly 30 years ago, and subsequent
reports in both the United States and Europe have documented additional cases. AAS users
have also contracted hepatitis B and C. The greatest risk for transmission of HIV and other
diseases in AAS users appears to arise from needle sharing and other unsafe needle
practices. This is likely because of the frequent use of injectable preparations, such as
testosterone and nandrolone, among long-term illicit AAS users. However, unsafe needle
practices represent only one possible risk factor for HIV and other infections in AAS users.
For example, a study of homosexual men in London gyms found that current AAS users
were significantly more likely than never-users to report unprotected anal intercourse with
partners of unknown serostatus, even in analyses adjusting for potential confounders. Given
that AASs are widely used by homosexual men, both illicitly and as prescribed treatments for
the wasting syndrome associated with HIV infection, there is a clear opportunity for the
spread of HIV both through needles and sexual practices. AAS users are also likely to have
spent time in prison, and prisoners, in turn, are well-documented to display an elevated risk
for hepatitis and HIV. Studies have linked community-acquired MRSA colonization and soft
tissue infection with competitive sports participants. Additional research has linked injection
of drugs with community-acquired MRSA infection. Studies have also reported soft tissue
abscesses related to anabolic-steroid injections [14426].
A recent study of anabolic steroid injectors in England and Wales, reported that 9 percent
had shared injecting equipment. Of considerable concern for public health is the identification
of 1.5 percent being HIV positive, 9 percent with antibodies to the hepatitis B core antigen
(anti-HBc) and 5 percent to hepatitis C virus (anti-HCV). In the case of hepatitis B, biological
samples were only tested for core antigen identifying those that had been exposed to
hepatitis B, rather than the level of users who are carriers of the virus. Still, it is noteworthy
that the results of the study indicate comparable levels of HIV prevalence between injectors
of anabolic steroids and intravenous heroin injectors in England and Wales [14437].
Tetanus
One case in the Intensive Care Unit was a patient with tetanus. It was diagnosed on the
basis of typical clinical picture consisting of the jaw, neck, back stiffness and difficulty in
swallowing. There was also associated history of sweating over face and neck. Later on, he
developed typical recurrent tetanic spasms, for which he was intubated and kept on
mechanical ventilation. Diazepam along with the atracurium infusion was given. He was
regularly taking AAS intramuscular injection as a self-prescribed and self-injected
medication, as part of bodybuilding activity. There was no evidence of mode of transmission
for tetanus other than intramuscular injection. This mode of transmission for tetanus is well
reported in the literature [150216].
1224
Injection policies
Anabolic-androgenic steroids (AAS) and other performance-enhancing drugs (PEDs) are

commonly misused to increase muscle size and strength, as well as improve physical
appearance. Many AAS and certain PEDs are administered via injection and therefore pose
a risk for transmission of infectious diseases such as human immunodeficiency virus (HIV),
hepatitis B virus (HBV), hepatitis C virus (HCV), and skin and soft tissue infections (SSTIs).
Further, AAS users may be more likely to take part in high-risk sexual behaviors than non-
AAS users. This review explores the prevalence of infectious diseases as well as risky
injection practices and sexual behaviors of AAS users in the current literature. A
comprehensive MEDLINE search (1984-17 April 2015) for English language reports was
performed on AAS users. Ten studies analyzed the prevalence of HIV infection, 6 studies
analyzed HBV infection, and 6 studies analyzed HCV infection; 20 studies analyzed injection
practices and 7 studies analyzed high-risk sexual behaviors of AAS users. HIV, HBV, HCV,
and SSTIs have been associated with AAS users. In particular, HIV infection seems much
higher among homosexual male AAS users. AAS users also take part in high-risk injection
practices but to a much lower extent than intravenous drug users. AAS users are also more
likely to engage in high-risk sexual behaviors than the general population. Clinicians and
health-policy leaders may utilize these findings to implement strategies to decrease the
spread of infectious diseases [150217].
High-performance sporting organizations should have strict policies in place around injection
therapy. Injection therapy in high-performance sport should be limited to the treatment of
illness or injury by the medical practitioner. There is no role for injection therapy as part of a
supplementation programme. If routine injection therapy is taking place as part of a
supplementation programme, one of the two things occurs:
1. The person doing the injecting is misleading the athlete and the sporting
organisation by injecting substances such as vitamins or other substances, for which
there is no scientific basis in the preparation of elite athletes.
2. The person doing the injecting is committing a doping violation.
No member of the support staff should be permitted to administer injections apart from the
medical practitioner. No one in a sporting organisation should be permitted to possess
injection equipment (syringes and hypodermic needles) other than the team doctor, the only
exception being those athletes who have certified medical conditions such as diabetes,
anaphylactoid reactions, etc [13027].
Gluteal mass
The use of anabolic steroids by bodybuilders is relatively common and associated with many
side effects. Local side effects include tissue necrosis and soft tissue infection at the injection
site. Systemic effects may be early epiphyseal closure in the immature skeleton, testicular
atrophy, sterility, acne, gynaecomastia and liver disorders such as hepatitis. It was report an
unusual case of a bodybuilder who developed a large painful inflammatory soft tissue mass
in his gluteal area. Multi-modality imaging showed direct evidence revealing the underlying
cause of the mass being depot steroid injections [02039].
The use of anabolic steroids by bodybuilders is relatively common and associated with many
side effects. Local side effects include tissue necrosis and soft tissue infection at the injection
site. Systemic effects may be early epiphyseal closure in the immature skeleton, testicular
atrophy, sterility, acne, gynaecomastia and liver disorders such as hepatitis. It was reported
1225
an unusual case of a bodybuilder who developed a large painful inflammatory soft tissue
mass in his gluteal area. Multi-modality imaging showed direct evidence revealing the
underlying cause of the mass being depot steroid injections [01070].
Sepsis
It was reported the case of a 30-year-old body builder who developed a gluteal abscess at
the site of injection of regularly self-administered anabolic steroids. After breaking the
abscess under general anaesthesia, the patient developed septic shock and fulminant adult
respiratory distress syndrome (ARDS). In addition to discussing the pathogenesis, differential
diagnosis, and treatment, it was focus on the immunomodulatory mechanisms of anabolic
substances that may have contributed to the course of the disease in this particular patient
[02040].
Pyomyositis
Pyomyositis, a deep bacterial infection of skeletal muscle, is due to the combination of

bacteremia and local muscle injury. It most commonly occurs in children and young adults,
with a male predominance; the lower extremities and pelvic girdle are typically involved, with
the quadriceps muscle being the most common site of infection. Recognized risk factors are
muscle trauma (including strenuous exercise), immunosuppression, diabetes mellitus,
malignancy, cirrhosis, rheumatologic disorders, and injections such as illicit drug abuse. The
infectious agent is S. aureus in 75-90 percent of cases, with group A Streptococcus a distant
second. Most patients will have a leukocytosis with neutrophilia and an elevated erythrocyte
sedimentation rate, but muscle enzymes such as creatinine kinase are typically normal.
Ultrasonography can be used to locate fluid collections within the musculature and assist
with needle aspiration, but CT imaging may better show muscle abnormalities such as
edema and abscess formation. Magnetic resonance imaging is thought to be even more
sensitive and is the preferred modality in early stages of the disease. A case was presented
regarding a previously healthy 45-year-old amateur bodybuilder who reported progressive
right thigh pain and swelling for 3 days. He admitted injecting anabolic steroids into the
lateral aspect of his right thigh 1 week prior. Although both thighs were quite large, the right
was clearly swollen; erythema, warmth, and tenderness were present but there was no
crepitus or fluctuance. Laboratory studies found an elevated white blood cell count of
19,000/mm3 with 86 percent neutrophils. A computed tomographic (CT) scan of the right
thigh revealed a loculated collection involving the vastus lateralis muscle from just below the
lesser trochanter to above the knee (17 cm length by 9 cm transverse by 6.5 cm anterior-
posterior), with fluid and inflammatory changes of the rectus femoris and tensor fascia lata
and overlying subcutaneous edema [13154].
Tuberculosis
It was presented a case report depicting masking of symptoms of intestinal tuberculosis by

anabolic androgenic steroids (AAS) causing delay in diagnosis which lead to a major
surgery. Negative tuberculosis skin test (TST) probably due to immunomodulating effects of
AAS also contributed to the delay. Patient also had early dependence on AAS and rapid
growth of scrotal sebaceous cysts, findings of which have not yet been reported. Patient
initially did not reveal his AAS abuse to physician. This is in line with previous studies which
report that majority of AAS abusers distrust medical professionals and do not disclose it to
their treating physicians. During hospitalisation he reported it probably due to seriousness of
his condition, impending major surgery and concurrent use of other medicines. The patient
had large sebaceous cysts on scrotum with history of rapid growth in last six months.
1226
Although this is not a known side effect of AAS use, increase in growth of sebaceous glands
and resulting acne have been documented. It is possible that this side effect might have
been missed in previous studies as patients are usually reluctant to reveal it unless
specifically asked. Further, most studies involved young age group who are more prone to
acne, a clearly visible symptom having significant importance in that age. Usually period for
development of AAS dependence is 9-12 months but this patient developed dependence in
just four months probably because AAS withdrawal symptoms were associated with
appearance of symptoms of tuberculosis [12149].
Invasive fungal rhinosinusitis
Invasive fungal rhinosinusitis is a potentially fatal infection that affects immunocompromised

patients. Prognosis is generally poor despite aggressive medical and surgical treatments. It
was presented the first reported case of invasive fungal sinusitis in a healthy 18-year-old
male athlete who was taking anabolic steroids. The effects of excessive AAS use on the
immune system are not fully understood, but there may be consequences at
supraphysiological concentrations. This case demonstrates potential immunomodulatory
effects of anabolic steroids and highlights a previously unknown cause of invasive fungal
sinusitis [14056].
Invasive fungal rhinosinusitis is a potentially fatal infection that affects immunocompromised

patients. Prognosis is generally poor despite aggressive medical and surgical treatments. We
present the first reported case of invasive fungal sinusitis in a healthy 18-year-old male
athlete who was taking anabolic androgenic steroids (AAS). The effects of excessive AAS
use on the immune system are not fully understood, but there may be consequences at
supraphysiological concentrations. This case demonstrates potential immunomodulatory
effects of anabolic steroids and highlights a previously unknown cause of invasive fungal
sinusitis [14455].
Gynecomastia
Androgen-deprivation therapy (ADT) is a key component of treatment for aggressive and

advanced prostate cancer, but it has also been associated with adverse effects on bone,
metabolic, cardiovascular, sexual, and cognitive health as well as body composition. To
review the current literature on the adverse effects of ADT and strategies for ameliorating
harm from ADT the Medline database (through PubMed) was searched from inception to
August 1, 2013, for studies documenting the side effects of ADT and for randomized and
prospective trials of interventions to mitigate those side effects. Adverse effects of ADT
include decreases in bone mineral density; metabolic changes such as weight gain,
decreased muscle mass, and increased insulin resistance; decreased libido and sexual
dysfunction; hot flashes; gynecomastia; reduced testicle size; anemia; and fatigue. Several
observational studies suggest an increased risk of diabetes and cardiovascular events,
although most published studies report that ADT is not linked to greater cardiovascular
mortality. Randomized trials have found value in treatments for some adverse effects
including bone loss (bisphosphonates, denosumab, selective estrogen receptor modulators),
markers of metabolic syndrome (exercise, diet, metformin), gynecomastia (tamoxifen,
prophylactic radiation), muscle loss (resistance and aerobic exercise), and hot flashes
(venlafaxine, medroxyprogesterone, cyproterone acetate, gabapentin). ADT is often a
necessary component of the treatment of aggressive prostate cancer, yet it has known
harms that can impair health and quality of life. Clinicians should be aware of interventions
that can help mitigate these adverse effects. Androgen deprivation therapy is a critical
1227
component of the management of aggressive and advanced prostate cancer, but it causes
adverse effects including bone loss, metabolic changes, gynecomastia, muscle loss, hot
flashes, and possibly increased cardiovascular events. Clinicians should be aware of
interventions that can help mitigate these adverse effects [14654].
Drugs are estimated to cause about 10-25 percent of all cases of gynecomastia. Over the
course of several decades, multiple medications have been implicated in the development of
gynecomastia mostly in the form of case reports and case series. However, these reports
suffer from a multitude of deficiencies, including poor quality of evidence. Studies were
selected for this review by performing an extensive electronic and hand-search using
BIOSIS, EMBASE and Medline, from 1940 to present, for all reported drug associations of
gynecomastia and their possible pathophysiology. Quality of evidence was assessed on a
three-point scale: good, fair and poor, and each of the drugs reported to cause gynecomastia
was assigned a level of strength. The pathophysiology of gynecomastia is also discussed in
detail for each of the drugs found to have a good or fair evidence of association with
gynecomastia. Most of the reported drug-gynecomastia associations were based on poor
quality evidence. The drugs definitely associated with the onset of gynecomastia are
spironolactone, cimetidine, ketoconazole, hGH, estrogens, hCG, anti-androgens, GnRH
analogs and 5-α reductase inhibitors. Medications probably associated with gynecomastia
include risperidone, verapamil, nifedipine, omeprazole, alkylating agents, HIV medications
(Efavirenz), anabolic steroids, alcohol and opioids [12143].
Testosterone is broken down to form estradiol in the peripheral tissues of the body. In
supratherapeutic doses of testosterone and its metabolites, the peripheral aromatization will
lead to a major increase in estradiol. Serum levels of estradiol are often seven times the
levels prior to onset of AAS use and approach medium levels of hormones found in normal
women. In male athletes these levels of estradiol will often lead to gynecomastia and a
heightened voice. Gynecomastia may cause breast pain and undesirable cosmetic affects for
athletes. Some athletes even resort to taking estrogen-blocking medications such as
tamoxifen to prevent these effects, although no scientific data support this practice. In most
cases, gynecomastia will remit upon discontinuation of the steroids. However, in more
prolonged use of AAS, gynecomastia can be permanent and thus require surgical resection
for cosmesis [06031].
Testosterone is converted to estrogens by aromatase and to DHT by way of 5-alpha

reductase. The estrogens lead to feminizing effects in men, such as gynecomastia and an
increase in voice pitch. Although the breast tissue that develops becomes softer and less
prominent after cessation of AAS use, this effect may be irreversible and require surgical
correction [07058].
Gynecomastia in males is a common adverse effect of chronic AAS abuse. These effects are
not readily reversible, particularly in adolescents. Adverse effects in women following chronic
AAS use include masculinization (male pattern baldness and hirsutism), acne, oily skin, and
breast atrophy. The virilizing effects of AAS use by women are similar to the clinical features
of the virilizing syndrome associated with congenital adrenal hyperplasia and adrenal
carcinoma [13003].
Gynecomastia, or painful breast enlargement, is a common and distressing complication of

AAS use occurring in as many as one-half of all users. Partially due to an imbalance of T/E2
signaling in breast tissue, symptoms may arise in the post-cycle period because of profound
ASIH or administration of hCG (and subsequent elevations in E2 secondary to aromatization)
along with a systemwide decline of endogenous androgen signaling. Alternatively, it may
occur while on-cycle, depending on the relative anabolic-to-androgenic effects as well as any
1228
progestin-like effects of medications used. AAS compounds that are susceptible to
aromatization are more likely to cause gynecomastia. Finasteride, used by as many as 10
percent of AAS users for alopecia, may potentiate this effect and should be discontinued in
ASIH users with gynecomastia. Herbal supplements such as tribulus terrestris and saw
palmetto extract have no proven benefit and might cause worsening gynecomastia. As such,
AAS users should be advised to discontinue these supplements. The use of hCG has been
reported in >40 percent of AAS users and may cause, or exacerbate, gynecomastia [14427].
Symptom duration is likely the best prognostic indicator for response to therapy with acutely
tender gynecomastia being the most amenable to medical treatment. Gynecomastia that has
persisted for >1 year is more likely to involve significant fibrosis and typically responds poorly
to drug therapy. In such nonpainful chronic cases, surgical treatment is the best option for
cosmetic improvement. Although large trials are lacking, tamoxifen appears to be the most
safe and effective agent for the medical management of AAS-associated gynecomastia.
Because tamoxifen has been used effectively to increase gonadotropin secretion and restart
the HPG axis in the setting of ASIH as well as idiopathic hypogondotropic hypogonadism the
authors recommend tamoxifen for the medical treatment of ASIH with concomitant AAS-
associated gynecomastia. Evidence that AIs are effective in the treatment of gynecomastia
exists, and the authors have used them previously with good success and minimal side
effects. However, it has been reported findings suggesting that suppression of circulating
estrogen levels with AIs decreases libido, worsens erectile dysfunction, and increases
percentage body fat in men with a chemically repressed HPG axis despite the administration
of TRT. Given that men with symptomatic ASIH may suffer from sexual dysfunction,
administration of AIs in this population may still be considered, but proper monitoring is
advised [14427].
AAS can also produce feminization (gynecomastia) in males, from the aromatization of
exogenous testosterone to estrogen metabolites [04018].
Another adverse effect is the occurrence of gynaecomastia in male athletes as a result of

AAS abuse. Besides the pain that may accompany gynaecomastia, the cosmetic implications
may be important for bodybuilders. Development of gynaecomastia is associated with the
peripheral conversion of AAS to estrogens, as a result of the huge amounts of exogenous
AAS administered. In the early stages spontaneous regression may be expected, but in long-
term cases surgical correction may be the only appropriate treatment. A common practice
among strength athletes is to accompany AAS abuse with self-administration of tamoxifen for
the prevention of gynaecomastia. Nevertheless, scientific data do not support the
effectiveness of this preventive method [00402].
Gynaecomastia is a common situation, with a proliferation of glandular component of male

breast secondary to an imbalance in sexual hormones in mammary tissue. A main cause of
gynaecomastia is anabolic steroids consumption. Few studies have been performed in
patients with high consumption of anabolic steroids, showing a high prevalence (52 %). In
this article we present a case of bilateral gynaecomastia secondary to anabolic steroids
intake, with a partial response to tamoxifen. In summary, a patient with sportive habits and
gynaecomastia, an early suspicious diagnosis is anabolic steroid intake. After suppression of
anabolic steroid intake and treatment with tamoxifen, a high remission rate can be achieved.
If gynaecomastia persisted, surgery is a good option [01066].
1229
Androgen and estrogen receptors in gynecomastia
In order to assess the relationship between anabolic steroid administration and

gynecomastia, we studied the effects produced by administering nandrolone decanoate and
a mixture of propionate, phenilpropionate, isocaproate and testosterone decanoate to
bodybuilders during a six month period. The following significant changes occurred: a 53
percent reduction in serum testosterone; LH and FSH levels were suppressed to 77 percent
and 87 percent, respectively, in comparison to control values; and although 45 percent of the
subjects showed an increase in serum estradiol levels, no statistically significant differences
were found compared with control estradiol levels. With regard to estradiol and androgen
receptors, 85 percent of gynecomastia tissue contained estradiol or androgen receptors,
while 40 percent contained both. The mean values of estradiol and androgen receptors in the
cytosol were 65 + 10 and 52 + 5 fmol/mg protein, respectively. Nuclear androgen and
estradiol receptor levels were 33 + 7 and 68 + 9 fmol/mg protein, respectively. The presence
of hormone receptors in gynecomastia receptive cells provides support for the hypothesis
that gynecomastia is steroid-dependent [01067].
Treatment
Use of anabolic steroids is one of many possible causes for gynecomastia. Optimal
surgical treatment for this entity involves a combination of liposuction and direct
excision. A series of 20 patients treated for anabolic steroid-induced gynecomastia
presented. Postoperative complications included two hematomas, one seroma
(bilateral), and three recurrences. Recurrence may be related hormonally active
retained subareolar tissue [01068].
Hyperplastic changes and receptor status in the breast
Anabolic androgenic steroids (AAS) are misused by athletes to improve their physical
performance. AAS with similar groups and configuration indicate that testosterone is the
base of this ability to stimulate anabolic activity. The effect of these compounds on the breast
tissue of males that consume them is a confirmation of its metabolic pathway. To confirm its
hormonal effects, the status of estradiol and progesterone receptors (ER, PgR) status was
determined in cytoplasmic and nuclear fractions (HRc, HRn) of 8 premalignant breast tissues
from 8 bodybuilders (aged 21 to 45 years) under AAS stimulation. The control group included
5 males with benign disorders of the breast, but not due to AAS administration. The
concentrations of ERc and ERn were significantly higher in males under AAS stimulation
than in males without these. The concentrations of PgRc and PgRn do not differ between
these two groups. The benign breast disease is remarkably similar in female and male
patients, suggesting a common origin. In the same way, the measurement of both HRc and
HRn is necessary to accurately report receptor concentration [00068].
Bone
Testosterone is the major gonadal sex steroid produced by the testes in men. Androgens
induce male sexual differentiation before birth and sexual maturation during puberty; in adult
men, they maintain the function of the male genital system, including spermatogenesis.
Testosterone is also produced in smaller amounts by the ovaries in women. The adrenal
glands produce the weaker androgens dehydroepiandrosterone, dehydroepiandrosterone
sulfate, and androstenedione. Because testosterone can be metabolized to estradiol by the
1230
aromatase enzyme, there has been controversy as to which gonadal sex steroid has the
greater skeletal effect. In this respect, there is increasing evidence that at least part of the
effects of androgens in men can be explained by their aromatization into estrogens. The
current evidence suggests that estradiol plays a greater role in maintenance of skeletal
health than testosterone, but that androgens also have direct beneficial effects on bone
[12142].
The definition of bone quality is evolving particularly from the perspective of anabolic agents
that can enhance not only bone mineral density but also bone microarchitecture,
composition, morphology, amount of microdamage, and remodeling dynamics. From a
MEDLINE search (1996-2010), articles were identified by the search terms "bone quality"
(1851 articles), "anabolic agent" (5044 articles), "PTH or parathyroid hormone" (32,229
articles), "strontium" or "strontium ranelate" (283 articles), "prostaglandin" (77,539 articles),
and "statin" or "statins" (14,233 articles). The search strategy included combining each with
the phrase "bone quality." Another more limited search aimed at finding more novel potential
agents. Parathyroid hormone is the only US Food and Drug Administration-approved bone
anabolic agent in the United States and has been the most extensively studied in in vitro
animal and human trials. Strontium ranelate is approved in Europe but has not undergone
Food and Drug Administration trials in the United States. All the studies on prostaglandin
agonists have used in vivo animal models and there are no human trials examining
prostaglandin agonist effects. The advantages of statins include the long-established
advantages and safety profile, but they are limited by their bioavailability in bone. Other
potential pathways include proline-rich tyrosine kinase 2 (PYK2) and sclerostin (SOST)
inhibition, among others. The ongoing research to enhance the anabolic potential of current
agents, identify new agents, and develop better delivery systems will greatly enhance the
management of bone quality-related injuries and diseases in the future [11325].
Effect on bone density
One study evaluated the relationship between anabolic androgenic steroid (AAS) use and
body constitution. Dual-energy x-ray absorptiometry was used to measure bone mineral
density (BMD, g/cm2) of the total body, arms, and legs. Total gynoid and android fat mass
(grams) and total lean mass (grams) were measured in 10 strength trained athletes (41 ± 8
years) who had used AASs for 5-15 years (Doped) and 7 strength trained athletes (29 ± 6
years) who had never used AASs (Clean). Seventeen sedentary men (30 ± 2 years) served
as controls. Doped athletes had significantly more lean body mass and a greater index of fat-
free/fat mass compared with Clean athletes and controls. Doped athletes also had
significantly less gynoid fat mass compared with that of Clean athletes. There were no
differences in BMD between the athletes but both groups had significantly higher BMDs at all
sites compared with that of controls. Thus, long-term AAS use seems to alter body
constitution, favoring higher muscle mass and reduced gynoid fat mass without affecting
BMD [12148].
Influence on muscle and tendon injury and injury healing
AAS may cause adverse musculoskeletal effects, especially tendon rupture attributable both
to the disproportionate strength of hypertrophied muscles, and to possible deleterious effects
of AAS on the architecture of the tendons themselves [14017]
In the past 20 years, there has been an increase in the incidence of upper extremity
tendinous injuries, especially in sports including strong physical activity, such as in weight
1231
lifting, as well as with the concurrent use of anabolic steroids. Today, there are more than
200 cases describing rupture of the pectoralis major muscle in athletes. Twenty athletes with
pectoralis major muscle (PMM) rupture were studied; 10 had surgical treatment, and the
other 10 were treated nonoperatively. The average follow-up was 36 months (range, 48-72
months). Injuries were diagnosed by history, physical examination, and subsidiary tests.
Functional evaluation and isokinetic evaluation were performed on all 20 patients. It was
concluded that total pectoralis major muscle rupture in athletes showed a better functional
result after surgical treatment than after nonsurgical treatment [10058].
The indiscriminate use of anabolic-androgenic steroids has been shown to induce pathologic
changes in the Achilles tendon in several situations. To study tendon remodeling in rats
treated with anabolic-androgenic steroids combined with an exercise program Wistar rats
were grouped as follows: sedentary (group I), injected with anabolic-androgenic steroids only
(group II), trained only (group III), and trained and injected with anabolic-androgenic steroids
(group IV). The trained groups performed jumps in water: 4 series of 10 jumps each, with an
overload of 50 to 70 percent of the animal's body weight and a 30-second rest interval
between series, for 6 weeks. Anabolic-androgenic steroids (5 mg/kg) were injected
subcutaneously. Activity of matrix metallopeptidases, a marker for tendon remodeling, was
analyzed in tissue extracts by zymography on gelatin-sodium dodecyl sulfate-polyacrylamide
gel electrophoresis. Morphological analyses of tendons showed that in group II, the most
external layer that covers the tendon was thicker with aggregation of the collagen fibers,
suggesting an increase in collagen synthesis. In group IV, an inflammatory infiltrate and
fibrosis in tendons as well as a pronounced increase of the serum corticosterone level were
observed. This training protocol upregulated matrix metallopeptidase activity, whereas
anabolic-androgenic steroid treatment strongly inhibited this activity. The appearance of lytic
bands with molecular masses of approximately 62 and 58 kDa suggests the activation of
matrix metallopeptidase-2. It was concluded that anabolic-androgenic steroid treatment can
impair tissue remodeling in the tendons of animals undergoing physical exercise by down-
regulating matrix metallopeptidase activity, thus increasing the potential for tendon injury.
Since the AAS abuse is so widespread, a better comprehension of the pathological effects
induced by these drugs may be helpful for the development of new forms of therapy of AAS-
induced lesions [06089].
Although testosterone administration elicits well documented anabolic effects on skeletal

muscle mass, the enhancement of muscle regeneration after injury has not been widely
examined. The purpose of one study was to determine if anabolic steroid administration
improves skeletal muscle regeneration from bupivacaine-induced injury. Male C57BL/6 mice
were castrated 2 weeks prior to muscle injury induced by an intramuscular bupivacaine
injection into the tibialis anterior muscle. Control mice received an intramuscular PBS
injection. Anabolic steroid (nandrolone decanoate, 6 mg/kg) or sesame seed oil was
administered at the time of initial injury and continued every 7 days for the study's duration.
Mice were randomly assigned to one of 4 treatment groups for 5, 14 or 42 days of recovery
as follows: 1) Control (uninjured); 2) nandrolone decanoate only (uninjured + nandrolone); 3)
Bupivacaine only (Injured); or 4) Bupivacaine + nandrolone. Tibialis anterior morphology,
protein and gene expression was analyzed at 14 and 42 days after injury, protein expression
was analyzed at 5 days after injury. After 14 days of recovery the Injury and Injury +
nandrolone treatments induced small diameter myofiber incidence, and also decreased
mean myofiber area. The increase in small myofiber incidence was 65 percent greater in
Injury + nandrolone muscle when compared to Injury alone. At 14 days, Injury + nandrolone
induced a 5-fold increased in muscle IGF-1 mRNA expression, which was greater than injury
alone. Muscle Akt activity and GSK3beta activity were also induced by Injury + nandrolone at
14 days of recovery, but not by Injury alone. Nandrolone had a main effect for increasing
muscle MyoD and Cyclin D1 mRNA expression at 14 days. After 42 days of recovery, Injury
1232
+ nandrolone increased large diameter myofiber incidence compared to Injury only. It was
concluded that nandrolone decanoate administration can enhance castrated mouse muscle
regeneration during the recovery from bupivacaine-induced injury [09063].
Experimental evidence exists that the use of AASs combined with intense exercise can
cause structural and biomechanical alterations of tendons resulting in rupture. Structurally,
the collagen fibril alignment is highly disorganized. From a biomechanical perspective, when
muscle strength is increased with AAS use, the tendon becomes stiffer, absorbs less energy,
and is more likely to fail during physical activity. Premature growth cessation due to physeal
closure in younger users has not been studied systematically. Such case reports of the
resultant permanent short stature have been described for several decades [07008].
Muscle mass seems to be affected greatly by AAS dosing. Higher doses have been shown to
garner increases in muscle mass. Muscle mass gains are larger when AAS use is combined
with strength training compared with AAS use alone. AASs increase the number of
myonuclei. Strenuous exercise seems to increase the number of androgen receptor sites on
the muscle. Body weight increases can be in the range of 2 to 5 kg after 10 weeks of AAS
use. With more androgen receptors present in the upper regions of the body, the neck,
shoulders, thorax, and upper arms gain the most new bulk. The thigh muscles require higher
doses to show measurable increases in mass and are not as likely to show increases in the
number of androgen receptors. Upon discontinuation, muscles shrink and strength declines
over a period of 6 to 12 weeks. Androgens stimulate osteoblast proliferation and
differentiation and inhibit the osteoclast. At the start of puberty, androgens stimulate bone
formation. At the end of puberty, they induce epiphyseal closure. In adulthood, the sex
hormones slow the rate of bone remodeling, protect against bone loss, encourage bone
formation, and increase bone density. The adolescent AAS user risks an increased rate of
muscle strains or ruptures. Unlike muscles, tendons do not increase in strength so with more
intense training, they have a greater risk for rupture. In a developing adolescent, the growth
plate cartilage is considered the “weakest link,” and generally is more prone to injury
compared with ligaments. A rapid increase in the intensity, frequency, or volume of training is
noted consistently in athletes who present with overuse injuries. Injury to the growth plate
from weight training has long been a subject of controversy; power lifting may increase the
risk for injury, even in adolescents not taking AAS [07058].
Long-standing rotator cuff tendon tearing is associated with retraction, loss of work capacity,
irreversible fatty infiltration, and atrophy of the rotator cuff muscles. Although continuous
musculotendinous relengthening can experimentally restore muscular architecture,
restoration of atrophy and fatty infiltration is hitherto impossible. Continuous relengthening
with pharmacological stimulation of muscle growth using an anabolic steroid or insulin-like
growth factor (IGF) can reverse atrophy and fatty infiltration as well as improve the work
capacity of chronically retracted rotator cuff muscles in sheep. Sixteen weeks after tenotomy
of the infraspinatus (ISP) tendon, atrophy and fatty infiltration had developed in the retracted
ISP muscle. The musculotendinous unit was continuously relengthened in 14 sheep during 6
weeks: Four sheep were treated without pharmacological stimulation, 4 with intramuscular
administration of an anabolic steroid, and 6 with IGF before final repair and rehabilitation (12
weeks). Changes were documented by intraoperative measurements of muscle work
capacity, histology, and computed tomography/magnetic resonance imaging.
Musculotendinous relengthening by continuous traction resulted in gains of length ranging
from 0.7 cm in the IGF group to 1.3 cm in the control group. Fatty infiltration progressed in all
groups, and the muscle's cross-sectional area ranged from 71 to 74 percent of the
contralateral side at sacrifice and did not show any differences between groups in weight,
volume, histological composition, or work capability of the muscle. The contralateral muscles
in the anabolic steroid group, however, showed significantly higher (mean ± standard
1233
deviation) muscle work capacity of 10 ± 0.9 N·m than the contralateral muscles of the control
group (6.8 ± 2.4 N·m). This was accompanied by an increased mean muscle fiber area as
well as by an unusual gain in the animals' weight after injection of the anabolic steroid. It was
concluded that subcutaneous continuous relengthening of a chronically retracted
musculotendinous unit is feasible and advances the retracted musculotendinous junction
toward its original position. This does not change the muscle work capacity. Whereas
anabolic steroids have been shown to be effective in preventing classic degenerative muscle
changes after tendon tears, neither an anabolic steroid nor IGF contributes to regeneration of
the muscle once degenerative changes are established. Thus, the findings demonstrate that
muscle cells lose reactiveness to an anabolic steroid and IGF once retraction has led to fatty
infiltration and atrophy of the muscle. Retraction of the muscle after tendon tears must be
avoided by early repair, particularly in an athlete, as no regeneration can be achieved by
mechanical or pharmacological means at this time [12147].
AASs may cause adverse musculoskeletal effects, especially tendon rupture, attributable
both to the disproportionate strength of hypertrophied muscles and to possible deleterious
effects of AAS on the architecture of the tendons themselves [14426].
The effect of anabolic androgenic steroids on tendons has not yet been fully elucidated. The
aim of one study was the evaluation of the impact of anabolic androgenic steroids on the
biomechanical and histological characteristics of Achilles tendons. Twenty-four male Wistar
rats were randomized into four groups with exercise and anabolic steroids (nandrolone
decanoate) serving as variables. Protocol duration was 12 weeks. Following euthanasia,
tendons' biomechanical properties were tested with the use of a modified clamping
configuration. Histological examination with light and electron microscopy were also
performed. In the group of anabolic steroids and exercise the lowest fracture stress values
were observed, while in the exercise group the highest ones. Histological examination by
light and electron microscopy revealed areas of collagen dysplasia and an increased
epitendon in the groups receiving anabolic steroids and exercise. These findings suggest
that anabolic androgenic steroids reverse the beneficial effect of exercise, thus resulting in
inferior maximal stress values [14256].
Pectoralis major ruptures
Rupture of the pectoralis major tendon is increasing in incidence, with a spike in the number
of reported cases in the last decade. This is commonly attributed to an increased interest in
health, fitness, and weight training combined occasionally with concomitant use of anabolic
steroids. It is essential for the diagnosis to be recognized and for the patient to be referred to
a surgeon with expertise in dealing with these injuries so that appropriate and informed care
can be implemented. Based on a comprehensive review of the literature and expert opinion,
we present a review of pectoralis major ruptures, including information pertaining to the
anatomy and biomechanics of the musculotendinous unit and how this relates to the injury
pattern and management; the clinical diagnosis and indications for additional imaging; and
the indications for nonoperative and operative management along with the authors' preferred
technique. A summary of outcomes is presented. It was concluded that the combination of
patient demographics and clinical features frequently yields an accurate diagnosis, but
further imaging is helpful. Magnetic resonance imaging with dedicated sequencing is the
investigation of choice and can aid in diagnosis, surgical planning, and providing important
information about prognosis and outcome. Early surgery is preferable, but good outcomes in
the chronic setting are achievable. With a detailed understanding of the anatomy, direct
repair to bone is possible with either transosseous or anchor repair techniques in acute and
1234
the majority of chronic cases. In chronic cases in which direct repair is not achievable,
autograft and allograft reconstruction should be considered [150221].
Muscle healing in power-lifters
Power-lifters have hypertrophic muscle fibers with fissures seen in cross-sections, called as
fiber splitting. Whether this phenomenon is due to real splitting or defective regeneration has
not been settled. To elucidate this matter,we have examined biopsies from the trapezius and
vastus lateralis of power lifters (P group) and power lifters self-administrating anabolic
steroids (PAS group). For this purpose, immunohistochemical staining of serial cross -
sections was used. The PAS group had significantly more fibers with fissures than the P
group in the vastus lateralis but not in the trapezius muscle (1.7 % in both groups). Serial
sections revealed that the fibers with fissures changed their profile profoundly over short
distances. Some such fibers had a mature staining profile, whereas other fibers indicated
recent degeneration and/or regeneration. Activation of satellite cells and formation of
aberrant segments were also evident. It was concluded that the so-called split fibers are due
to defect regeneration. Some fibers with fissures are the results of old events of segmental
muscle fiber damage, whereas the others reflect an ongoing process. The normal
regenerative process is most likely disturbed in power-lifters by their continuous training with
repeated high mechanical stress on the muscles [06090].
Tendon adaptation
Combined androgenic-anabolic steroids (AAS) and overloading affects tendon collagen

metabolism and ultrastructure and is often associated with a higher risk of injury. The aim of
this prospective study was to investigate whether such effects would be reflected in the
patellar tendon properties of individuals with a history of long-term resistance training and
AAS abuse (RTS group), compared with trained (RT) and untrained (CTRL) nonsteroids
users. Tendon cross-sectional area (CSA), stiffness, Young's modulus, and toe limit strain
were measured in vivo, from synchronized ultrasonography and dynamometry data. The
patellar tendon of RT and RTS subjects was much stiffer and larger than in the CTRL group.
However, stiffness and modulus were higher in the RTS group (26 % and 30 %, respectively)
than in the RT group. Conversely, tendon CSA was 15 percetn larger in the RT group than in
RTS, although differences disappeared when this variable was normalized to quadriceps
maximal isometric torque. Yet maximal tendon stress was higher in RTS than in RT (15 %),
without any statistical difference in maximal strain and toe limit strain between groups. The
present lack of difference in toe limit strain does not substantiate the hypothesis of changes
in collagen crimp pattern associated with AAS abuse. However, these findings indicate that
tendon adaptations from years of heavy resistance training are different in AAS users,
suggesting differences in collagen remodeling. Some of these adaptations (e.g, higher
stress) could be linked to a higher risk of tendon injury [13121].
Tendon injuries
Accumulating case reports have described tendon rupture in men who use anabolic-
androgenic steroids (AAS). However, no controlled study has assessed the history of tendon
rupture in a large cohort of AAS users and comparison nonusers. Medical histories were
obtained from 142 experienced male bodybuilders aged 35 to 55 years recruited in the
course of 2 studies. Of these men, 88 reported at least 2 years of cumulative lifetime AAS
use, and 54 reported no history of AAS use. In men reporting a history of tendon rupture, the
circumstances of the injury, prodromal symptoms, concomitant drug or alcohol use, and
details of current and lifetime AAS use (if applicable) were recorded. Surgical records were
obtained for most participants. Nineteen (22 %) of the AAS users, but only 3 (6 %) of the
1235
nonusers, reported at least 1 lifetime tendon rupture. The hazard ratio for a first ruptured
tendon in AAS users versus nonusers was 9.0 (95 % confidence interval 2.5 to 32.3).
Several men reported 2 or more independent lifetime tendon ruptures. Interestingly, upper-
body tendon ruptures occurred exclusively in the AAS group (17 % AAS users vs 0
nonusers; risk difference, 0.17), whereas there was no significant difference between users
and nonusers in risk for lower-body ruptures (7 % AAS users, 6 %). Of 31 individual tendon
ruptures assessed, only 6 (19 %) occurred while weightlifting, with the majority occurring
during other sports activities. Eight (26 %) ruptures followed prodromal symptoms of
nonspecific pain in the region. Virtually all ruptures were treated surgically, with complete or
near-complete ultimate restoration of function. It was concluded that AAS abusers, compared
with otherwise similar bodybuilders, showed a markedly increased risk of tendon ruptures,
particularly upper-body tendon rupture [150220].
Tendon rupture has been linked with AAS use on the basis of a small number of published
case reports, and it has been suggested that these drugs predispose to tendon rupture by
altering collagen structure. AASs appear to induce reversible changes in the biomechanical
properties of tendon producing a stiffer, less elastic tendon, but the ultimate strength of the
tendon is unaffected. Although AASs increase tendon stiffness, no consistent AAS-induced
ultrastructural or biochemical alterations have been found to account for the changes in
biomechanical properties, and distinction should be made between loss ofelasticity and
actual tendon rupture. It is possible that the rapid strength adaptations produced by AAS in
skeletal muscle are not simultaneously matched by slower adapting, less vascular tendon
structures, making tendons the weakest link in the chain [04018].
Effect of supraphysiological doses on collagen metabolism

It was examined the effect of supraphysiological doses of anabolic androgenic steroids
(AAS) on collagen metabolism and whether the changes reflect the alterations in muscle,
bone, and tendon collagen metabolism, possibly in a tissue-specific manner. Serum
carboxyterminal propeptide of type I procollagen (PICP), carboxyterminal telopeptide of type
I collagen (ICTP), aminoterminal propeptide of type III procollagen (PIIINP), urine
hydroxylysylpyridinoline (HP), and lysylpyridinoline (LP) as well as urine creatinine were
determined from 17 men abusing AAS. Measurements were made twice during the intake of
AAS and twice during the subsequent withdrawal period. When the volunteers were on
steroids, their serum PIIINP concentrations and urine HP/LP ratio were significantly higher
and their serum ICTP concentrations were significantly lower than during the withdrawal
period. Serum PIIINP correlated with total cumulative doses of injectable intramuscular
steroids, and serum ICTP correlated with the duration of the steroid intake period. The
results suggest that high doses of AAS decrease the degradation and seem to increase the
synthesis of type I collagen. Furthermore, high doses of AAS are suggested to enhance soft
tissue collagen metabolism on the basis of increased type III collagen synthesis and elevated
HP/LP ratio during the steroid administration period. Although the tissue-specific turnover of
collagen of soft connective tissues remains unknown, the turnover of bone collagen seems
not to change following the use of high doses of AAS, at least within the time interval of the
present study [00067].
Compartment syndrome
Anabolic steroid accelerated multicompartment syndrome following trauma

Anabolic steroids such as nandrolone are used recreationally by some athletes to achieve a
rapid increase in muscle bulk from the anabolic effects on muscle tissue. Chronic
compartment syndrome has been described in athletes who undergo rigorous training
regimes; it may be treated by conservative methods or subcutaneous fasciotomy. Acute
compartment syndrome is an emergency, presenting with severe pain in the affected limb. If
1236
surgical decompression is not performed quickly, muscle necrosis ensues accompanied by
systemic metabolic effects. Delay in treatment is usually because of failure to recognise the
condition, particularly in the unconscious patient with multiple injuries. A 23 year old man was
airlifted to the casualty department of Hull Royal Infirmary after falling from his motorcycle. All
injuries were confined to the right side of the patient, and consisted of an undisplaced right
radial head fracture and fractures of the right iliac crest, right femoral shaft, and a compound
(Gustilo 3B) fracture of the right tibia and fibula. On the left, all muscle compartments were
soft and the limbs pain-free on passive and active movement. In theatre the patient's right
foot was noted to be cold and dusky with absent pulses. An on table angiogram showed
patent vessels proximal to the ankle, with arterial spasm, but no other vascular compromise.
Fasciotomies were performed of the right thigh and all four compartments of the lower leg,
which resulted in a warm pink foot. Chronic, acute on chronic, and rarely acute compartment
syndrome has been recognised in athletes and soldiers who undergo rigorous training, which
is due to an increase in volume of muscle in a tight fascial compartment, or repeated trauma.
The pressure within that compartment can further increase because of compromise of the
microvascular circulation which leads to an accumulation in tissue fluid both intracellularly
and extracellularly. Inflammatory mediators accumulate because lymphatic and venous
return are impeded producing further oedema, and a vicious cycle occurs leading to muscle
necrosis.This patient had taken nandralone (Deca-Durabolin®) before the accident and was
returning from a heavy exercise session when his injury occurred. Although it is common for
compartment syndrome to occur in the presence of a fracture, severe contusion, or a crush
injury, we postulate that the increase in muscle bulk due to the exercise along with the use of
anabolic steroids brought about the severe compartment syndrome seen in this patient,
affecting three of his four limbs including areas apparently not affected by the trauma
[00071].
In the upper limb

Acute compartment syndrome, a surgical emergency, is defined as increased pressure in an
osseofascial space. The resulting reduction of capillary perfusion to that compartment
requires prompt fasciotomy. Treatment delay has a poor prognosis, and is associated with
muscle and nerve ischemia, resultant infarction, and late-onset contractures. It was reported
a case of traumatic bilateral upper limb acute compartment syndrome associated with
anabolic steroids, requiring bilateral emergency fasciotomies. A 25-year-old male
bodybuilder taking anabolic steroids, with no past medical history, presented to the
Emergency Department 25 min after a road traffic accident. Secondary survey confirmed
injuries to both upper limbs with no distal neurovascular deficit. Plain radiographs
demonstrated bilateral metaphyseal fractures of the distal humeri. Within 2 h of the accident,
the patient developed clinical features that were consistent with bilateral upper arm
compartment syndrome. Bilateral fasciotomies of both anterior and posterior compartments
were performed, confirming clinical suspicion [13122].
In the lower limb

Acute exertional compartment syndrome is the result of muscle ischemia within a tight fascial
compartment. It was reported a 22-year-old boxer, with recent intake of anabolic steroids,
who developed acute exertional compartment syndrome of the lower legs following an
assault from which he had to run away. He presented with bilateral footdrop. Nerve
conduction studies (NCS) and electromyography (EMG) were consistent with bilateral deep
and superficial peroneal neuropathies, but magnetic resonance imaging (MRI) demonstrated
hemorrhagic necrosis of the pretibial muscles. This case illustrates that the differential
diagnosis for footdrop includes not only central and peripheral nervous system and muscle
causes, but also compartment syndromes [05058].
1237
Rhabdomyolysis
Rhabdomyolysis, or acute skeletal muscle destruction, may be accompanied by

myoglobinaemia, myoglobinuria, and an elevated serum creatine kinase level. This disorder
has many potential causes. In one article, the authors describd a case of rhabdomyolysis
occurring after vigorous weight lifting by a man who was supplementing his weight-training
programme with the intake of anabolic androgenic steroids dispensed to him by a colleague
[01072].
Rhabdomyolysis is a clinical syndrome in which the contents of injured muscle cells leak into
the circulation. This leakage results in electrolyte abnormalities, acidosis, clotting disorders,
hypovolemia, and acute renal failure. More than 100 conditions, both traumatic and non-
traumatic, can lead to rhabdomyolysis. Intervention consists of early detection, treatment of
the underlying cause, volume replacement, urinary alkalinization, and aggressive diuresis or
hemodialysis. Patients with rhabdomyolysis often require intensive care, and critical care
nurses are instrumental in both the early detection and the ongoing management of this life-
threatening syndrome [03068].
One case in an Intensive Care Unit was a patient of rhabdomyolysis leading to acute kidney
injury. It was suspected on the basis of high-intensity exercise in otherwise routine gym
visitor, followed by pain in both thigh with tenderness, deranged lab reports which include
very high level of CPK, alanine and aspartate aminotransferases, low level of calcium and
after exclusion of other common causes including severe sepsis. Rhabdomyolysis may
develop in an individual after strenuous activity even who are athlete as it was in our patient.
He did more than 500 sit-ups at stretch that too first time along with his daily gym activity.
Moreover, he took analgesics for muscle pain, which in association with poor oral intake
worsens acute kidney damage. Exertional rhabdomyolysis is more likely to occur when
strenuous exercise is performed under high temperatures and humidity. Other factors include
improper hydration, inadequate recovery between bouts of exercise, intense physical
training, and inadequate fitness levels for beginning high-intensity workouts. As he did his
activity in the gym, high temperature seems to be unlikely contributor. Intense physical
training and inadequate fitness level for beginning high-intensity workouts appear to be more
logical explanation for rhabdomyolysis in our patient. Rhabdomyolysis is an important cause
of acute renal failure (ARF), and main pathophysiological mechanisms are renal
vasoconstriction, intraluminal cast formation, and direct myoglobin toxicity. Around 33
percent of the episodes of rhabdomyolysis lead to ARF [150216].
AAS-users engaged in heavy weightlifting can display rhabdomyolysis, sometimes with

massive elevations of serum creatine kinase (CK) levels leading to myoglobinemia,
myoglobinuria, elevated creatinine levels, decreased glomerular filtration rate (GFR), and the
occasional progression to acute renal failure. Notably, one recent case series has
documented 10 cases of focal segmental glomerulonephritis among frequent AAS users
[14017].
Muscular AAS users engaged in heavy weightlifting can display rhabdomyolysis, sometimes
with massive elevations of serum creatine kinase levels, leading to myoglobinemia,
myoglobinuria, elevated creatinine levels, decreased glomerular filtration rate, and the
occasional progression to acute renal failure. Notably, one recent case series has
documented 10 cases of focal segmental glomerulonephritis among frequent AAS users
[14426].
A 34-year-old bodybuilder presented at the emergency room with fever, vomiting and muscle
cramps that had started during a bodybuilding session. Several days before he started
1238
training he had used tablets and intramuscular injections containing the anabolic steroids:
dehydro-chloro-methyltestosterone, boldenone and trenbolone. In addition, he had taken
clenbuterol tablets, liothyronine tablets and subcutaneous injections of phosphatidylcholine.
Laboratory investigations revealed massive rhabdomyolysis. The patient was treated with
intravenous fluid replacement and sodium bicarbonate to alkalinize the urine. He recovered
quickly and his renal function remained unaffected. Doping'among amateur athletes in the
Netherlands occurs frequently. Apart from long term side-effects, doping can also cause
acute health problems. Therefore it is important to ask about doping use during history taking
in amateur athletes [06091].
The use of supraphysiologic doses of testosterone increases muscle mass and maximal
voluntary strength in a dose-dependent manner, but there is no definitive evidence that
testosterone improves performance in endurance events. Rare case reports associate a
general necrotizing myopathy with the chronic use of AAS. A 23-year old man developed
diffuse myalgias and severe rhabdomyolys is with anuria. Although he developed a viral
upper respiratory tract infection 1 week prior to the development of rhabdomyolysis, muscle
biopsy and serum testing revealed no evidence of inflammation or immune disease. He had
a history of chronic AAS abuse. Other case reports associate localized rhabdomyolysis of the
deltoid muscles with chronic AAS injections in the same location. There are few data in the
medical literature regarding the effect of chronic AAS abuse on the function of connective
tissue. Several case reports associate the chronic abuse of AAS with disruption of
connective tissues including spontaneous rupture of ligaments and muscles [13003].
Rhabdomyolysis (breakdown of skeletal muscle tissue) may be caused by mechanical,

physical, chemical, or biological factors. It was presented the unique case of a bodybuilder
who developed localized rhabdomyolysis of the deltoid muscle after injection of steroids into
the shoulder region. Polymyositis and dermatomyositis, mild injury, infectious myositis
without phlegmon or abscess formation, radiation therapy, subacute denervation,
compartment syndrome, early myositis ossificans, rhabdomyolysis, and sickle cell crisis were
differential diagnoses. The patient was treated with intravenous fluid replacement and
sodium bicarbonate to alkalinize the urine. Four days after admission, his pain had
decreased, he had regained range of motion, and his renal function remained unaffected.
This was the first description of localized rhabdomyolysis in the area of an AAS injektion
[09062].
AAS users also are at risk for rhabdomyolysis or acute skeletal muscle destruction.
Rhabdomyolysis has been reported after vigorous weight lifting and may be more likely in
patients escalating and supplementing weight training with AASs. Physicians should consider
the creatine phosphokinase and gamma-glutamyl transpeptidase levels as essential
elements in distinguishing muscle damage from liver damage when evaluating enzyme
elevations in patients who use anabolic steroids [07058].
Effects on kidneys
Renal side effects of AAS are uncommon and have been documented in a few isolated case
reports. These reports noted a minimal effect of AAS use on renal function with a mild
elevation in serum creatinine. The combination of AAS and creatine supplementation,
commonly abused by weight lifters, may increase renal damage. One case of membrano-
proliferative glomerulonephritis has been cited in an athlete with prolonged AAS abuse. An
unusual cancer in adults, Wilms’ tumor, has been seen in a few athletes who were self-
administering AAS over several years. In one case report, a 23-year-old bodybuilder on
cycles of 8 weeks of oral stanozolol and oxymetholone and parenteral nandrolone,
1239
testosterone, and boldenone, along with a high-protein diet, developed profound symptoms
after only 6 months of use. Of note, the patient was also limiting sodium and water intake and
taking the diuretic torasemide. The patient presented with acute confusion, asthenia, and
anorexia of 1 month’s duration [06031].
Anabolic steroids and creatine supplementation is one of the current abuse used by body
builders. It is less known that this combination beside of many deleterious effects may also
cause renal damage. Authors report a case of diffuse membranoproliferative
glomerulonephritis type I in a 22-year-old man who had been taking continuously methandion
in a large quantity and 200 grams of creatine daily, and was sent to the outpatient
nephrologic unit with typical clinical signs of nephrosis syndrome. They also call attention to
the role of the continuously consumed creatine in the renal failure [03060].
Collapsing glomerulopathy
Collapsing glomerulopathy (CG) is a proliferative podocytopathy, increasingly recognized in a

variety of disease conditions. It was reported a case of CG in a 16-year-old boy with IgA
nephropathy (IgAN) who presented with acute kidney injury, marked proteinuria and
hypertension following a short period of anabolic steroid use. Although CG has been
associated with long-term anabolic steroid use among body builders, there is no data on the
effect of anabolic steroid use in persons with underlying renal disease like IgAN. It was
postulated that development of CG in our patient could be temporally linked to intake of
body-building steroids along with a predisposing background renal disease of IgAN [150217].
Acute kidney injury
It was earlier reported 10 body builders who developed renal insufficiency and focal
segmental glomerulosclerosis (FSGS) while taking anabolic steroids and protein and creatine
supplements with a daily protein intake of 300–550 g/day. The high-protein intake has been
of concern to nephrologists because it increases glomerular filtration rates and is
experimentally associated with glomerular hyperfiltration and FSGS. Recent evidence
indicates that anabolic steroids are directly toxic to glomeruli and that segmental sclerosis is
the result of podocyte loss mediated by apoptosis through a podocyte androgen receptor.
Creatine powder is marketed as a muscle building supplement. Loading doses of 20-25
g/day for 5 days and then maintenance doses of 5 g/day are considered safe and to
effectively contribute to exercise endurance and muscle strength. Creatine is used by a large
number of competitive as well as casual athletes. The number of reported adverse events is
small and usually associated with exercise-induced acute renal failure and rhabdomyolysis
during intense training but even with moderate exercise. In the literature it was found three
case reports of creatine-associated acute kidney injury not related to rhabdomyolysis. One
body builder developed acute tubular necrosis after taking loading doses of 20 g/day for 5
days. In two patients, kidney injury was associated with acute interstitial nephritis. Four
bodybuilders who injected anabolic steroids and ingested commercial protein (78-104
g/day)and creatine (15 g/day) products presented with serum creatinine levels between
229.84 and 335.92 µmol/L (2.6-3.8 mg/dL). Renal biopsies revealed acute tubular necrosis.
Four weeks after discontinuing injections and supplements, serum creatinine was in the
normal range and estimated glomerular filtration rate > 1.00 mL/s (60 mL/min), including two
patients with biopsies showing >30 percent interstitial fibrosis and tubular atrophy. The
findings highlight a risk for acute and potentially chronic kidney injury among young men
abusing anabolic steroids and using excessive amounts of nutritional supplements [150042].
1240
End-stage renal disease in a bodybuilder
In the United States, as many as 1 million individuals are estimated to use drugs for athletic
achievements or for gaining a more muscular appearance. Reported untoward negative
effects of AAS‐supported bodybuilding include an increase in coronary risk factors, acute
myocardial infarction, cholestatic jaundice, abnormal liver function, rhabdomyolytic
complications, and severe mood and psychotic disorders. It was reported on a possible renal
side‐effect in a patient using the beta2 adrenoreceptor agonist clenbuterol and AAS. A
27‐year‐old man was referred to our department by a general practitioner because of
azotaemia, with s‐creatinine 754 μmol/l, s‐urea 26.3 mmol/l, s‐uric acid 444 micromol/L, a
semiquantitative urine protein excretion of >500 mg/L, and haematuria of >250 RBC/microL.
He reported a sudden short syncopal status during his work as a coach‐builder, with an
elevated systolic BP of 200 mmHg. Moreover, he complained about frequent fits of dizziness,
fatigue and vision loss during the previous 3 months. There was no family history of chronic
illness, hypertension, or renal or systemic diseases. His past medical history revealed
recurrent episodes of pharyngitis in childhood more than three times a year. He was a
non‐smoker, not exposed to animals, and did not travel. The last documented clinical status
was his military examination 9 years previously, with no indication of any physical disability.
In his spare time he had been an active bodybuilder for 5 years with an extensive physical
exercise programme three to four times a week for 3-4 h. He had regularly taken a
high‐protein commercial diet (Designer protein®, BMS‐Sporternährung Gronau, Germany)
with more than 2 g/kg body weight/day containing L‐glutamine 33.3 g, taurine 33.3 g, and
creatine 210 g. Additionally he had taken AAS (testosterone/Depot‐Rotex Medica®) 750–
1000 mg/week at 6‐week intervals as well as clenbuterol (Spiropent® tablets) t.d.s. according
to official guidelines [2] for 18 months. The sources of supply were often unknown.
Sometimes he got them from unchecked dealers, mostly from East‐European countries. He
reported on progressive peripheral oedema and an attack of gout during drug intake, which
began 1 year before presentation. He denied the use of analgesic drugs, non‐steroidal
anti‐inflammatory drugs (NSAID), laxatives, or diuretics. Physical examination showed an
athletic (81 kg, 1.86 m) man with appropriate muscular tone, trifling peripheral oedema, no
signs of cardiac or pulmonary decompensation, with a regular heart beat of normal frequency
without any murmurs or pericardial rubbing. The blood pressure was 190/110 mmHg without
any side difference by single measurement. During ambulatory 24‐h registration (Spacelabs®
model 90207, Redmond Inc., Washington, USA), a hypertensive profile was obvious with
98.1% of systolic and 92.6% of diastolic and 100% of night‐time values above the
well‐defined threshold respectively. MAPNIGHT/DAY‐ratio was 0.9. Pertinent laboratory findings
included s‐creatinine (1030 μmol/l) with a creatinine clearance of 10.2 ml/min/1.73 m2, and
elevated s‐urea (31.6 mmol/l), moderately increased s‐uric acid (485 μmol/l),
hyperphosphataemia (2.54 mmol/l), high intact parathyroid hormone (iPTH) level (281 ng/l)
with low 1.25‐(OH)2D3 (<2.0 pg/ml), hypoproteinaemia (55.0 g/l), increased s‐IgG (4.4 g/l),
total cholesterol (6.42 mmol/l), low‐density lipoprotein (LDL) cholesterol (4.53 mmol/l),
LDL/high‐density lipoprotein (HDL) ratio (4.04), cholesterol/HDL cholesterol ratio (5.73),
lipoprotein (a) (Lp(a)) (37.7 mg/dl), and haemoglobin (Hb)A1C (6.6%). Red blood cell count
showed a decreased Hb (5.5 mmol/l), haematocrit (0.27), and reticulocyte count (6%), i.e.
normochromic normocytic anaemia. Serum electrolytes (Na+, K+, Ca2+, PO4, C1−, Mg2+),
acid–base‐status, liver function parameters, and serological findings (hepatitis B and C
viruses, HIV, leptospira, hantavirus) revealed no pathological findings. No histological
similarities to cyclosporin toxicity or simple arteriosclerotic lesions were observed. The
patient commenced haemodialysis treatment three times a week and was prepared for renal
transplantation. In the case, several possible pathogenic factors for the development of
end‐stage renal disease (ESRD) may be considered. Due to the advanced chronic damage
1241
the histological findings were not definite enough to allow an exact differentiation or definition
of the primary underlying aetiological factors. An isolated effect of beta‐adrenergic
substances on the acceleration of a hypertensive renal damage process may be considered,
although this has not yet been documented. Especially under intermittent treatment with
unphysiological to toxic doses of clenbuterol in rats, an acute increase in muscle blood flow
and hypertensive reaction hints at a transient intrinsic blood pressure activity of clenbuterol
which could not be sustained by chronic treatment because of a remarkable beta‐adrenergic
receptor down‐regulation. However, the observation of increased plasma glucose and serum
insulin levels in patients with essential hypertension after mabuterol suggests a role of
beta‐2‐adrenoreceptor in the pathogenesis of insulin resistance and its negative renal
sequelae]. Other data argue for a possible blood‐pressure‐independent role of the
sympathetic nervous system in renal damage in experimental hypertension. The
echocardiographic findings seems to underline this link, although there is no proof of a
pathogenic association between AAS and cardiohypertrophic effects. Although “hypertensive
nephrosclerosis” is quite often a misdiagnosis (because of the scarce available histological
evidence) a considerable portion of hypertensives – with constitutional, socio‐economic,
ethnic, and environmental varieties – appears to suffer from true’hypertensive
nephrosclerosis, even if only a small percentage of these patients develops ESRD.
Nevertheless, the accelerating effects of a high protein consumption in the process of chronic
renal failure has been well known for more than a century. Bodybuilders often prefer a
high‐protein and creatine‐supplemented diet to achieve maximum skeletal muscle
hypertrophy and training adaptations during intense exercise. Furthermore, attention should
also be paid to the possibility of interstitial nephritis as an adverse effect of clenbuterol,
although there are no reports on a direct link or renal complications during application. The
observation of prevention of cyclosporin‐induced nephrotoxicity in rats by the
beta2‐adrenoreceptor agonist clenbuterol due to isolated renal cortical vasodilatation caused
by reduced intracellular calcium argues against a pathogenic role of the adrenoreceptor
agonist in inducing renal damage. Lifestyles which lead to chronic hypovolaemia, especially
in subjects with a bodily‐stressed personality, may magnify renal damage processes, and are
frequently found among bodybuilders (or ballet dancers) and are often exacerbated by an
on‐the‐spot use of diuretics. It has been] clearly demonstrated recently that AAS such as
testosterone at different doses plays a permissive role for renal compensatory growth in rats.
They mentioned that the magnitude of renal risks in adults depend on gender and may be
presumably mediated via sex hormones. This is presumably mediated by renal testosterone
receptors and may set the stage for accelerated progression of renal disease in the organism
exposed to male sex hormones. The growing number of papers investigating the atherogenic
effects of AAS caused by marked HDL‐level depression suggests an additional independent
risk factor. Bodybuilding is increasingly popular and doping more than a phrase since the
Tour de France 1998. In view of the reported case, the following quotation out of an official
guide book for the use of AAS in bodybuilding seems to be nearly sarcastic: ‘If the claims
about the risks of physical damage were true, no professional bodybuilders would exist any
more since all of them would have died long ago. In fact, the non‐stop use of steroids is quite
usual in ambitious athletes and results in constant increase rates … E.g. if a 24‐year‐old
athlete becomes the world champion, he has a good physical predisposition and looks back
on an almost non‐stop steroid consumption of several years’. In conclusion, it was
suggested that the use of clenbuterol and AAS provides a scenario that may increase the
risk of renal failure especially in pre‐existing kidney diseases. Renal failure due to
consumption of (intermittent) high to toxic doses of beta‐adrenergic substances and/or AAS
should be recognized in general. Bodybuilders, usually exposed to forced muscle gain and
hypertensive situations, should be considered a high‐risk group in particular [01071].
1242
Other side effects
AAS may affect the immune system, the lungs, and possibly other organ systems, and might
cause acne, although knowledge in these areas remains limited [14017].
Anabolic-androgenic steroids (AAS) are synthetic drugs derived from testosterone. Illegally,
these drugs are regularly self-administered by body builders and power lifters to enhance
their sportive performance. Adverse side effects of AAS include sexual dysfunction,
alterations of the cardiovascular system, psyche and behavior, and liver toxicity. However,
severe side effects appear only following prolonged use of AAS at high dose and their
occurrence is limited. Occasionally, AAS abuse may be linked to certain social and
psychological traits of the user, like low self-esteem, low self-confidence, suffered hostility,
childhood conduct disorder, and tendency to high-risk behavior. The overwhelming
stereotype about AAS is that these compounds cause aggressive behavior in males.
However, the underlying personality traits of a specific subgroup of the AAS abusers, who
show aggression and hostility, may be relevant, as well. Use of AAS in combination with
alcohol largely increases the risk of violence and aggression. The dependence liability of
AAS is very low, and withdrawal effects are relatively mild. Based on the scores for acute
and chronic adverse health effects, the prevalence of use, social harm and criminality, AAS
were ranked among 19 illicit drugs as a group of drugs with a relatively low harm [10059].
Side effects of anabolic steroids with relevance in forensic medicine are mainly due to life-
threatening health risks with potential fatal outcome and cases of uncertain limitations of
criminal liability after steroid administration. Both problems are typically associated with long-
term abuse and excessive overdose of anabolic steroids. Side effects may be due to direct
genomic or nongenomic activities (myotrophic, hepatotoxic), can result from down-regulation
of endogenous biosynthesis (antiandrogenic) or be indirect consequence of steroid
biotransformation (estrogenic).Logically, there are no systematic clinical studies available
and the number of causally determined fatalities is fairly limited. A compilation reviews typical
abundant observations in cases where nonnatural deaths (mostly liver failure and sudden
cardiac death) were concurrent with steroid abuse. Moreover, frequent associations between
structural characteristics and typical side effects may be explained [10060].
Regarding the health risks associated with the abuse of performance-enhancing drugs in
sport data from randomized clinical trials may not be sufficient to identify the complete range
of adverse effects possible with the abuse; more specific studies are necessary to assess
their actual and full toxic potential [08139].
Thyroidal effects
The most prominent effect on human thyroid function of anabolic steroids is reduction of
thyroxine binding globulin (TBG), with consequent reductions of total serum T3 and T4,
depending however on the susceptibility of the drug to aromatization and subsequent
transformation into estrogen. In rats, anabolic steroids also act in the peripheral metabolism
of thyroid hormones and seem to exert an important proliferative effect on thyroid cells
[08145].
The use of anabolic steroids to increase physical performance and for aesthetic ends has
reached alarming indices in the last three decades. Besides the desired actions, several
collateral effects have been described in the literature, such as the development of some
1243
types of cancer, gynecomasty, peliosis hepatis, renal insufficiency, virilization, amongst
others. The most prominent effect on human thyroid function is the reduction of thyroxine
binding globulin (TBG), with consequent reductions of total serum T3 and T4, depending
however on the susceptibility of the drug to aromatization and subsequent transformation into
estrogen. In rats, anabolic steroids also act in the peripheral metabolism of thyroid hormones
and seem to exert an important proliferative effect on thyroid cells. Thus, the aim of the
present paper was to review data on the effect of supraphysiological doses of anabolic
steroids on thyroid function, showing the danger that indiscriminate use of these drugs can
cause to health [07073].
Thyroid cells have androgen receptors, and AASs may directly influence thyroid function.
Some studies have shown effects on thyroid function, including a decrease in total
triiodothyronine, thyroxine (T4), and thyroid-binding globulin. Some studies have shown an
increase in thyrotropin and free T4, whereas others have shown no change in these
concentrations. It is unclear if this relative impairment in thyroid function leads to a clinical
effect. These changes may be due to direct block of thyroid hormone release or synthesis or
some other mechanism [07058].
Adrenal effect (on cortisol production)
Complementary to the competitive antagonism with the estrogen receptors, a similar

competitive antagonism has been described with respect to glucocorticoid receptors.
Glucocorticoids are substances with catabolic properties that will be released in the serum as
a result of (strong) physical or mental stress, e.g. exercise training, surgery and
psychological problems. By binding to the glucocorticoid receptors, the AAS are able to
counteract the breakdown of proteins by the glucocorticoids. This competitive antagonism
may also play a role in the treatment of osteoporosis through reduction of bone breakdown
and stimulation of bone formation. Recently, evidence has become available that AAS
stimulate proliferation and differentiation of osteoblastic cells and have the capacity to
counteract bone breakdown. Testosterone has also been found to correct calcium balance
and bone formation, playing a role in the reduction of bone resorption [04002].
Hematological
AASs increase renal synthesis of erythropoietin. They also promote erythropoietic stem cell
differentiation. Subsequently, hemoglobin and hematocrit may become elevated, which could
result in erythrocytosis or sludging. Two adult cases of intramuscular testosterone–induced
polycythemia were reportedly reversed by switching to transdermal testosterone; however, a
65-year-old man developed hypertension and polycythemia during daily testosterone
application to his scrotum for 5 years (estimated dose 10 mg/d). Polycythemia and
hypertension resolved when testosterone was discontinued. Mild, but significant, increases in
mean red blood cell, hematocrit, hemoglobin, and white blood cell concentrations in 33 men
were reported following intramuscular testosterone enanthate, 200 mg every 3 or four weeks
for 24 weeks. The men remained asymptomatic. Increased platelet count and aggregation
also may occur. AASs may potentiate platelet aggregation and be thrombogenic in humans;
however, another study found only non-significant trends, including thrombocytosis and
increased aggregation [07058].
AAS use is associated with dose-related increases in hemoglobin and hematocrit, and
polycythemia is a frequent adverse event of AAS use. Androgens stimulate erythropoiesis by
increasing sensitivity to erythropoietin, suppressing hepcidin transcription, and increasing
iron availability for erythropoiesis [14426].
1244
AAS are considered to influence the haematological system via two main pathways. First,
anabolic steroids stimulate erythropoiesis directly and erythropoietin synthesis in the kidney.
Secondly, the effects of androgens have been demonstrated to promote erythropoietic stem
cell differentiation and to increase the sensitivity of erythroid progenitors. Since the
introduction of recombinant human erythropoietin in the 1980s, the administration of AAS for
the these effects has been relegated to the background both by clinicians and athletes
[04002].
Testosterone treatment induces erythrocytosis that could potentially affect blood viscosity
and cardiovascular risk. We thus investigated the effects of testosterone administration on
blood viscosity and erythrocyte deformability using mouse models. Blood viscosity,
erythrocyte deformability, and hematocrits were measured in normal male and female mice,
as well as in females and castrated males after short-term (2 wk) and long-term (5-7 mo)
testosterone intervention (50 mg/kg, weekly). Castrated males for long-term intervention
were studied in parallel with the normal males to assess the effect of long-term testosterone
deprivation. An additional short-term intervention study was conducted in females with a
lower testosterone dose (5 mg/kg). The results indicate no rheological difference among
normal males, females, and castrated males at steady-state. Short-term high-dose
testosterone increased hematocrit and whole-blood viscosity in both females and castrated
males. This effect diminished after long-term treatment, in association with increased
erythrocyte deformability in the testosterone-treated mice, suggesting the presence of
adaptive mechanism. Considering that cardiovascular events in human trials are seen early
after intervention, rheological changes as potential mediator of vascular events warrant
further investigation [150218].
Polycythemia
Supraphysiologic levels of plasma androgens stimulate erythropoietin production in a dose-
dependent manner and may lead to clinically significant secondary polycythemia. The
increase in plasma viscosity may be a contributing factor to adverse cardiovascular events in
AAS users, especially in those patients with preexisting coronary risk factors; however, this
potential relationship has not been definitely demonstrated by meta-analysis. Nevertheless,
correction of severe polycythemia in AAS users should be attempted by phlebotomy.
However, ultimately the discontinuation of AAS and a restoration of normal endogenous
hormone levels are paramount for reducing the patient's risk for potential polycythemia-
associated complications [14214].
Intussusceptions are generally associated with mechanical lead points or localized

inflammation that function as foci for intestinal telescoping. It was presented a case of a
patient whose abuse of anabolic steroids resulted in the development of multiple
simultaneous intussusceptions. Our patient had no additional identifiable risk factors for
intussusception. Consistent with previous reports, corticosteroid induced polycythemia and
its consequent hyperviscosity led to intravascular sludging and mesenteric ischemia with
associated bowel wall thickening. The localized intestinal induration then served as
mechanical foci for intussusception. Due to the illicit nature of anabolic androgenic steroid
(AAS) abuse, the physiologic effects of supraphysiologic doses are sparsely reported and
poorly understood. The scope of AAS abuse and its consequences are likely under-reported
and under-recognized within the medical community. The case presented a unique
diagnostic and therapeutic challenge with which aimed to increasing awareness and clinical
suspicion for AAS among healthcare personnel [150011].
Unilateral sudden sensorineural hearing loss due to an infarct in the vertebrobasilar system
has been widely reported. Most patients have a background of traditional coronary risk
1245
factors related to these cerebrovascular episodes. A 32-year-old male, a regular user of
anabolic steroids, presented to the emergency department with unilateral sensorineural
hearing loss and symptoms suggestive of an infarct of the anterior inferior cerebellar artery
but in the absence of risk factors for ischaemic stroke. Magnetic resonance imaging
confirmed the presence of infarction in the region supplied by the anterior inferior cerebellar
artery. Polycythaemia was found on haematological analysis, which we believe was
secondary to the use of anabolic steroids. The patient was commenced on aspirin as per the
stroke management protocol. There was resolution of neurological symptomatology six
weeks after the episode, but no improvement in hearing. This is the first case report of
unilateral sensorineural hearing loss secondary to the use of anabolic steroids causing
polycythaemia. This cause should be considered in the differential diagnosis of patients
presenting with sensorineural hearing loss, especially in young males, when no other risk
factors can be identified [150219].
Hypercalcemia
A 26-year-old male bodybuilder was admitted to the surgical department of a Danish

community hospital for hematemesis. During the clinical interview, he revealed that he had
recently finished a course of anabolic steroids and erythropoietin. The patient also had a
previous history of infections and chronic ulcers due to paraffin-oil injections in both upper
arms one year before. Over the course of the next few hours, the patient developed signs of
multi-organ dysfunction, including pancreatitis, hemorrhagic gastritis, nephropathy with
temporary anuria, and respiratory insufficiency, and was transferred to the ICU. After
manometric monitoring on the patient's upper arms proved difficult, invasive blood pressure
monitoring was used and revealed that the patient was in a state of hypertensive crisis. This
case of multi-organ dysfunction was possibly caused by multi-substance-induced
hypercalcemia [11067].
Persistent hiccups
Hiccups have been classified as a neurologic reaction triggered by a multitude of factors.

There are only a few reports of persistent hiccups associated with oral and intravenous
corticosteroid use in the medical literature. It has been proposed that corticosteroids lower
the threshold for synaptic transmission in the midbrain and directly stimulate the hiccup reflex
arc. There is a recent report of progesterone-induced hiccups, which were thought to occur
secondary to the glucocorticoid-like effects of progesterone on the brainstem. It was reported
the first case of anabolic steroid-induced hiccups occurring in an elite power lifter. The
hiccups occurred within 12 hours of the individual increasing his doses of oral anabolic
steroids and persisted for 12 consecutive hours until medical attention was sought. In this
report the pathophysiology of anabolic steroid-induced hiccups is discussed, and the
postulated relationships of steroids and the hiccup reflex arc reviewed [01073].
Effect on inflammation
Aging can alter the skeletal muscle growth response induced by overload. The initiation of
overload induces muscle extracellular matrix expansion, increased cellularity, and
inflammatory gene expression, which are all related to processes important for myofiber
growth. These remodeling processes are also biological targets of testosterone. It is not
certain how aging affects the inflammatory response to functional overload and whether
anabolic steroid administration can alter this response. The effect of anabolic steroid
administration on inflammatory processes during functional overload is not known. The
purpose of this study was to determine if age altered the skeletal muscle inflammatory
1246
response at the onset of functional overload and whether anabolic steroid administration
would modulate this response in young or older animals. Five-month and 25 month F344 x
BRN rats were given nandrolone decanoate (ND) (6 mg/kg bw/wk) or sham injections for 3
weeks, and then the soleus muscle was overloaded (OV) for 3 days by synergist ablation.
ND alone induced a 230% increase in ED1(+) cells in 5 month muscle. Three days of OV had
no effect on ED1(+) cell number at either age. OV combined with ND induced a 90%
increase in ED2(+) cells in 5 month muscle, while there was no effect of either treatment
alone at this age. In 25 month muscle, OV induced a 40 percent increase in ED2(+) cells.
Regardless of age, OV induced muscle TNF-alpha mRNA expression (300%) and IL-6
mRNA expression (900 %). ND attenuated OV-induced IL-6 mRNA but not TNF-alpha
expression in both age groups. The overload induction of IL-1beta mRNA was 3-fold greater
in 25 month muscle (1400 %), compared to 5 month muscle (400 %). ND administration
ablated the overload IL-1beta mRNA induction in 25 month muscle. Anabolic steroid
administration can suppress inflammatory cytokine gene expression at the onset of overload
and this effect is age dependent [06088].
In basket ball players
It was analyze the outcome on testosterone (T) and cortisol (C) responses in 12 professional
basketball players during a season of competition. Serum adrenocorticotropic hormone
(ACTH), C, total testosterone (TT), and free testosterone (FT) levels were analyzed in
October, December, March, and April. A day after the games, blood samples were taken.
Serum ACTH levels were maintained at the initial levels during the season. However, basal
C significantly changed during the season, with lower levels in December and in April. Basal
serum TT levels increased during the season until a maximum in March. No differences were
presented in the TT values in December, March, and April. Basal FT presented high levels in
October and December, followed by a low level in March, remaining low in April. The T/C
increased during the season, attaining a maximum level in December, followed by a
significant decrease in March. Free T/C ratio decreased during the season (lower level in
March). In conclusion, the players maintained a good anabolic-catabolic balance [10062].
Side effects in elderly
Testosterone supplementation has been shown to increase muscle mass and strength in
healthy older men. The safety and efficacy of testosterone treatment in older men who have
limitations in mobility have not been studied. Community-dwelling men, 65 years of age or
older, with limitations in mobility and a total serum testosterone level of 100 to 350 ng per
deciliter (3.5 to 12.1 nmol per liter) or a free serum testosterone level of less than 50 pg per
milliliter (173 pmol per liter) were randomly assigned to receive placebo gel or testosterone
gel, to be applied daily for 6 months. Adverse events were categorized with the use of the
Medical Dictionary for Regulatory Activities classification. The data and safety monitoring
board recommended that the trial be discontinued early because there was a significantly
higher rate of adverse cardiovascular events in the testosterone group than in the placebo
group. A total of 209 men (mean age, 74 years) were enrolled at the time the trial was
terminated. At baseline, there was a high prevalence of hypertension, diabetes,
hyperlipidemia, and obesity among the participants. During the course of the study, the
testosterone group had higher rates of cardiac, respiratory, and dermatologic events than did
the placebo group. A total of 23 subjects in the testosterone group, as compared with 5 in the
placebo group, had cardiovascular-related adverse events. The relative risk of a
cardiovascular-related adverse event remained constant throughout the 6-month treatment
period. As compared with the placebo group, the testosterone group had significantly greater
improvements in leg-press and chest-press strength and in stair climbing while carrying a
1247
load. It was concluded that in a population of older men with limitations in mobility and a high
prevalence of chronic disease, the application of a testosterone gel was associated with an
increased risk of cardiovascular adverse events [10329].
Dental health
One study aimed to evaluate periodontal microbiological differences between systemically

healthy nonsmoker males taking anabolic androgenic steroids (AASs) and non-AAS users
and to find associations between disease severity and AAS use. Ninety-two men practicing
bodybuilding were included in the study. They were divided into AAS users and a matched
control nonuser group and subgrouped based on their most severe periodontal condition.
Pooled subgingival samples from each individual were cultured to evaluate specific
periodontopathogen infection. AAS users had significantly higher prevalence of severe
periodontitis. AAS users had greater gingival inflammation and clinical attachment loss of ≥ 3
mm than nonusers (odds ratio (OR) 2.4; 95 % confidence interval 0.8-6.4). AAS users were
4.9 times more likely to be infected with Prevotella intermedia than AAS nonusers. The OR of
presenting subgingival Aggregatibacter actinomycetemcomitans was 8.2 times higher in AAS
users. AAS users were 5.6 times more likely to present subgingival Candida spp. than
nonusers. AAS users were 14.8 times more likely to present subgingival Candida
parapsilosis than nonusers. The likelihood of AAS users presenting subgingival Candida
tropicalis was 4.3 times higher than nonusers. A. actinomycetemcomitans was mostly
isolated in individuals with severe periodontitis and was associated with subgingival
Porphyromonas gingivalis, P. intermedia, and Candida spp. AAS use may increase the risk
for severe periodontitis and may cause a subgingival selection of certain Candida species.
Specific periodontopathogens, such as Candida dubliniensis and Candida albicans, seem to
be negatively affected by AAS use. The higher risk for disease progression in AAS users
may be explained by the significantly higher proportions of A. actinomycetemcomitans, P.
gingivalis, P. intermedia, and Candida species as compared to controls. Data on the
influence of AAS on subgingival periodontopathogens and disease progression are scarce.
Higher proportions of specific periodontopathogens are plausible in AAS users. AAS users
had a higher prevalence of severe periodontitis, gingival inflammation, and clinical
attachment loss. Men taking AAS are at greater risk of periodontitis and specific
periodontopathogen infection [14456].
Effect on gingival tissues
Anabolic androgenic steroid (AAS) is the familiar name for synthetic derivatives of the male
sex hormone, testosterone. A large number of young adults abuse AAS to enhance
performance and physical appearance. The aim of one study was to evaluate the effects of
AAS abuse on the gingival tissues in a group of bodybuilders and weight lifters. The test
group was composed of 24 athletes aged between 17 and 29 years who had been using
AAS for >1 year. All subjects were clinically examined for plaque levels (plaque index),
gingival inflammation (gingival index), and gingival enlargement. The results were compared
to a control group of 20 bodybuilders who had never used AAS drugs and who matched for
age, educational level, and oral habits according to the data obtained from the test group.
Although there were no statistical differences between the plaque index and gingival index
scores of the study group and the control group, the AAS abusers had statistically higher
scores of gingival thickness, extent of gingival encroachment, and total gingival enlargement
scores compared to non-users. It was concluded that the results of this study have revealed
that the prolonged use of AAS is closely associated with significant levels of gingival
enlargement. Because recreational abuse and abuse in non-competitive sports seem to
1248
increase despite legislation, dentists and periodontists should be familiar with the adverse
effects of these synthetic derivatives of testosterone on the gingival tissues [06087].
Peridontit
One study aimed to evaluate periodontal microbiological differences between systemically
healthy nonsmoker males taking anabolic androgenic steroids (AASs) and non-AAS users
and to find associations between disease severity and AAS use. Ninety-two men practicing
bodybuilding were included in the study. They were divided into AAS users and a matched
control nonuser group and subgrouped based on their most severe periodontal condition.
Pooled subgingival samples from each individual were cultured to evaluate specific
periodontopathogen infection. AAS users had significantly higher prevalence of severe
periodontitis. AAS users had greater gingival inflammation and clinical attachment loss of
≥3 mm than nonusers (odds ratio 2.4;). AAS users were 4.9 times more likely to be infected
with Prevotella intermedia than AAS nonusers (OR 4.9). The OR of presenting subgingival
Aggregatibacter actinomycetemcomitans was 8.2 times higher in AAS users (OR 8.2). AAS
users were 5.6 times more likely to present subgingival Candida spp. than nonusers
(OR 5.6). AAS users were 14.8 times more likely to present subgingival Candida parapsilosis
than nonusers (OR 14.8). The likelihood of AAS users presenting subgingival Candida
tropicalis was 4.3 times higher than nonusers. A. actinomycetemcomitans was mostly
isolated in individuals with severe periodontitis and was associated with subgingival
Porphyromonas gingivalis, P. intermedia, and Candida spp. It was concluded that AAS use
may increase the risk for severe periodontitis and may cause a subgingival selection of
certain Candida species. Specific periodontopathogens, such as Candida dubliniensis and
Candida albicans, seem to be negatively affected by AAS use. The higher risk for disease
progression in AAS users may be explained by the significantly higher proportions of A.
actinomycetemcomitans, P. gingivalis, P. intermedia, and Candida species as compared to
controls. Data on the influence of AAS on subgingival periodontopathogens and disease
progression are scarce. Higher proportions of specific periodontopathogens are plausible in
AAS users. AAS users had a higher prevalence of severe periodontitis, gingival
inflammation, and clinical attachment loss. Men taking AAS are at greater risk of periodontitis
and specific periodontopathogen infection [13155].
Persistent hiccups
Hiccups have been classified as a neurologic reaction triggered by a multitude of factors.

There are only a few reports of persistent hiccups associated with oral and intravenous
corticosteroid use in the medical literature. It has been proposed that corticosteroids lower
the threshold for synaptic transmission in the midbrain and directly stimulate the hiccup reflex
arc. There is a recent report of progesterone-induced hiccups, which were thought to occur
secondary to the glucocorticoid-like effects of progesterone on the brainstem. It was reported
the first case of anabolic steroid-induced hiccups occurring in an elite power lifter. The
hiccups occurred within 12 hours of the individual increasing his doses of oral anabolic
steroids and persisted for 12 consecutive hours until medical attention was sought. In this
report the pathophysiology of anabolic steroid-induced hiccups is discussed, and the
postulated relationships of steroids and the hiccup reflex arc reviewed [01073].
Toxicity
To study the doping substances used in sport and their toxicity a retrospective analysis from
January 1992 to December 2000 of the cases of use of doping substances in sport reported
by telephone to the anti-poison center in Marseilles was performed. Fifty-one cases were
reported concerning 48 men and 3 women with a mean age of 30, ranging from 10 to 55
1249
years. Sixty-three percent of cases were reported over the last four years. The sport
practiced was bodybuilding, except in 2 cases (cycling in one case and running in the other).
The products used were mainly anabolizing hormones (15 times), clenbuterol (14 times) and
creatine (7 times). A third of cases concerned associations of substances and 19 cases
presented with symptomatology. It was concluded that the diversity in nature and status of
the substances mentioned and their association requires enhanced vigilance with regard to
the use of drugs in sport. The recent measures voted within the framework of the anti-doping
law dated 23/3/99 are aimed at increasing surveillance with the development of anti-doping
antennae [01074].
Genotoxicity (cancer risks)
AASs may affect the immune system, the lungs, and possibly other organ systems and might
cause acne, although knowledge in these areas remains limited. Notably, there is little
evidence of an association between AAS use and cancer, with the exception of rare reports
of hepatic cancers, intratesticular leiomyosarcoma, and renal cell carcinoma. Conspicuous
by their absence are reports of prostate cancer in AAS users. To date, there is no clear
evidence that androgen administration causes prostate cancer; we are aware of only 2 case
reports of prostate cancer in bodybuilders, both published more than 20 years ago. However,
the possibility remains that high doses of AAS administered during the peripubertal period
may exert long-term epigenetic effects and may increase the risk of prostate-related events
later in life. Given that older AAS users (who started AAS use in their peripubertal years in
the 1980s) are just now entering the fifth decade of their life, we may have more evidence
regarding AAS use and prostate cancer in the coming years [14426].
Notably, there is little evidence of an association between AAS use and cancer, with the
exception of rare reports of hepatic cancers, intratesticular leiomyo-sarcoma, and renal cell
carcinoma. Conspicuous by their absence are reports of prostate cancer in AAS users. To
date there is no clear evidence that androgen administration causes prostate cancer. Only
two case reports of prostate cancer in bodybuilders, both published more than 20 years ago.
However, the possibility remains, that high doses of AAS administered during the peri-
pubertal period may exert longterm epigenetic effects and may increase the risk of prostate-
related events later in life. Given that older AAS users (who started AAS use in peri-pubertal
years in the 1980s) are just now entering the fifth decade of their life, we may have more
evidence regarding AAS use and prostate cancer in the coming years [14017].
The abuse of anabolic steroids for doping raises concerns. Many of these compounds have
never been examined for their toxicological properties. Aside from hormonal (androgenic)
activity, anabolic steroids may also exert genotoxic effects. In one study, it was determined
the potencies of the "designer steroid" madol and the anabolic prohormone 19-
norandrostenedione to induce micronuclei in V79 cells in vitro. CREST analysis was used to
differentiate between aneugenic and clastogenic mechanisms of micronucleus induction.
Cytotoxicity of the steroids and their influence on the cell cycle were assessed in parallel. In
addition, the ability of the drugs to increase production of reactive oxygen species and to
induce apoptosis were studied. Both agents caused a concentration-dependent increase in
the rates of micronuclei in V79 cells, exceeding a doubling of the background micronucleus
rates of untreated controls, which was evident at 27 muM and 29 muM for madol and 19-
norandrostenedione, respectively. The steroid-induced micronuclei were predominantly
kinetochor (CREST)-negative, pointing to a clastogenic mode of action. As cytotoxicity of
both compounds is weak, cytotoxicity was unlikely to contribute to their genotoxicity. The
observed genotoxicity of both compounds was due neither to apoptosis induction nor to
production of reactive oxygen species. However, the ability of both steroids to induce
micronuclei appears related to their lipophilicity. Therefore, a "non-specific" chromosomal
1250
genotoxicity of madol and 19-norandrostenedione, based on hydrophobic interactions,
appears likely. This could well result in biologically relevant increases in chromosomal
damage as soon as critical concentrations of the agents are reached in vivo. Regarding the
current misuse of the steroids for doping, the uncontrolled administration of very high doses
must be considered. Therefore it cannot be ruled out that Thess drugs present genotoxic
hazards under current misuse conditions by athletes in sports or in body building [08149].
To evaluate genotoxicity of anabolic androgenic steroids (AAS) in male bodybuilders by a

micronucleus assay in buccal mucosa cells 11 male bodybuilders volunteered to participate
in a study and two groups were formed: group 1 (n=6), without AAS consumption and group
2 (n=5), with AAS consumption. A sample of buccal epithelium was taken from each
participant once a week for 6 weeks. Samples were fixed, stained and analysed by a light
microscope, and 2000 cells were counted from each slide. Results are expressed as
micronucleated cells (MNC) per 1000 cells and were analysed by the Mann-Whitney U test
and Wilcoxon's test. A marked increased in MNC was seen in bodybuilders with AAS
consumption compared with those without AAS consumption (mean 4.1 MNC/1000 cells vs
0.4 MNC/1000 cells, respectively). Intragroup comparisons showed no differences in the
MNC frequencies during the sampling time in group 1, whereas the MNC frequency in group
2 varied significantly, reaching the highest MNC frequencies in the third and fourth week of
sampling; frequency in the first sampled week was 1.1 MNC/1000 cells. Significant
differences in all sampled weeks were found between the two groups. It was concluded that
AAS consumption increased the frequency of MNC from buccal mucosa in bodybuilders
[07078].
In recent years, increasing attention has been paid to the role of hormones in breast cancer
etiology, following reports that heightened levels of endogenous hormones and exposure to
exogenous hormones and other endocrine-disrupting chemicals through food and the
environment are associated with increased breast cancer risk. Seven hormone drugs
(testosterone propionate, trenbolone acetate, estradiol, zeranol, progesterone, melengestrol
acetate, and bovine somatotropin) are approved by the U.S. Food and Drug Administration
for use in food animals. There is concern that these drugs or their biologically active
metabolites may accumulate in edible tissues, potentially increasing the risk of exposure for
consumers. To date, the potential for human exposure to residues of these compounds in
animal products, as well as the risks that may result from this exposure, is poorly understood.
In this paper, we discuss the existing scientific evidence examining the toxicological
significance of exposure to hormones used in food animal production in relation to breast
cancer risk. Through a discussion of US federal regulatory programs and the primary
literature, we interpret the state of surveillance for residues of hormone drugs in animal
products and discuss trends in meat consumption in relation to the potential for hormone
exposure. Given the lack of chronic bioassays of oral toxicity of the seven hormone
compounds in the public literature and the limitations of existing residue surveillance
programs, it is not currently possible to provide a quantitative characterization of risks that
result from the use of hormonal drugs in food animal production, complicating our
understanding of the role of dietary hormone exposure in the population burden of breast
cancer [150224].
Chromosome damage
The aim of one study was to evaluate the potential of the androgenic anabolic steroids (AAS)
for inducing chromosome damage, apoptosis, and necrosis, using the micronucleus test on
exfoliated cells from the oral mucosa of AAS users. The sample consisted of 55 male
individuals, practitioners of physical exercise divided into two groups: 25 individuals who
were users of AAS and 30 individuals in the control group. Cytological analysis included, in
addition to micronuclei, counting of broken eggs and degenerative nuclear changes
1251
indicative of apoptosis (karyorrhexis, condensed chromatin, and pyknosis) and necrosis
(karyolysis in addition to these changes). The statistical analysis did not show differences in
occurrences of micronuclei, karyolysis, and broken eggs between the groups. The
occurrence of apoptosis was significantly higher in cells from control subjects. The results
obtained showed that inhibition of apoptosis was induced by AAS, suggesting that this may
be one of the mechanisms contributing toward the association that has been described
between use of AAS and the carcinogenic process [150225].
Fatal events
Anabolic-androgenic steroids (AAS) are widely abused, but the potential for dependence and
addiction remains unclear. Recent studies from our laboratory have shown that male and
female hamsters will voluntarily self-administer testosterone and other AAS. Furthermore, it
was observed fatal androgen overdose during self-administration. This suggests that AAS
are potentially addictive, independent of their effects on muscle mass or athletic performance
[06093].
It was reported a 42-year-old male amateur body builder and user of anabolic androgenic
steroids, who developed ARDS, acute kidney injury, and refractory supraventricular
tachycardia. He required extracorporeal membrane oxygenation, continuous veno-venous
hemodialysis, and catheter ablation. It was believed that long-term anabolic androgenic
steroid abuse predisposed the patient to multiple organ dysfunction syndrome, from its
immunomodulatory effects in an otherwise healthy patient. Anabolic androgenic steroid use
should be part of the history taking process, since it may complicate diagnosis, disease
progression, and prognosis [13156].
Side effects of topical anabolic steroids
Androstanolone is an androgen with effects and efficacy comparable to dihydrotestosterone.

Topical sexual steroid hormones may easily penetrate human skin and cause systemic
effects. Topical androgens like testosterone have been used for decades to treat vulvar
lichen sclerosus et atrophicus with a doubtful efficacy. Due to systemic absorption of
testosterone virilism expressed as hirsutism may develop. Topical application of testosterone
causes also systemic effects. Indeed, sexual steroids including androgens may show a
significant percutaneous absorption leading to changes in the serum levels. Precursors like
pregneolone or androstendione fail to alter serum levels because of rapid metabolisation. In
controlled trials carefully monitored low-dose testosterone therapy in women efficiently
increased female sexual interest and desire in the postmenopausal period. An up to two
years administration in clinical trials did not cause serious side effects. It is, however,
recommended that testosterone levels should be carefully monitored. It was reported five
cases with different patterns of adverse reaction due to non-critical and/ or unmonitored use
of anabolic steroids: deepening of the voice due to topical use of AAS in an anti-cellulite
cream; circumscribed hypertrichosis and late onset acneiform eruptions due to testosterone
replacement therapy after ovariectomy in women. Homolateral gynecomastia and infertility,
acne and striae distensae was noted in males using injectable AAS. Cellulite is frequently
observed in middle aged women and is characterized by furrowed and edematous skin on
thighs, hips and buttocks. It seems to be caused by weakened muscular septa and a diffuse
pattern of extrusion of underlying adipose tissue into thedermis. An increase of hypodermal
1252
adipose tissue and weakened fibrous septa extending perpendicularly from the bones to the
skin surface, have been observed. A higher body mass index is associated with an increased
grade of cellulite. Life-style drugs may bear a significant risk of adverse effects, some
irreversible or fatal [07013].
Anabolic steroids combindes with other abuses
Testosterone and opiate use
Abuse of anabolic androgenic steroids (AAS) and opioids intersects in athletics. Evidence
from humans and animals suggests that AAS may act in the brain through opioidergic
mechanisms, and may potentiate effects of opioids. To determine whether AAS enhance
motivation for opioid intake, in this study, male rats were treated chronically for 6 weeks with
high levels of testosterone (7.5 mg/kg) or vehicle subcutaneously, and they were tested for
morphine self-administration under fixed-ratio (FR) and progressive-ratio (PR) schedules.
Initially, rats received chronic morphine infusion (16.8-50 mg/kg/day) over 7 days.
Subsequently, rats were tested for morphine self-administration (3.2 mg/kg) 6 h/day for 3
days under an FR1 schedule, and for 7 days under a PR 9-4 schedule. Under the FR1
schedule, controls self-administered more morphine (96 ± 9 mg/kg) than testosterone-treated
rats (63 ± 7 mg/kg). Under the PR schedule, there was no effect of testosterone on morphine
intake or operant responding (27 ± 6 responses vs 31 ± 6 responses for vehicle). To
determine whether testosterone enhances morphine sedation, additional rats were treated
with testosterone or vehicle and evaluated for locomotor behavior and rearing activity over 30
min in response to saline or 10 mg/kg morphine. Morphine inhibited locomotor activity and
rearing; testosterone selectively reduced rearing behavior, but did not alter locomotor
behavior. These results suggest that testosterone does not increase motivation for morphine
[14252].
Anabolic-androgenic steroids and heroin use
Anabolic-androgenic steroids (AAS) are commonly used drugs by young males. There are
known associations with other drugs, including heroin. Current explanations for the
association with heroin include evidence that both heroin and AAS users come from similar
social groups and that both heroin and AAS have similar effects on certain neurochemical
pathways. The purpose of this study was to determine what additional socio-cultural
explanations might account for the association between AAS and heroin use. One study was
conducted with eight focus groups of 30 individuals including both heroin and non-heroin
users, and individual semi-structured interviews with two key informants. AAS were used to
reverse the weight loss associated with heroin use. Because of the stigma, hiding weight loss
from heroin, or demonstrating recovery by increased size, were important both for the heroin
user himself and for reassurance to others. Increased size and increased muscularity and
strength were important in intimidating others for roles such as drug dealing. It was
concluded that the need to demonstrate weight gain in recovery from heroin use, and the
advantages of increased muscularity for intimidation purposes, provide further explanations
for the link between heroin and AAS use [14253].
Reversibility of the effects in former anabolic-androgenic steroid abusers
In contrast to the acute effects of anabolic-androgenic steroid (AAS) abuse, the long-term
risk profile of former long-term abusers (ExA) is less clear. Blood parameters of 32 male
1253
bodybuilders and powerlifters were studied. Fifteen ExA had not been abusing AAS for at
least 12-43 months on average (mean dosage 700 mg for 26 weeks per year over 9 years),
17 athletes (A) were still abusing AAS (750 mg for 33 weeks per 8 years). Hemoglobin (+5
%), leucocytes (+33 %) and platelets (+38 %) were significantly higher in A. Alanine
aminotransferase (ALT) and aspartate aminotransferase (AST) were higher, cholinesterase
activity (CHE) lower in A compared to ExA with normal values for gamma-glutamyl
transpeptidase (gamma-GT) and bilirubin. ALT, AST and CHE correlated significantly with
the extent (duration and weekly dosage, expressed as a point score) of AAS abuse in A.
Total and LDL-cholesterol were similar, HDL-cholesterol was distinctly lower in A than in ExA
and correlated negatively with the extent of AAS abuse. Testosterone and estradiol were
significantly higher, while LH, FSH and the sexual-hormone-binding (SHB) protein were
lower in A than in ExA (each P<0.001). Two ExA had testosterone levels below the normal
range. The alterations in cell counts, HDL-cholesterol, liver function and most hormones of
the pituitary-testicular axis induced by a long-term abuse of AAS were reversible after
stopping the medication for over 1 year. In some ExA, an increased ALT activity and a
depressed testosterone synthesis were found [03069].
In contrast to the acute effects of anabolic-androgenic steroid (AAS) abuse, the long-term
risk profile of former long-term abusers (ExA) is less clear. Blood parameters of 32 male
bodybuilders and powerlifters were studied. Fifteen ExA had not been abusing AAS for at
least 12-43 months on average (mean dosage 700 mg for 26 weeks per year over 9 years),
17 athletes (A) were still abusing AAS (750 mg for 33 weeks per 8 years). Hemoglobin (+5
%), leucocytes (+33 %) and platelets (+38 %) were significantly higher in A. Alanine
aminotransferase (ALT) and aspartate aminotransferase (AST) were higher, cholinesterase
activity (CHE) lower in A compared to ExA with normal values for gamma-glutamyl
transpeptidase (gamma-GT) and bilirubin. ALT, AST and CHE correlated significantly with
the extent (duration and weekly dosage, expressed as a point score) of AAS abuse in A.
Total and LDL-cholesterol were similar, HDL-cholesterol was distinctly lower in A than in ExA
and correlated negatively with the extent of AAS abuse. Testosterone and estradiol were
significantly higher, while LH, FSH and the sexual-hormone-binding (SHB) protein were
lower in A than in ExA. Two ExA had testosterone levels below the normal range. The
alterations in cell counts, HDL-cholesterol, liver function and most hormones of the pituitary-
testicular axis induced by a long-term abuse of AAS were reversible after stopping the
medication for over 1 year. In some ExA, an increased ALT activity and a depressed
testosterone synthesis were found [03070].
Long-term effects on social-medicine demography
The health risks associated with long-term therapeutic doses of testosterone and chronic
supraphysiologic doses of AAS are unknown. With chronic AAS use, doses tend to increase
and cycles become longer and more frequent, until some athletes take the drugs almost
continuously. The most severe consequences of long-term AAS use may be on the
cardiovascular system. Pathological AAS-induced left ventricular hypertrophy, impaired
diastolic filling, and arrhythmia may lead to an increased risk of myocardial infarction and
sudden death. The risk of mortality among chronic AAS users is reported to be 4.6 times
higher than non-AAS users. Although AASs have been proposed as etiologic factors for
some cancers, case reports linking these drugs with hepatic tumors, renal carcinoma, and
testicular tumors are rare. There are no reports linking AAS with prostate cancer, and
androgen treatment in older men does not induce significant increases in prostate-specific
antigen [018].
1254
Swedish data
The acute effects of AAS-use on mental health are well described in the literature, for
example, increased aggression and irritability, anxiety and depressive symptoms, cognitive
impairment, suicidal behaviours, hypomanic symptoms, enthusiasm and increased self-
confidence. There are studies showing minor or no alterations in aggressive behaviours in
past AAS-abusers who had been abstinent for a year or longer. On the other hand, one study
found that past (abstinent period not defined) AAS-abusers had significantly more psychiatric
diagnoses, as diagnosed by DSM-IV, than current abusers. A Finnish study, concerning a
12-year follow-up study of 62 male-elite Finnish powerlifters, where there was a high
suspicion of AAS-use, reported a 4.6 times higher death rate compared with the general
population. The main causes of death were myocardial infarctions and suicides. The high
occurrence of suicide in populations with a strong suspicion of previous AAS-use is in line
with preliminary results from our own study group, showing that death risk from suicide was
increased by 2-4 times. One study aimed to investigate whether previous AAS-use affects
mental health, present sociodemographic data, sport activity and substance abuse in a
retrospective 30-year follow-up study of former elite athletes. During 2004, a questionnaire
including structured questions concerning sociodemographic variables, previous and past
sport activity, lifetime prevalence of seeking professional help for mental-health problems
and previous and past substance use was sent to 996 Swedish male-elite power sport
athletes on the top 10 national ranking lists during any of the years 1960-1979 in wrestling,
Olympic lifting, powerlifting and the throwing events in track and field answered a
questionnaire. At least 20 percent of the former athletes admitted previous AAS-use.. A
reminder to fill in the questionnaire was sent to those athletes who had not answered, and
finally 683 (69 %) subjects had answered the questionnaire including the specific questions
in the questionnaire concerning whether they had ever used AAS, and if so, when in relation
to their sport career. Regarding their past sport activity, the former AAS-users were
significantly older when they started training in the sport discipline within which they reached
their highest ranking and they spent more hours per week training during their sport active
years compared with non-AAS-users. There was no difference in mean age between the
groups when they discontinued elite power sports. Compared with non-AAS-users, former
AAS-users had significantly more often sought professional help for depression (13 % vs 5
%), anxiety (13% vs 6 %), melancholy (13 % vs 4 %), concentration deficit (4 % vs 1 %) and
worry for mental health (8 % vs 3 %). The two groups did not differ regarding frequency of
present alcohol consumption. Concerning tobacco use, the former AAS-users were less
often present tobacco users compared with the non-AAS-users. Regarding previous tobacco
use, the two groups did not differ. The former AAS-users showed higher lifetime prevalence
of illicit drug use compared with the non-AAS-users but AAS-users had more often been
offered AAS compared with the non-AAS-users (87 % vs 20). All the former AAS-users
(n=143) reported having used AAS during their active sports career. The percentage of AAS-
users in specific sport disciplines were: powerlifting (57 %), Olympic lifting (47 %), track and
field (28 %) and wrestling (6 %). Forty-two percent (n=60) had administrated AAS by tablets,
3 percent (n=5), by injections and 55 påercent (n=78) by both tablets and injections. The
main reasons for using AAS were: to achieve better sport results (81 %), to train harder (56
%), a suspicion that their competitors used AAS (45 %) and faster recovery (43 %). There
were no significant differences between former high and low AAS-users concerning
sociodemographic variables and present and past sport activity. Furthermore, there were no
differences in past and present substance use (tobacco, alcohol and illicit drugs). However,
the former high AAS-users significantly more often combined AAS with the use of illicit drugs
and with the use of alcohol compared with the former low AAS-users. The AAS-users also
differed in former sport activity pattern compared to non AAS-users. It was concluded that a
relationship exists between use of AAS and mental-health problems. Thus, the results from
this study of former male-elite athletes in sport disciplines, where increased muscle strength
1255
has a marked influence on performance, showed that at least 20% admitted AAS-use during
their active sport career. The study indicates that the former AAS-users had a higher
frequency of lifetime prevalence of seeking professional expertise for several mental
problems. Furthermore, former AAS-users more often had used illicit drugs. Former AAS-
users were significantly older when they started training in their sport discipline in which they
had been most successful and they also spent more hours per week training. Furthermore, if
the former AAS-using athletes used AAS for a longer time than 2 years, they had more often
sought professional expertise for anxiety, irritation and anger and they had also more often a
combined use of alcohol and illicit drugs together with AAS. These high-consumers of AAS
reported having experienced more side effects of AAS, compared with those athletes having
used AAS no longer than 2 years. A former use of AAS does not seem to have a negative
long-term effect on either present substance abuse and present sport activity or on whether
they presently lived in a relationship or not. The present results can be compared with
previous results regarding lifetime prevalence of AAS-use among elite athletes active in the
60s and 70s. For example, in 1972, it was estimated that one-third of the Swedish-elite track
and field athletes used AAS. The present study is, however, based on a larger number of
athletes and in four power sports. In the same year's Olympic Games, 68 percent of the
participants in the track and field events reported prior steroid abuse. The former elite
athletes admitting AAS-use were significantly younger compared with the non-users. This
age difference might explain why the former AAS-users are, to a higher degree, in their
present employment. However, this difference disappeared when the old-age pensioners,
that is, above 65 years of age, were excluded from the analysis [13157].
Increased mortality in former users of anabolic steroids
Physical training has been shown to reduce mortality in normal subjects, and athletes have a
healthier lifestyle after their active career as compared with normal subjects. Since the
1950s, the use of anabolic androgenic steroids (AAS) has been frequent, especially in power
sports. The aim of the present study was to investigate mortality, including causes of death,
in former Swedish male elite athletes, active 1960-1979, in wrestling, powerlifting, Olympic
lifting, and the throwing events in track and field when the suspicion of former AAS use was
high. Results indicate that, during the age period of 20-50 years, there was an excess
mortality of around 45 percent. However, when analyzing the total study period, the mortality
was not increased. Mortality from suicide was increased 2-4 times among the former athletes
during the period of 30-50 years of age compared with the general population of men.
Mortality rate from malignancy was lower among the athletes. As the use of AAS was
marked between 1960 and 1979 and was not doping-listed until 1975, it seems probable that
the effect of AAS use might play a part in the observed increased mortality and suicide rate.
The otherwise healthy lifestyle among the athletes might explain the low malignancy rates
[13158].
Tour de France (1947-2012)
In the context of recent concerns regarding performance enhancing techniques and potential
negative health effects of high-level physical activity, data on the long-term outcomes and
causes of death in elite endurance cyclists are of particular interest. Characteristics and vital
status of all French participants in the Tour de France were collected for the 1947-2012
period. Causes of death were obtained from 1968. Overall and disease-specific mortalities
were compared with the French male population using overall and specific standardized
mortality ratios (SMRs) with their 95 percent confidence intervals (CIs). Among the 786
French cyclists who participated at least once between 1947 and 2012, 208 (26 %) died by 1
September 2012. Neoplasms and cardiovascular diseases accounted for 61 percent of
1256
deaths. It was observed a 41 percent lower mortality in French cyclists (SMR: 0.59, 95 %
confidence interval 0.51 to 0.68), which did not change over time. It was observed for main
mortality causes: for neoplasms (SMR: 0.56; 95 % confidence interval 0.42 to 0.72) and for
cardiovascular death (SMR: 0.67; 95 % confidence interval 0.50 to 0.88), except mortality
related to external causes (SMR: 1.06, 95 % confidence interval 0.71 to 1.53). It was
observed a substantially and significantly lower mortality in participants in the Tour de
France, compared with the general male population. However, the results do not allow us to
assess in detail the balance between positive effects of high-level sports activity and
selection of healthy elite athletes, versus any potential deleterious effects of excessive
physical exercise or alleged doping [13159].
Finland
One article focused on anabolic steroid adverse effects on the cardiovascular system and
mental health issues as well as the possible increase in the incidence of neoplasms in
anabolic steroid users. On the basis of findings in the literature, the authors consider these
three issues as the most significant concerning morbidity and mortality among anabolic
steroid users. A Finnish study has shown an increased incidence of premature mortality
among power lifters. Anabolic steroids and other concomitantly used drugs are the probable
cause of this increased mortality, as power training itself does not increase health risks and
all types of physical activity promote health [02049].
1257
TESTOSTERONE
Testosterone is more than a "male sex hormone". It is an important contributor to the robust
metabolic functioning of multiple bodily systems. The abuse of anabolic steroids by athletes
over the years has been one of the major detractors from the investigation and treatment of
clinical states that could be caused by or related to male hypogonadism. The unwarranted
fear that testosterone therapy would induce prostate cancer has also deterred physicians
form pursuing more aggressively the possibility of hypogonadism in symptomatic male
patients. In addition to these two mythologies, many physicians believe that testosterone is
bad for the male heart. The classical anabolic agents, 17-alkylated steroids, are indeed,
potentially harmful to the liver, to insulin action, and to lipid metabolism. These substances,
however, are not testosterone, which has none of these adverse effects. The current
evidence, in fact, strongly suggests that testosterone may be cardioprotective. There is
virtually no evidence to implicate testosterone as a cause of prostate cancer. It may
exacerbate an existing prostate cancer, although the evidence is flimsy, but it does not likely
cause the cancer in the first place. Testosterone has stimulatory effects on bones, muscles,
erythropoietin, libido, mood and cognition centres in the brain, penile erection. It is reduced in
metabolic syndrome and diabetes and therapy with testosterone in these conditions may
provide amelioration by lowering LDL cholesterol, blood sugar, glycated hemoglobin and
insulin resistance. The best measure is bio-available testosterone which is the fraction of
testosterone not bound to sex hormone binding globulin. Several forms of testosterone
administration are available making compliance much less of an issue with testosterone
replacement therapy [08063].
Testosterone is the principal male sex hormone. As with all natural steroids, it is
biosynthesized from cholesterol. Phase I metabolism employs some very specific enzymes
and pathways. Phase II metabolism and excretion follow more general patterns. Synthetic
and endogenous steroids differ in this measure. Numerous xenobiotic compounds have been
derived from testosterone. The modifications typically aim at a reduction of the androgenic
properties while maintaining the anabolic potential. Most of these compounds have been
withdrawn from the legal market. However, they are found to be illicitly added to otherwise
inefficient nutritional supplements. These products represent a major problem to doping
control. Recently, clinical trials with selective androgen receptor modulators have been
started [10078].
Testosterone causes the hypertrophy of muscle fibers and an increase in the number of
myonuclei and satellite cells. This metabolic action follows from the binding of testosterone to
the androgen receptor. Its ability to transduce the binding of testosterone in the cytoplasm of
the cell to its anabolic action (altering gene function in the nucleus) is inversely proportional
to the number of CAG repeats in the first exon of the androgen receptor, a member of the
nuclear transcription factor family of receptors and located in virtually all tissues.There are
many reports of muscle hypertrophy and increases in strength in athletes who take anabolic
steroids. Virtually none are controlled trials of just testosterone or one of other anabolically-
active steroids, for many ingest and inject multiple potentially active drugs [10001].
The primary screening method for the detection of doping by athletes using synthetic
versions of endogenous steroids such as testosterone relies on measurement of the ratio of
testosterone (T) to epitestosterone (E) in urine. In 2005 the World Anti-Doping Agency
(WADA) lowered the T/E value at which samples undergo further investigation from six to
four. This has resulted in a large increase in the number of athletes with naturally elevated
T/E ratios undergoing investigation without a corresponding increase in the number of proven
1258
doping offences involving testosterone. The objective was to develop a new simple screening
protocol that can, with high probability, not only distinguish athletes whose natural T/E values
exceed four from those whose T/E values have been elevated by testosterone doping but
also detect those athletes with naturally low T/E values that do not exceed four despite being
administered testosterone. Testosterone (250 mg Sustanon) was administered weekly to a
group of 47 young adult males for five weeks in a double-blind placebo controlled study and
urine samples collected. The samples were analysed for steroid concentrations using GC/MS
and for luteinizing hormone (LH) by immunoassay. The elevation of T/E that occurred in all
subjects was accompanied by a significant reduction in urinary LH concentrations to levels
that are rare in normal subjects. The appropriate measurement of urinary LH, with the
measurement of T/E values, can markedly improve the efficiency of detection of doping with
testosterone by male athletes, particularly those who have low natural T/E ratios [09085].
Theoretical, overviewing, aspects
Testosterone is regarded as an attractive supplement for obtaining masculinity and sexuality;

however, there have been pros and cons regarding its application as a treatment. In addition,
there is also conventional repulsion on adoption of testosterone to any kind of exercise to
anyone with concern with sports. However, we should keep in mind that in terms of
rehabilitation, our main concern is not fairness but efficiency. And there are obvious
advantages of testosterone in recovery and rejuvenation. It was here aimed to introduce the
possibility of testosterone in recovery and rejuvenation and are to bring up a topic the
application of testosterone in exercise rehabilitation. Considering the light and darkness in
testosterone, moderate use of testosterone under professional medication counseling might
be an effective possibility to those with sickness and illness and should be considered as a
possible option to assist the recovery from frailty and illness. Since the doping test was first
adopted in 1968, the 10th Winter Olympic game in France, there has been increasing
awareness of the need to intensify the efforts to fight unfairness in sports. Testosterone has
been a representative forbidden drug and unrecoverable stigma has been attached to the
athlete detected as a drug user by doping test, no matter how famous he/she is or how great
his/her achievement. Therefore, usage of androgenic hormones like testosterone might be a
very sensitive topic to those having any relation with any kinds of sports. This ergogenic aids
have been considered as unfairness and immorality in any society governed by the rule of
law in the physical competition filed like sports. Therefore, caution is required when
introducing the efficacy of an androgenic hormone like testosterone for improving the
exercise capacity to those specializing in a field of exercise. We are very sensitive to that.
Paradoxically, it has been efficient that way. However, as a matter of fact, you are not an
Olympic team coach, but an expert in exercise rehabilitation. Not for breaking a new record,
but for efficient recovery and early reversion of an object, such a minor foul should be
admitted, of course. Could we not make this forbidden fruit for athletes, testosterone, as a
divine blessing for exercise rehabilitation? [150226].
It is suggested that the sex steroid hormones testosterone and estrogen (SSH) provide
receptor cells with reliable information on protein synthesis and on the level of oxidative
metabolism in the cells of the gonads. The SSH are derived from the oxidation of cholesterol.
This oxidation is a side reaction of the oxidative processes in the mitochondria that generate
most of the energy to the organism. The amount of SSH that is synthesized is correlated to
the partial pressure of oxygen at the synthesizing cells. The amount of free SSH that a cell
can hold is checked by the damage that free steroids may cause. This damage is prevented
by proteins that bind with SSH. As a result, SSH levels are correlated also with the ability of
the SSH synthesizing cell to produce proteins that bind with them. A cell can only synthesize
1259
SSH in relation to the oxidative processes within it and to its ability to produce the binding
proteins necessary to prevent the damage caused by SSH. As a result, the information
conveyed by SSH is reliable. It was examined the specific damage caused by testosterone
and estrogen, and suggest why each of them is best suited for its function. Although both
SSH can provide similar information on the metabolism in the cells that synthesize them,
there are secondary reasons why testosterone and estrogen were selected to serve
particular functions. Testosterone improves the efficiency of the proton pump at the
mitochondria in producing ATP, but increases oxidative damage. Estrogen on the other hand
decreases oxygen damage but also decreases the efficiency of the proton pump. These
differences between the two SSH may explain why females use estrogen to inform the body
about the activity of the cells in their gonads while males do it by testosterone. The increased
oxidative damage may also explain why in males the testosterone that reaches the brain is
turned into estrogen. It was also suggested why fish use 11-keto testosterone and why
insects do not use these two steroids [11326].
Testosterone is among the oldest drugs in medicine. It has a long efficacy and safety record
for its prime role of androgen replacement therapy in men with androgen deficiency.
Testosterone and synthetic analogue androgens have also been used in pharmacological
androgen therapy (PAT) to produce androgenic effects on marrow, muscle or bone. Although
PAT is increasingly being superseded by newer, more expensive drugs, androgens remain
cost-effective in many older applications. Androgen misuse is the systematic over-prescribing
for unproven medical indications. Misuse is increasingly evident for male ageing
("andropause") and some other clinical conditions. Further trials for new indications for
androgens require reliable safety data, but rising costs may make it increasingly attractive to
circumvent the need for evidence by promoting off-label mass marketing. Androgen abuse is
the illicit self-administration of often massive doses of androgens for non-medical purposes -
notably power sports and body building. In parallel with effective detection reducing
androgen abuse in elite sports, more focus is needed on non-sporting cosmetic, recreational
and occupational androgen abuse. Despite ongoing androgen misuse and abuse,
testosterone remains under-prescribed for younger men with classical androgen deficiency
that frequently remains undiagnosed [06050].
Testosterone therapy is prescribed for millions of men each year, and the number is
increasing rapidly. Prescription sales of testosterone increased by 500 percent in the United
States between 1993 and 2000. Most testosterone prescriptions are written to treat
nonspecific symptoms, such as fatigue or sexual dysfunction, when accompanied by
testosterone levels below the laboratory reference range. Currently, testosterone levels that
are at least 2 SD below the mean value for healthy young adults are classified as low.
Although convenient, this classification fails to consider the physiological consequences of
specific testosterone levels. More than 80 percent of circulating estradiol in men is derived
from the aromatization of testosterone. Thus, as serum testosterone levels decline, there is a
concomitant decline in serum estradiol levels. Nevertheless, the consequences of male
hypogonadism are routinely attributed solely to androgen deficiency; the potential role of the
concomitant decline in estrogens is typically ignored. It has become clear, however, that
estrogen deficiency may be important in the pathogenesis of some consequences of male
hypogonadism, such as bone loss. The potential role of estrogen deficiency in the
pathogenesis of other consequences of hypogonadism, such as alterations in body
composition or sexual function, is largely unknown. Information on the role of estrogens in
male hypogonadism may help identify men at risk for specific manifestations of the condition
and may provide a rationale for novel approaches to its management. We sought to
determine the relative degree of testosterone deficiency, estradiol deficiency, or both at
which undesirable changes in body composition, strength, and sexual function begin to occur
and whether those changes are due to androgen deficiency, estrogen deficiency, or both. It
1260
was recruited two cohorts of men who were 20 to 50 years of age and healthy. All the men
had normal serum testosterone levels. All participants received goserelin acetate
(Zoladex®), at a dose of 3.6 mg subcutaneously at weeks 0, 4, 8, and 12, to suppress
endogenous gonadal steroids. Participants were then randomly assigned to receive 0 g
(placebo), 1.25 g, 2.5 g, 5 g, or 10 g of a topical 1 percent testosterone gel (AndroGel®) daily
for 16 weeks. Participants in cohort 2 also received anastrozole (Arimidex®) at a dose of 1
mg daily to block the aromatization of testosterone to estrogen. Participants were unaware of
the study-group assignments. Participants were seen every 4 weeks. At each visit, fasting
blood samples were obtained to measure gonadal steroid levels, and questionnaires were
administered to assess physical function, health status, vitality, and sexual function. At
baseline and week 16, body fat and lean mass were assessed by means of dual-energy x-
ray absorptiometry (DXA); subcutaneous- and intraabdominal-fat areas and thigh-muscle
area were measured by means of computed tomography (CT); and lower-extremity strength
was determined by means of a leg press. Data on bone homeostasis (bone-turnover markers
and bone mineral density), risk factors for cardiovascular disease (blood pressure, lipids, and
insulin sensitivity), and levels of leptin and prostate-specific antigen were also collected but
are not included in the present report. It was enrolled 198 men in cohort 1 and 202 men in
cohort 2. There were no significant differences in baseline testosterone levels among dose
groups or between cohorts. In the study, it was found that the dose of testosterone required
to prevent adverse changes in a variety of measures varies considerably. When
aromatization was intact, fat accumulation began with mild gonadal steroid deficiency (a
testosterone level of approximately 300 to 350 ng per deciliter), whereas lean mass, thigh-
muscle area, and muscle strength were preserved until gonadal steroid deficiency was more
marked (a testosterone level ≤200 ng per deciliter). Sexual desire and erectile function, the
two major domains of sexual function, showed distinct patterns of change as serum
testosterone levels were reduced. The variation in tissue sensitivity to androgens could be
due to polymorphisms affecting polyglutamine repeat length in the androgen-receptor gene,
tissue-specific differences in androgen-receptor expression or local hormone metabolism, or,
as shown in the study, variation in the roles of androgens and estrogens in the regulation of
target-tissue responses. Observational studies have shown that lean mass and strength are
reduced and fat mass is increased in men with low testosterone levels. Men with
hypogonadism report less sexual activity, fewer sexual thoughts, and fewer spontaneous
erections than men with normal testosterone levels. Moreover, testosterone replacement
increases lean mass, decreases fat mass, and can improve sexual function in men with
hypogonadism. These observations have led to the widespread belief that undesirable
changes in body composition and sexual dysfunction in men with hypogonadism are due to
androgen deficiency. However, because estradiol is a metabolite of testosterone, it is difficult
to distinguish the effects of androgens from those of estrogens in observational studies, or
even in randomized, controlled trials if aromatizable androgens are used without the
administration of an aromatase inhibitor. By administering a variety of testosterone doses
with and without concomitant aromatase inhibition, it was found that changes in lean mass,
thigh-muscle area, and leg-press strength were attributable to changes in testosterone
levels, whereas changes in fat measures were primarily related to changes in estradiol
levels. Both androgens and estrogens contributed to the maintenance of normal libido and
erectile function. Although these results may be surprising, they are consistent with studies
showing that body fat is increased in humans and male mice with null mutations of the
aromatase gene or the estrogen-receptor α gene and that sexual function is markedly
impaired in mice and humans with these genetic defects. These observations may have
important clinical implications. First, they provide a physiological basis for interpreting
testosterone levels in young and middle-aged men and identifying the adverse
consequences that are most likely to occur at various gonadal steroid levels. Second,
because increases in visceral fat reduce insulin sensitivity and are associated with diabetes
and the metabolic syndrome,27 the marked increase in intraabdominal fat with aromatase
1261
inhibition could portend an increase in cardiovascular disease with long-term estrogen
deficiency. Finally, because lean mass, thigh-muscle area, and erectile function were
reduced at a testosterone dose (1.25 g per day) that elicited a mean serum level of
approximately 200 ng per deciliter, testosterone supplementation seems justified in men with
testosterone levels in this range. However, some men have alterations in these functional
outcomes at lower or higher testosterone levels, and other consequences of hypogonadism,
such as increases in body fat and loss of sexual desire, routinely develop at higher mean
testosterone levels. Thus, each person's specific clinical scenario should be considered
when interpreting the clinical significance of the circulating testosterone level. These findings
may also have implications for older men. Serum testosterone levels decline modestly as
men age, such that 20 percent of men older than 60 years of age and 50 percent of men
older than 80 years of age have testosterone levels at least 2 SD below the mean level in
young men. The finding that estrogens have a fundamental role in the regulation of body fat
and sexual function, coupled with evidence from prior studies of the crucial role of estrogen
in bone metabolism,6-8 indicates that estrogen deficiency is largely responsible for some of
the key consequences of male hypogonadism and suggests that measuring estradiol might
be helpful in assessing the risk of sexual dysfunction, bone loss, or fat accumulation in men
with hypogonadism. For example, in men with serum testosterone levels of 200 to 400 ng per
deciliter, sexual-desire scores decreased by 13 percent if estradiol levels were 10 pg per
milliliter or more and by 31% if estradiol levels were below 10 pg per milliliter. The findings
also suggest that treatment with aromatizable androgens would be preferable to treatment
with nonaromatizable androgens in most men with hypogonadism [13188].
There is a widespread perception that testosterone supplementation adversely affects the

plasma lipoprotein profile and increases the risk of atherosclerotic heart disease; this
premise is not supported by data. Thus, the long-term consequences of testosterone
supplementation on the risk of atherosclerosis progression remain unknown. Although
supraphysiological doses of testosterone and nonaromatizable androgens frequently used by
body builders undoubtedly decrease plasma high-density lipoprotein (HDL) cholesterol
levels, physiological testosterone replacement in older men has been associated with only a
modest or no decrease in plasma HDL cholesterol. Cross-sectional studies of middle-aged
men have found a direct, rather than an inverse, relationship between serum testosterone
levels and plasma HDL cholesterol concentrations, as well as an inverse correlation between
serum testosterone levels and visceral fat volume. Testosterone supplementation in middle-
aged men who have truncal obesity is associated with a reduction in visceral fat volume,
serum glucose concentration, and blood pressure and an improvement in insulin sensitivity.
These data suggest that serum testosterone levels in the range that is midnormal for healthy
young men are consistent with an optimal cardiovascular risk profile at any age and that
testosterone concentrations either above or below the physiologically normal male range
may increase the risk of atherosclerotic heart disease. In light of these data, the present
review will discuss the rationale for the use of testosterone supplementation in HIV-infected
men with fat redistribution syndromes. In spite of the widespread belief that testosterone
supplementation increases the risk of atherosclerotic heart disease, evidence to support this
premise is lacking. Although supraphysiological doses of testosterone, such as those used
by athletes and recreational body builders, decrease plasma high-density lipoprotein (HDL)
cholesterol concentrations, replacement doses of testosterone have had only a modest or no
effect on plasma HDL in placebo-controlled trials. In epidemiological studies, serum total and
free testosterone concentrations have been inversely correlated with intra-abdominal fat
mass, risk of coronary artery disease, and type 2 diabetes mellitus. Testosterone
administration to middle-aged men is associated with decreased visceral fat and glucose
concentrations and increased insulin sensitivity. Testosterone infusion increases coronary
blood flow. Similarly, testosterone replacement retards atherogenesis in experimental models
of atherosclerosis. However, the long-term risks and benefits of testosterone administration
1262
in human immunodeficiency virus-infected men with fat redistribution syndrome have not
been studied in randomized clinical trials [03078].
It was examined the relationship between serum free testosterone levels and visceral fat
measured by CT scan in healthy men aged 30-70 years. Serum free testosterone levels were
inversely correlated with visceral fat mass. However, free testosterone concentrations were
measured by a tracer analog method, which is affected by sex hormone–binding globulin
(SHBG). Because SHBG is decreased by obesity, it is possible that the apparent relationship
between visceral fat and free testosterone is a reflection of the relationship between SHBG
and visceral fat volume. Similarly, healthy hypogonadal men have a higher fat mass than
age-matched eugonadal controls. Most conclusive information has emerged from the elegant
studies, which demonstrated that the experimental induction of androgen deficiency in
healthy young men by administration of a gonadotropin-releasing hormone (GnRH) agonist is
associated with a decrease in fat-free mass and an increase in fat mass. Thus, androgen
deficiency, whether it occurs spontaneously or is induced experimentally, is associated with
increased fat mass, particularly in the visceral fat compartment [03078].
Metabolites
Controversial results have been reported in the literature regarding the behavior of two
testosterone (T) metabolites (3alpha-glucuronide-6beta-hydroxyandrosterone and 3alpha-
glucuronide-6beta-hydroxyetiocholanolone) excreted after T administration. Due to their
potential as biomarkers of T misuse, a UHPLC-MS/MS method for the direct quantification of
these glucuronides was developed and validated. In addition, the main phase II metabolites
of T that compose the steroid profile used for doping control purposes (glucuronides of T,
epitestosterone, androsterone and etiocholanolone) were included. The method was found to
be linear and with suitable LODs and LOQs for all metabolites. The average accuracies were
between 86 and 120 percent, the RSDs for the intra- and inter-day precision were below 15
and 25 percent respectively. The method showed low matrix effect. Samples obtained before
and after the administration of T were analyzed by both the developed UHPLC-MS/MS
method and the GC-MS/MS method currently used by anti-doping laboratories. Relevant
disagreements between the results obtained for 3alpha-glucuronide-6beta-
hydroxyandrosterone and 3alpha-glucuronide-6beta-hydroxyetiocholanolone quantitation
were observed. These markers seemed to be more suitable for the screening of T misuse
when detected by UHPLC-MS/MS. These discrepancies were further investigated in 50 urine
samples from healthy volunteers. The two methods gave highly correlated results for all
metabolites that are currently included in the athlete's steroid profile confirming the reliability
of the UHPLC-MS/MS method. However, the quantification of 3alpha-glucuronide-6beta-
hydroxyandrosterone and 3alpha-glucuronide-6beta-hydroxyetiocholanolone, was only
possible by using the UHPLC-MS/MS method since three interfering compounds were
observed when performing the GC-MS/MS analysis with the most intense ion transitions.
These results confirm the potential of the resistant glucuronides as biomarkers of T misuse.
Additionally, they suggest that previously reported reference ranges for these metabolites
should be reevaluated [150227].
The detection of testosterone (T) misuse by doping control laboratories is mainly based on
monitoring urinary T phase I metabolites released after enzymatic hydrolysis of the
corresponding phase II glucuronide metabolites by gas chromatography (tandem) mass
spectrometry (GC-MS(/MS)) methods. However, this strategy fails to properly determine two
recently reported phase II metabolites of T conjugated with glucuronic acid that remained
mostly conjugated after the hydrolysis step. These metabolites were identified as
1263
glucuronides of 6-hydroxyandrosterone (6beta-OH-And) and 6beta-hydroxyetiocholanolone
(6beta-OH-Etio) but their exact conjugation site remained undetermined. In this study, the
four possible glucuronides of 6beta-OH-And and 6beta-OH-Etio were synthesized and
characterized by nuclear magnetic resonance (NMR) spectroscopy. Moreover, their
chromatographic properties and MS spectra were compared to those obtained for the urine
samples collected after administration of T. Results confirmed that the recently reported
metabolites were the 3alpha-glucuronides of 6beta-OH-And and 6beta-OH-Etio. The
synthesis and the elucidation of the exact structure of the metabolites presented in this study
are crucial steps for the development of analytical methods in order to explore their role in T
metabolism and their potential usefulness as biomarkers of T misuse [150228].
The implementation of the athlete steroidal passport in doping control analysis aims to detect
intra-individual changes in the steroid profile related to the abuse of anabolic steroids. In this
context, the study of intrinsic variations associated with each marker is of utmost importance.
In the present work, the influence of several factors in the excretion of the recently reported
testosterone metabolites conjugated with cysteine (delta1-AED; 1,4-androstadien-3,17-dione,
delta6-AED; 4,6-androstadien-3,17-dione, delta6-T; 4,6-androstadien-17beta-ol-3-one, and
delta15-AD; 15-androsten-3,17-dione) is evaluated for the first time. Degradation experiments
at 37 °C proved that, although the cysteinyl moiety is released, the variation for urinary
delta1-AED/delta6-AED, delta1-AED/delta6-T ratios is less than 30 percent. Moreover,
freeze/thaw cycle testing resulted in RSDs values below 15 percent for all the analytes.
Regarding infradian variability, moderate variations (below 40 %) were observed.
Additionally, notable alterations in the excretion of these compounds have been observed in
the earliest stages of pregnancy. UGT2B17 polymorphism, responsible for the low T/E ratio
found in some population, does not influence the excretion of cysteinyl compounds whereas
the intake of exogenous substances (alcohol or 5alpha-reductase inhibitors) dramatically
affects their excretion. The urinary concentrations of delta1-AED, delta6-AED, and delta15-AD
decreased (<50 %) after the ethanol intake, whereas after the administration of dutasteride,
an important increase was observed for the concentrations of delta6-AED, delta6-T and
delta15-AD. Overall, the presented data describes the stability of the urinary cysteinyl steroids
under the influence of many factors, proving their potential as suitable parameters to be
included in the steroidal module of the athlete's biological passport [150229].
Influence of exercise on growth hormone and testosterone in prepubertal boys
The purpose of this study was two fold a) to determine the levels of hormonal parameters
which are related to growth and sexual maturation (T, SHBG, FAI, GH) in 66 pre-pubertal
and early-pubertal boys (11-13 years old) who systematically engage in individual and team
sports activities of endurance, strength, speed and skill, respectively, and b) to investigate
the effect of two different forms of exercise namely aerobic (AG) and weight training (WG) on
androgen levels in 19 sedentary pre-pubertal boys. The control groups (CG) consisted of
boys of the same age who attended only the school physical education programmes. The
individuals included in the study participated voluntarily after their parents had been informed
and had given their written consent. Hormonal concentrations were determined using
radioimmunoassay and immunoradiometric assays. No differences were observed among
the various athletes' groups as regards Tanner stages, height and weight. The mean T and
FAI values of the control group did not differ from those of the corresponding athletes group.
Significant differences were observed among the groups regarding BMI, percent body fat, T,
SHBG, FAI and GH. T and FAI values in the WG group were significantly higher than the
corresponding concentrations: a) in the AG group by 338 and 609 percent and b) in the
control group CG by 91 and 96 percent, respectively. The hormonal differences detected
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among the various groups of athletes must be attributed as much to the type of physical
exercise and to developmental factors as to the selection criteria used for the different
athletic talents. The importance of the specificity of training stimulus in the hormonal
adaptations of pre-pubertal sedentary subjects was demonstrated [03079].
Normal values
Normal total plasma testosterone levels in males are in the range of 300 to 1,000 ng/dL.
Most is bound by sex hormone-binding protein and is inactive; free testosterone, the active
form, makes up only 2 to 3 percent of circulating testosterone. Testosterone is metabolized
into dihydrotestosterone, which is 10 times more potent than testosterone, and estradiol,
which has feminizing effects [07031].
Reference ranges are essential for partitioning testosterone levels into low or normal and
making the diagnosis of androgen deficiency. It was established reference ranges for total
testosterone (TT) and free testosterone (FT) in a community-based sample of men. TT was
measured using liquid chromatography tandem mass spectrometry in nonobese healthy
men, 19-40 years old, in the Framingham Heart Study Generation 3; FT was calculated.
Values below the 2.5th percentile of reference sample were deemed low. It was determined
the association of low TT and FT with physical dysfunction, sexual symptoms (European
Male Aging Study, EMAS, only), and diabetes mellitus in three cohorts: Framingham Heart
Study generations 2 and 3, EMAS, and the Osteoporotic Fractures in Men Study. In a
reference sample of 456 men, mean (SD), median (quartile), and 2.5th percentile values
were 724 (221), 699 (297), and 348 ng/dL for TT and 142 (45), 134 (60), and 70 pg/mL for
FT, respectively. In all three samples, men with low TT and FT were more likely to have slow
walking speed, difficulty climbing stairs, or frailty and diabetes than those with normal levels.
In EMAS, men with low TT and FT were more likely to report sexual symptoms than men
with normal levels. Men with low TT and FT were more likely to have at least one of the
following: sexual symptoms (EMAS only), physical dysfunction, or diabetes. It was concluded
that the reference ranges generated in a community-based sample of men provide a rational
basis for categorizing testosterone levels as low or normal. Men with low TT or FT by these
criteria had higher prevalence of physical dysfunction, sexual dysfunction, and diabetes.
These reference limits should be validated prospectively in relation to incident outcomes and
in randomized trials [11327].
Problems in evaluating serum testosterone values
Testosterone (T) and other androgens are incorporated into an increasingly wide array of
human sexuality research, but there are a number of issues that can affect or confound
research outcomes. One review addressed various methodological issues relevant to
research design in human studies with T; unaddressed, these issues may introduce
unwanted noise, error, or conceptual barriers to interpreting results. Topics covered are (1)
social and demographic factors (gender and sex; sexual orientations and sexual diversity;
social/familial connections and processes; social location variables), (2) biological rhythms
(diurnal variation; seasonality; menstrual cycles; aging and menopause), (3) sample
collection, handling, and storage (saliva vs. blood; sialogogues, saliva, and tubes; sampling
frequency, timing, and context; shipping samples), (4) health, medical issues, and the body
(hormonal contraceptives; medications and nicotine; health conditions and stress; body
composition, weight, and exercise), and (5) incorporating multiple hormones. Detailing a
comprehensive set of important issues and relevant empirical evidence, the review provided
a starting point for best practices in human sexuality research with T and other androgens
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that may be especially useful for those new to hormone research [13194].
Time of day is of importance for testosterone levels
The aim of one study was to determine the effect of time of day on performance, pacing, and
hormonal and metabolic responses during a 1000-m cycling time-trial. Nine male,
recreational cyclists visited the laboratory four times. During the 1st visit the participants
performed an incremental test and during the 2nd visit they performed a 1000-m cycling
familiarization trial. On the 3rd and 4th visits, the participants performed a 1000-m TT at
either 8 am or 6 pm, in randomized, repeated-measures, crossover design. The time to
complete the time trial was lower in the evening than in the morning (88 ± 9 vs 95 ± 11 s,
respectively), but there was no significant different in pacing. However, oxygen uptake and
aerobic mechanical power output at 600 and 1000 m tended to be higher in the evening.
There was also a main effect of time of day for insulin, cortisol, and total and free
testosterone concentration, which were all higher in the morning (+60 %, +26 %, +31 % and
+22 %, respectively). The growth hormone was twofold higher in the evening. The plasma
glucose was about 11 percent lower in the morning. Glucagon, norepinephrine, epinephrine
and lactate were similar for the morning and evening trials, but the norepinephrine response
to the exercise was increased in the morning (+46 %), and it was accompanied by a 5-fold
increase in the response of glucose. Muscle recruitment, as measured by electromyography,
was similar between morning and evening trials. The findings suggest that performance was
improved in the evening, and it was accompanied by an improved hormonal and metabolic
milieu. There is thus limited research connecting circadian variations in hormone levels with
pacingl and testosterone peak at 8:00 am, but do not seem to respond to exercise. On the
other hand, growth hormone (GH) seems to be slightly reduced in the morning, but its
response to exercise is not altered. In addition, the effect of circadian rhythm on insulin and
glucagon, two hormones that are strongly linked with metabolic milieu and could influence
performance during different times of day, is unknown. Given that performance during a
short-distance cycling TT is dependent on the ability of athletes to produce and maintain a
high level of power output throughout the test, the coordinate action of these hormones might
be associated with an improved or impaired performance. For example, epinephrine and
norepinephrine prepare the body for an immediate action increasing several physiological
markers (e.g. heart rate, blood pressure, metabolic rate, and glycogenolysis and glycolysis),
while cortisol increases general physiological stress and, together with glucagon and GH,
increases blood glucose. Elevated testosterone concentration may be involved with an
increased neuromuscular efficiency and force output. Thus, an improved performance at a
given time of day might be associated with hormonal and metabolic at rest and/or during the
exercise. However, while circadian rhythms for some hormones have been relatively well
characterized, their integrated responses and association with TT performance have not
been clarified [14761].
Plasma and urinary markers of oral testosterone
Orally administered testosterone undecanoate (TU), an anabolic, androgenic steroid, can

potentially be abused by athletes. Indirect evidence for detecting oral TU intake could be
deduced from the changes in steroid profile post-administration. Direct evidence could be
obtained by detection of unchanged TU in plasma. To this end, both urinary and plasma
steroid profiles of six healthy male subjects given a single oral dose of 120 mg of TU were
studied by gas chromatography/mass spectrometry (GC/MS) and gas chromatography/
tandem mass spectrometry (GC/MS/MS). The increased concentration of glucuronidated
testosterone in plasma appears to be the most characteristic sign of oral TU intake. The
testosterone glucuronide (TG)/nonconjugated testosterone (T) ratio, TG/17-
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hydroxyprogesterone (17OHP) ratio, and TG/luteinizing hormone (LH) ratio were observed to
be significantly elevated above their basal levels for 10 h, 10 h, and 6 h, respectively. Urinary
ratios of TG/epitestosterone glucuronide (EG) were found to be higher than the cut-off value
of 6 for the period 4 approximately 8 h post-administration, but only in three subjects. One
subject failed to respond with respect to all of the above-mentioned indirect markers, as TG
was not significantly increased in either plasma or urine. Unchanged TU was directly
detected in plasma of all six subjects from 1 approximately 1.5 h to 4 approximately 6 h after
oral TU intake by GC/MS/MS, providing unequivocal proof of exogenous testosterone intake.
Distinct and complementary markers for detecting oral TU intake could be obtained from
plasma and urine, respectively [02055].
Detection of injected testosterone in blood
Injections of synthetic esters of testosterone are among the most common forms of
testosterone application. In doping control, the detection of an intact ester of testosterone in
blood gives unequivocal proof of the administration of exogenous testosterone. The aim of
the current project was to investigate the detection window for injected testosterone esters as
a mixed substance preparation and as a single substance preparation in serum and plasma.
Furthermore, the suitability of different types of blood collection devices was evaluated.
Collection tubes with stabilizing additives, as well as non-stabilized serum separation tubes,
were tested. A clinical study with six participants was carried out, comprising a single
intramuscular injection of either 1000 mg testosterone undecanoate (Nebido(®) ) or a mixture
of 30 mg testosterone propionate, 60 mg testosterone phenylpropionate, 60 mg testosterone
isocaproate, and 100 mg testosterone decanoate (Sustanon(®) ). Blood was collected
throughout a testing period of 60 days. The applied analytical method for blood analysis
included liquid-liquid extraction and preparation of oxime derivatives, prior to TLX-sample
clean-up and liquid chromatography-tandem mass spectrometry (LC-MS/MS) detection. All
investigated testosterone esters could be detected in post-administration blood samples. The
detection time depended on the type of ester administered. Furthermore, results from the
study show that measured blood concentrations of especially short-chained testosterone
esters are influenced by the type of blood collection device applied [150230].
Candida albicans in urine can produce testosterone
The International Olympic Committee-approved drug test to detect testosterone

administration is based on the concentration ratio of urinary testosterone to epitestosterone
(T/E). Usually the T/E is ∼1, but after administration, the urinary excretion rate of
testosterone increases and hence the ratio will be augmented; International Olympic
Committee-accredited laboratories report T/E >6. Constant refrigeration of competitors’
samples in transport is logistically difficult; hence, samples are usually transported at ambient
temperature and may take several days to reach these laboratories. Preservative is not
added because it is reasoned that adulteration with a foreign substance may lead to legal
challenges. Concern has been raised about maintaining the integrity of the sample,
particularly because possible urinary microbial production of testosterone may cause an
adverse finding, and several appeals have been made on that basis, the case of a British
athlete who had competed internationally. A wide range of microorganisms can contaminate
urine. Thus, testing the general hypothesis that microbial production of testosterone is
possible simply by incubating a limited number of untreated urine samples at ambient
temperature has not been fruitful. It was decided that a better approach was to perform a
relatively large-scale study in which urine samples from females were inoculated with a
single potential candidate organism. To rationalize the choice of organism, a fresh approach
was used that involved searching a comprehensive protein database to identify putative
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organisms that are most likely to produce enzymes able to convert steroid precursors to
testosterone. Unlike prokaryotes (e.g. Escherichia coli), many eukaryotic microorganisms are
capable of both de novo synthesis of steroids and transformation of steroid substrates.
Essential enzymes in the conversion of the precursor steroids, androstenedione (androst-4-
ene-3,17-dione) and androstenediol (androst-5-ene-3beta,17beta-diol), to testosterone in
humans are 17beta-hydroxysteroid dehydrogenase (17beta-HSD) and 3beta-hydroxysteroid
dehydrogenase/4,5-isomerase (5-ene-3beta-HSD), respectively. A search of the SWall
protein sequence database revealed several yeasts and fungi that express proteins
homologous with human 17beta-HSD and 5-ene-3beta-HSD; such proteins have been
putatively identified as having these enzymatic activities. This, together with some direct
experimental evidence suggests that some eukaryotic microbes may synthesize
testosterone. The choice of experimental organism was narrowed to Candida albicans
because it is found in the typical vaginal flora. No clear cutoff for candiduria has been
established, and there is inadequate information regarding asymptomatic candiduria in
healthy individuals. Urinary contamination in sports samples is unlikely to exceed 10 000
colony-forming units/mL (CFU/mL), but to account for contrary speculation, experiment also
incorporated inoculation of urine at 100 000 CFU/mL. A total of 134 women (age range, 18-
40 years; mean, 23 years) volunteered a urine specimen. Ethical permission and written
informed consent were obtained in accordance with our institutions. A criterion for inclusion
was good health and for exclusion was the use of antifungal preparations in the previous 2
weeks. For the T/E, compared with the 134 nontreated urines, positive changes were
observed in 92 of the 10K-treated urines and 99 of the 100K-treated urines. The box plot in
Fig. 1⇓ summarizes the differences in the T/E ratios observed between the paired samples
(treated minus nontreated). Compared with nontreated urine samples, the largest increases
in the T/E observed for 10K-treated urine were 0.23, 0.27, and 0.42 and for 100K-treated
urine were 0.44 and 0.51 (see extreme values in Fig. 1⇓ ); further analysis by GC-tandem
MS (GC-MS/MS) under identical chromatographic conditions showed no coeluting substance
present that would interfere with testosterone quantification, as judged by peak height and
area ratios of ion abundance. The data demonstrated that statistically significant amounts of
testosterone can be produced in urine in the presence of C. albicans. However, because
testosterone was increased but only to a minor extent, the resulting augmentation of the T/E
ratios was also minor. Thus, in comparison with the laboratory reporting threshold (T/E = 6),
these increases are of little evidential value for any individual case. The mechanism appears
to be by conversion of urinary androstenedione rather than by synthesis de novo. If this is the
case, augmentation of the T/E ratio will be limited by the microbial rate of formation of
testosterone and the concentration of androstenedione originally present in a spot sample;
we would expect the latter to be typically <10 μg/L based on excretion rate data [Ref. (10)
and references therein]. However, the maximum possible concentration of androstenedione
that can be present in single urine specimens collected from healthy individuals is not known,
nor are the number of strains of C. albicans. As a consequence, it is difficult to comment
further as to the possibility of how much testosterone can be formed by different strains of
this microbe [02062].
Normal production
Testosterone is the primary male hormone synthesized in the testes. It serves distinct
functions at different stages of life. During embryonic life, androgen action is central to the
development of the male phenotype. At puberty, the hormone is responsible for the
secondary sexual characteristics that transform boys into men. Testosterone regulates many
physiologic processes in the adult male including muscle protein metabolism, sexual and
cognitive functions, erythropoiesis, plasma lipids, and bone metabolism. During adult life, the
average male produces approximately 7 mg of testosterone daily, about 2500 mg of
testosterone each year, and a total of 130 g by 75 years of age. The normal range of plasma
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testosterone in males is 300 to 1000 ng/dL, but the average value declines by age 80 to
approximately 50 percent of that at age 20 years. In females, the circulating testosterone
levels are typically about 10 of those observed in men [04018]
Synthetic derivatives
Anabolic steroids are synthetic derivatives of the male hormone testosterone, manufactured
to maximize anabolic and minimize androgenic effects. The active ingredient, testosterone,
has several possible metabolic fates. First, it binds to the androgen receptor (AR) in target
tissues to exert its androgenic and anabolic effects. Second, it is 5alpha-reduced in some
target tissues (including skin and liver) to dihydrotestosterone, which also acts on the AR.
Finally, it can be aromatized to estradiol to exert estrogenic activities. Chemical modifications
of testosterone have been useful pharmacologically to alter the relative anabolic-androgenic
potency, slow the rate of inactivation, change the pattern of metabolism, or decrease the
aromatization to estradiol. Most orally active AAS preparations are 17alpha-alkylated
derivatives of testosterone that are relatively resistant to hepatic degradation. Esterification of
the 17alpha-hydroxyl group makes the molecule more soluble in lipid vehicles used for
injection and, hence, slows the release of the injected steroid into the circulation.
Testosterone has an anabolic:androgenic ratio of 1, whereas the ratio for nandrolone is 10
and that for stanozolol is 30. However, all AASs are virilizing if administered for long enough,
at high enough doses [04018].
Half life
Designer AAS are altered so as to increase the bioavailability and prolong the desired
effects. For example, testosterone’s half-life is measured in minutes, whereas fluoxy-
mesterone, a synthetic AAS, has a half-life of 9.2 hours [06031].
Testosterone/epitestosterone concentration ratio (T/E)
It has been reported that after oral, rectal, or intramuscular T administration, the excretion of
testosterone glucuronide (TG) increased more than other T metabolites. Epitestosterone (E)
was found not to be a metabolite of T because deuterated T administration did not result in
significant deuterated EG excretion. The origin of E is still discussed. Although it has been
shown that half of total E production is of testicular origin, the remaining 50 percent is still
debated. Administration of adrenocorticotrophic hormone (ACTH) results in an increased EG
production, indicating an adrenal origin. Also, adrenal insufficiency as observed in Addison's
disease correlates to significantly decreased T and E excretion rates. Also peripheral
production is possible. The mean T/E ratio of urine samples of Caucasian males and females
in the first population study was 1-2. The values showed a logarithmic normal distribution
with an upper limit value lower than 6. Using these data, the Medical Commission of the
International Olympic Committee (IOC) banned the use of T in 1982 and stated that a T/E
ratio above 6 was sufficient proof of T abuse. When applying this criterion in research and
routine analyses, cases of naturally occurring T/E ratios above 6 appeared. It was
administered testosterone enanthate in several doses intramuscularly to healthy men over a
period of six months. They found via linear interpolation between doses that the T/E ratio
exceeded the cutoff point of 6 when natural production (around 45 mg/week) was doubled by
weekly administration of a comparable dose of exogenous T. Nowadays, the IOC states that
a follow-up investigation is needed for T/E ratios above 6. In the follow up, possible elevated
T/E ratios due to physiological or pathological circumstances should be proven. This proof
may be supplied by review of previous tests, endocrinological investigations, or
unannounced testing over several months. The aim of multiple tests is the establishment of
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an individual reference range of an athlete, depending on the intraindividual T/E variability. A
single T/E ratio that is higher than the upper limit of the individual reference range (mean + 3
times standard deviation) indicates T abuse. Since its introduction, critics have put forward
several cases in which the T/E ratio was up for discussion because of the assumed risk of
false-positive or false-negative results. Despite this criticism, the T/E ratio has been the most
frequently applied method to detect T abuse. A lot of research has been done to investigate
the factors that could influence the outcome of a T/E ratio analysis. The ratio of the
concentration of testosterone glucuronide to the concentration of epitestosterone glucuronide
(T/E ratio) as determined in urine is the most frequently used method to prove testosterone
abuse by athletes. A T/E ratio higher than 6 has been considered as proof of abuse in the
past; however, cases of naturally occurring higher T/E ratios have been described. Since the
introduction of the T/E ratio in doping analysis, the parameters that may or may not influence
the T/E ratio, possibly leading to false-positive results, have been debated. To achieve more
insight on the influencing circumstances, an overview is given to obtain an objective view on
the merits of the urinary T/E ratio. Relevant analytical aspects of the T/E ratio, potential
parameters of endogenous and exogenous origins, as well as some alternative methods to
determine testosterone abuse, such as the urinary testosterone/luteinizing hormone ratio,
gas chromatography-combustion-isotope-ratio mass spectrometry, hair analysis, and high-
performance liquid chromatography-mass spectrometry, are discussed [00072].
Determining the origin of testosterone and other steroids in human urine is a major issue in
doping control. According to the latest published laboratory statistics of 2009 from WADA, 65
percent of reported adverse and atypical analytical findings by the accredited anti-doping
laboratories belonged to the substance group of anabolic agents. Out of these, testosterone
is by far the most common agent reported, constituting as much as 70 percent of the
findings. An atypical analytical finding of testosterone, however, must not be confused with
an anti-doping rule violation. The detection of the administration of testosterone is primarily
based on a population reference interval of a urinary ratio of testosterone to epitestosterone,
excreted as glucuronide, referred to as the T/E ratio. Since it is well known that natural
outliers of a normal steroid profile exist, a further investigation is mandatory after a finding of
an elevated T/E ratio. For this reason, samples with a T/E ratio that equals or exceeds 4 are
amongst other parameters recommended to be submitted to isotope ratio mass
spectrometric (IRMS) analysis. The IRMS analysis is based on differences in isotope ratio
between endogenous and synthetic testosterone and provides the basis for confirmatory
analysis. The 13C/12C ratio in natural compounds, such as steroids, is determined by the
pathway by which they were produced. Consequently, synthetic testosterone is generally
less enriched in 13C and shows a different 13C/12C ratio than human endogenous
testosterone. Out of the samples with T/E ratios above 4 submitted to IRMS analysis in one
laboratory, about 8 percent were reported as adverse analytical findings for the application of
synthetic testosterone or testosterone prohormones [11574].
The use of biomarkers of doping is not new. For example, the testosterone/epitestosterone
concentration ratio (T/E) was introduced by several sports organizations in the 1970s to deter
the administration of anabolic steroids. Because epitestosterone is only a minor product of
testosterone metabolism and does not increase after exogenous testosterone administration,
the net effect of the latter is an increase in T/E. In 1983, a T/E in excess of 6.0 was
considered indicative of steroid doping by the International Olympic Committee. The
introduction of this rule was mitigated, however, by the discovery a few years later that some
individuals may have naturally increased T/E, a phenomenon that has recently been
attributed to the discovery of genetic polymorphisms that are associated with the metabolism
of anabolic steroids. Currently, in addition to the T/E, a urinary steroid profile that includes
multiple testosterone metabolites and precursors is used to detect steroid doping, in addition
to doping with other anabolic agents, such as designer steroids, gonadotropins, estrogen
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antagonists, aromatase inhibitors, androgen precursors, and selective androgen receptor
modulators [11426].
T/E in police-seized drugs

One possible explanation for some of the negative findings at IRMA in T/E-positiva cases
might be the use of testosterone with endogenous-like delta values. The isotopic ratio
depends on the manufacturing process and on the carbon feed stocks of the starting
materials. Hence, it could be possible to produce testosterone products with carbon isotope
ratios in, or close, to the endogenous range, by using 13C enriched starting materials. In one
study, the content of a number of black market testosterone products collected in Austria
were analyzed. Additionally, 13C/12C ratios were measured for testosterone in the products
after cleavage of the testosterone ester. The aim was to determine whether some of these
products had similar 13C/12C ratios to those normally found for endogenous testosterone,
which could prevent a positive isotopic ratio mass spectrometric (IRMS) finding in doping
control. Moreover, it was investigated to what extent the preparations contained the masking
agent epitestosterone, in order to lower the testosterone/ epitestosterone (T/E) ratio in
urinary steroid profiles. Out of 30 analyzed products, the declared ingredients differed from
the actual content in 10 cases. Epitestosterone, however, could not be found in any of the
products. The products displayed d13CVPDB values between 24 and 29 percent. Formore
than half of these products, the values were within a range reported for endogenous urinary
steroids [11574].
Androsterone to epitestosterone ratio

The conspicuous interindividual differences in metabolism and urinary excretion of
testosterone and its metabolites make it challenging to reveal testosterone doping. The
variation in testosterone glucuronide excretion is strongly associated with a deletion
polymorphism in the uridine diphosphate-glucuronosyltranferase (UGT) 2B17 gene. The
objective of one study was to identify additional biomarkers to detect testosterone abuse and
to elucidate alternative pathways for testosterone elimination in individuals devoid of the
UGT2B17 enzyme. For this purpose a new ultraperformance liquid chromatographic tandem
mass spectrometric method for simultaneous determination of 10 different sulfo- and
glucuronide-conjugated steroids was developed. Fifty-four healthy male volunteers with two,
one, or no allele (ins/ins, ins/del, or del/del) of the UGT2B17 gene participated in the study.
Intervention included a single im dose of 500 mg testosterone enanthate. Urinary sulfo- and
glucuronide-conjugated steroids were measured. Testosterone sulfate levels decreased in all
individuals after the dose. The individual differences in the excretion of all sulfated
metabolites were large. Thus, these metabolites will not serve as appropriate biomarkers for
testosterone abuse. However, androsterone glucuronide excretion increased in all of our
study subjects after the testosterone dose. Etiocholanolone sulfate was excreted at
significantly higher levels in UGT2B17 del/del individuals. It was proposed that the
androsterone glucuronide to epitestosterone glucuronide ratio may serve as a
complementary biomarker to reveal testosterone abuse [11575].
Free testosterone/cortisol ratio
In golf
The purpose of one investigation was to study the effects of 36 continuous holes of
competitive golf on salivary testosterone, cortisol, and testosterone-to-cortisol ratio and their
relation to performance in eight elite male collegiate golfers (age 20 years). Thirty-six holes
of a 54-hole NCAA golf tournament were played on the first day of the competition. A saliva
sample was taken 45 minutes prior to the round and immediately following each hole for a
total of 37 samples per subject. Time matched baseline samples were collected on a
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different day to account for circadian variation. Six-hole areas under the curve (AUC) values
were calculated for endocrine measures. Significant increases were noted for cortisol during
competition, however, testosterone did not change during competition compared to baseline.
Testosterone-to-cortisol (T/C) ratio was significantly lower throughout the competition
compared to baseline measures. Thirty-six-hole AUC testosterone-to-cortisol ratio response
was correlated to 36-hole score. There was a high correlation between pre-round
testosterone, T/C ratio response, and 36-hole score. CSAI-2 somatic anxiety was correlated
to pre-round cortisol and testosterone response. These results indicate a significant
hormonal response during 10 hours of competitive golf. Good golf performance (low golf
scores) in this competition was related to low T/C ratio. Additionally, results from this
investigation validated CSAI-2 somatic anxiety with physiological measures of anxiety
[06059].
In soccer
The following up of some hormonal parameters during the professional soccer training
process could be one of the indicators of the training effects. On the other hand,
overreaching and overtraining as an opposite adaptation of supercompensation could be
detected by following up some hormonal changes. The aim of one study was to evaluate the
changes in some hormonal parameters in professional soccer players during a half-season
competition. It was included 30 professional soccer players from a soccer club of our
National Soccer League in this study. All sport medical examinations were conducted tree
times: before the preparation phase, before the competition phase (after previous phase) and
after finishing the competition phase. There were significant differences in all evaluated
hormones between three phases of soccer training process, including significant decrease in
T/C of more than 30 percent at the end of the competition phase (phase III). The decrease in
muscle mass after the preparation phase and the increase in fat mass at the end of
competition phase were insignificant. The hormonal changes indicated that some indices
could indicate overreaching and overtraining at the end of professional soccer competition
season. Although insignificant, the decrease in muscle mass after the preparation phase and
the increase in fat mass at the end of competition phase were undesirable effects for us
[06060].
The free testosterone:cortisol ratio (FTCR) is widely used for studying and preventing
overtraining syndrome in various sports. The use of FTCR for following overtraining
syndrome was proposed originally with two approaches: FTCR lower than 0.35x10-3,
calculated on free testosterone (FT) in nanomoles per liter (nmol/L) and on cortisol (C) in
micromoles per liter (mmole/L) or a decrease of the ratio of 30 percent or more in
comparison with the previous value. In our experience, the use of an absolute value as a
threshold is not useful, whereas the evaluation of the concentrations of hormones and their
ratio in comparison with previous ones is more useful. These classical approaches are not,
however, sufficient to describe the various possible physiological modifications linked to
training excess and/or incomplete recovery. It was collected samples from 32 professional
soccer players of an Italian First Division team, during the period 2001- 2003. It was
analyzed the values of 21 athletes during the season 2001-2002 and of 11 athletes during
the season 2002-2003 (6 out of 11 were examined also during the previous one) always
present when the 4 (first season) or 5 (second season) blood drawings have been
performed. We applied an original, pragmatic and easy-to-use classification of FTCR values,
in association with classical interpretations based on decreases of the values in comparison
with previous athlete's result. It was used the traditional approaches in two consecutive
seasons in a professional soccer team: the evaluation of the decrease >30% of the
parameter in comparison with the previous value or with the basal (preseason) value are
shown. The statistical differences between the FTCR values of the six athletes followed in
both seasons were not significant. Thus, the classification method that was proposed is
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advantageous in comparison with traditional interpretative schemes, because identify
different risk categories, stratifying the interval between the values 0.35-0.8 [06061].
Time-course of testosterone action
The classic model of steroid action is that steroid hormones have a relatively slow time-
course of action by acting as transcription factors after binding to intracellular receptors
Athletes know that the anabolic gains realized while on a pre-competition steroid “cycle” will
persist for weeks after AAS use is discontinued. However, research in animals has
demonstrated behavioral effects of testosterone that occur within minutes. In the 1980s, East
German scientists developed an androgen nasal spray to enhance aggression and
competitiveness without systemic effects. Similarly, intranasal 4,16-androstadien-3-one
induces an amphetamine-like “high” in human volunteers [12100].
Stability in the urine
The stability of testosterone glucuronide (TG), epitestosterone glucuronide (EG) and the T/E
ratio in urine has been studied. Samples were analyzed by gas chromatography coupled to
mass spectrometry (GC/MS). Urine samples were submitted to a solid-liquid cleanup
followed by extraction of unconjugated testosterone (T) and epitestosterone (E) with tert-
butyl methyl ether (free fraction). The remaining aqueous phase was hydrolyzed with beta-
glucuronidase and extracted at alkaline pH with n-pentane. Analytes were analyzed by
GC/MS as their enol-trimethylsilyl (TMS) derivatives. The urine for stability testing was
obtained from an excretion study after the administration of T to healthy volunteers. The
homogeneity of the sample was verified before starting the stability study. The stability of TG
and EG was evaluated at different storage conditions. For long-term stability testing, analyte
concentration in urine stored at 4 degrees C and -20 degrees C was determined at different
time intervals for 22 months. For short-term stability testing, analyte concentration was
evaluated in urine stored at 37 degrees C for 3 and 7 days. The effect of repeated freezing
(at -20 degrees C) and thawing (at room temperature) was studied for up to three cycles.
Data obtained in this work demonstrated the stability of TG, EG and the T/E ratio in sterilized
urine samples stored at 4 and -20 degrees C for 22 months and after going through repeated
freeze/thaw cycles. Decreases in concentration were observed after 7 days of storage at 37
degrees C due to the partial cleavage of the glucuronide conjugates; however, the T/E ratio
was not affected. These results show the feasibility of preparing reference materials
containing TG and EG to be used for quality control purposes [06051].
Effects of anabolic precursors on serum testosterone concentrations
The effects of androgen precursors, combined with herbal extracts designed to enhance
testosterone formation and reduce conversion of androgens to estrogens was studied in
young men. Subjects performed 3 days of resistance training per week for 8 weeks. Each
day during Weeks 1, 2, 4, 5, 7, and 8, subjects consumed either placebo (PL; n=10) or a
supplement (ANDRO-6; n=10), which contained daily doses of 300 mg androstenedione, 150
mg DHEA, 750 mg Tribulus terrestris, 625 mg Chrysin, 300 mg Indole-3-carbinol, and 540
mg Saw palmetto. Serum androstenedione concentrations were significantly higher in
ANDRO-6 after 2, 5, and 8 weeks, while serum concentrations of free and total testosterone
were unchanged in both groups. Serum estradiol was elevated at Weeks 2, 5, and 8 in
ANDRO-6, and serum estrone was elevated at Weeks 5 and 8. Muscle strength increased
similarly from Weeks 0 to 4, and again from Weeks 4 to 8 in both treatment groups. The
acute effect of one third of the daily dose of ANDRO-6 and PL was studied in 10 men (23 + 4
years). Serum androstenedione concentrations were elevated in ANDRO-6 from 150 to 360
1273
min after ingestion, while serum free or total testosterone concentrations were unchanged.
These data provide evidence that the addition of these herbal extracts to androstenedione
does not result in increased serum testosterone concentrations, reduce the estrogenic effect
of androstenedione, and does not augment the adaptations to resistance training [00079].
The term prohormone strictly refers to a post-translational peptide that is cleaved by

convertases into a variety of bioactive hormones. In the supplement context, prohormones
refer to androgenic precursors which, when ingested, become enzymatically activated to
testosterone derivatives. An understanding of the biochemical pathways emphasises the
similarity between testosterone and its precursors. Users see prohormones as a natural
means to improve muscle strength, body composition and general well-being with fewer side
effects than testosterone or synthetic androgenic steroids and a more practical (capsule)
form of intake. The marketing strategy of commercial websites is to promote prohormones as
“legal alternatives” to testosterone with similar anabolic effects. Of course, many consumers
are unaware that these prohormones are included on the WADA list of prohibited substances
as well as being illegal for sale or importation in many countries. Prohormones have another
concerning role in sports nutrition as contaminants in other sports supplements which
account for a large proportion of inadvertent doping offences. From cholesterol,
pregnenolone is produced which converts to testosterone via dihydroepiandosterone
(DHEA). The path via DHEA produces androstenedione (DIONE) and androstenediol (DIOL)
which convert to testosterone. Importantly, however, these precursors can also be converted
to the estrogens, which may cause effects such as gynaecomastia and liver dysfunction. To
counteract this effect, some users of prohormones alternate between 1 month on and 2
months off, allowing restoration of intrinsic function within each cycle. Users also often stack
differing prohormones of differing oestrogenicity within each cycle, and take N-acetyl
cysteine to prevent liver dysfunction. In addition, selective estrogen receptor modulators or
aromatase inhibitors are taken to mitigate oestrogenic effects, and androgenic herbal
compounds taken to reduce the low period between cycles. Despite these sophisticated
multidrug regimens and marketing claims, research fails to demonstrate any anabolic or
ergogenic effects of taking DHEA, DIONE or DIOL, and confirms the risk of adverse side
effects for DIONE and DIOL. For example Broeder et al gave 200 mg/day DIONE or DIOL to
middle-aged men over a 12-week resistance training programme, and showed a significant
16 percent increase in testosterone levels after 1 month of use which had returned to
prestudy levels by 12 weeks. DIOL did not significantly increase blood testosterone levels.
The major fate of the ingested DIONE and DIOL appears to be aromatisation, since blood
estrogen levels were increased by about 63 percent. There was no enhancement in muscle
strength during resistance training above placebo but, conversely, an 11 percent increase in
the LDL-Cholesterol/HDL-Cholesterol lipid ratio, corresponding to a significant increase in the
cardiovascular disease risk. Twelve weeks of supplementation reduced blood luteinising
hormone levels, which may serve to decrease inherent testicular and adrenal testosterone
production. The last major literature review, by Brown et al, confirmed the findings that
DIONE, DIOL and DHEA do not augment muscle size and strength gains observed from
resistance training alone, and that use of DIONE and DIOL may predispose users to serious
health risks [12459].
An area for concern is the abuse of a precursor to AAS known as prohormones, such as
androstenediol. Many prohormone synthetic chemicals have been banned, but numerous
clones have been made with a minor change in the formulation. These prohormones
increase hormone levels within the body leading to many of the same benefits and side
effects as the AAS [12119].
The first steroids introduced on the prohormone market were dehydroepiandrosterone

(DHEA) and androst-4-ene-3,17-dione in 1996, shortly thereafter followed by androst-4-ene-
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3beta,17beta-diol, androst-5-ene-3beta,17beta-diol and androst-5-ene-3,17-dione. These
steroids can be regarded as prohormones of testosterone as they are claimed to metabolise
to testosterone after oral administration. Several studies on the metabolism of DHEA
androst-4-ene-3,17-dione, androst-4-ene-3beta,17beta-diol, androst-5-ene-3beta,17beta-diol
and androst-5-ene-3,17-dione have indicated the possibilities of using alterations in the
steroid profile to detect misuse of these steroids. Based upon these studies, monitoring the
concentration and ratios of several steroids can be used for the detection of the
administration of a testosterone prohormone. A similar approach was already successfully
implemented for testosterone and dihydrotestosterone misuse. Amongst the most powerful
indicators, besides the increase in the individual urinary concentrations of the administered
steroids, were the ratio of androsterone/etiocholanolone and testosterone/ epitestosterone.
These parameters were already well established in doping control analysis to detect the
administration of testosterone. Besides these parameters, other steroids have been
proposed for the detection of misuse of androst-4-ene-3,17-dione, including 6alpha-OH-
androstenedione, 6beta-OH-androsterone, 6beta-OH-etiocholanolone, 6beta-OH
epiandrosterone and 4-OH-androstenedione. For the detection of DHEA administration,
7beta-OH-DHEA, 7-keto-androsterone, 16alpha-OH-androsterone and 16alpha-OH-
etiocholanolone can be used as indicative parameters. 5alpha-Androstane-3beta,17beta-diol
is a prohormone of dihydrotestosterone (DHT) and is marketed as such. Administration of
this steroid resulted in elevated urinary levels of endogenous 5alpha-steroids, similarly as
after DHT administration as well as in high urinary concentrations of epiandrosterone
sulphate. It was therefore suggested that for the detection of the abuse of 5alpha
androstane-3beta,17beta- diol, the same criteria as for DHT-misuse could be applied, in
addition to the increase in urinary 5alpha-androstane-3beta,17beta- diol glucuronide
concentration [06004].
5alpha-Androstane-3,17-dione is an endogenous steroid and an intermediate in the

conversion of androstenedione to androsterone and etiocholanolone and is naturally present
in urine in low concentrations. In microbiologically contaminated urine however, the
concentration of this steroid might rapidly increase. 5alpha-Androstane- 3,17-dione is listed
as a controlled substance in the 2004 ASCA but was in spite of that detected as a
contaminant in a nutritional supplement [06004].
Testosterone concentrations are blunted after resistance exercise in men
One study examined the hypothesis that exercise-induced changes in circulating

testosterone would be centrally mediated via hypothalamic-pituitary release of luteinizing
hormone (LH). It was tested this hypothesis by examining overnight LH, total and free
testosterone (TT and FT), and cortisol (C) concentrations in 10 young healthy men (21 + 1
years) during two experimental sessions: a control and an acute heavy-resistance exercise
bout (50 total sets consisting of squats, bench press, leg press, and latissimus dorsi pull-
down). Exercise was performed from 1500 to 1700, and blood sampling began at 1700 and
continued until 0600 the next morning. Blood was sampled every 10 min for LH and every
hour for TT, FT, and C. Hormonal concentrations were determined via RIA, and the secretion
characteristics of LH were analyzed with deconvolution analysis. When overnight
postexercise concentrations were compared with control concentrations, no statistically
significant differences were observed for LH half-life, LH pulse frequency, interpulse interval,
pulse amplitude, or pulse mass. Significant differences were observed for LH production rate.
For the ANOVA marginal main effect means due to condition, C was significantly elevated,
while TT and FT were significantly decreased for the exercise condition. These data
demonstrate that the decline in overnight testosterone concentrations after acute heavy-
resistance exercise is accompanied by a blunted LH production rate and elevated C
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Stress from exercise in the below sea level environment
A comparative study (n=20) of serum levels of leutinizing hormone (LH) and testosterone (T)
between male trained athletes (high-school students, 16-18 years old) living in North and
South Shouna in the Jordan Valley (JV), 320-360 meters below sea level, and those living in
Ramtha and Irbid, 550-650 meters above sea level, was conducted in November, 1999.
Serum levels of LH and T were also measured in these athletes following a 20 Km
noncompetitive run. The air temperatures on the day of the experiment was comparable (25
degrees C in North and South Shouna versus 23 degrees C in Ramtha and Irbid). Before
exercise, serum levels of LH and T in athletes of the below sea level environment (North and
South Shouna) were similar to those levels in athletes of the above sea level environment
(Ramtha and Irbid). Exercise caused a significant increase in serum levels of both LH and T
only in athletes of North and South Shouna. It seems likely that the higher secretion of LH
contributes, at least in part, for the higher serum levels of T following exercise. Taken
together, these data suggest that exercise has an effect on LH and T secretion that is similar
to that of fasting. And finally, the below sea level environmental factors, such as the high
barometric pressure, as well as the genetic background of the athletes affect the pituitary and
adrenal cortex as well as testicular tissue, resulting in the secretion of more LH and T
[01078].
Effect of rowing
In one study, it was examined anabolic and catabolic hormone responses to a single
endurance rowing training session in 12 male competitive single scull rowers. A work
intensity eliciting a blood lactate concentration of 4 mmol/L was determined on a rowing
ergometer during an endurance rowing training session lasting about 2 h (distance covered
23 + 3 km). Venous blood samples were obtained before and after on-water rowing. Cortisol,
testosterone and sex hormone binding globulin were measured and free testosterone and
the free testosterone: cortisol ratio calculated. Blood lactate concentration did not change
significantly during training; however, body mass was reduced and was related to the
distance covered. The concentrations of cortisol and testosterone did not change significantly
during rowing or in the first 2 h of recovery. Free testosterone was reduced in the first 2 h of
recovery, but no significant changes were observed in the free testosterone: cortisol ratio.
Immediately after rowing, the concentrations of cortisol and free testosterone were related to
the distance covered. The findings indicate that a prolonged low-intensity training session
results in a similar anabolic and catabolic hormone stimulus for trained rowers [01079].
The purposes of one study were to investigate the resting levels and the acute hormonal
response of total testosterone (Ttot), free testosterone (Tfree), cortisol (C) and Tfree:C ratio to
the 6-minute all-out rowing ergometer test. It was performed a cross-sectional study to
investigate the responses of blood hormones to the maximal rowing ergometer test in college
rowers with 8 college level male rowers (22 years) in a graded exercise test at the intensities
of 150, 200 and 250 W (6-minute each) and a 6-minute all-out test on a rowing ergometer
(Concept II, Morrisville, USA) were performed at separate measurement sessions. Heart rate
(HR) was recorded at the end of each load during a graded exercise test. Individual physical
working capacity of rowers was calculated at the maximum HR recorded during a 6-minute
all-out test. Venous blood samples were obtained 3 minutes before and immediately after the
6-minute all-out test to determine the concentration of Ttot, Tfree, Tfree:C ratio, glucose and
lactate (LA) in blood. Mean power was 354.3 + 26.8 W and HR 190.9 + 5.1 beats/min during
the all-out test. Mean blood LA concentration at the end of the all-out test was 13.4 + 1.44
mmol/L. The all-out test on the rowing ergometer did not change significantly the
1276
concentration of Ttot, Tfree, C, and Tfree:C ratio. The transformation of these hormone
values to effect sizes (ES) demonstrated that all-out test only moderately influenced these
hormone levels. Significant relationships were observed between the resting levels of Ttot
and Tfree, and mean power of the 6-minute all-out test. There was a non-significant increase
in the level of Ttot following the all-out rowing ergometer test which was significantly related
to mean power and covered distance. The results suggest that there are no significant
changes in hormone levels during a 6-minute all-out rowing ergometer test. However, rowing
performance is positively related to the resting values of Ttot and Tfree, and the non-
significant changes in Ttot level following a 6-minute all-out test [01080].
Biphasic dose responses to prolactin
Testosterone was reported to affect a variety of reproductive endpoints. More notable were
the effects of dihydrotestosterone on cell proliferation in the prostate cancer cell model
LNCaP and Sertoli cell function. Testosterone production was also biphasically affected by
prolactin that was administered to adult testicular cells in vitro [01081].
Seasonal variations
Humans' endogenous testosterone concentrations vary over a number of temporal scales,

with little known about variation longer than monthly cycles. Past studies of seasonal or
circannual variation have principally used male participants and have produced inconsistent
results. Thus, little is known about how testosterone concentrations fluctuate throughout the
year, whether such variation differs between men and women, and whether there are
influences of hormonal contraceptive use. The present study collected saliva samples from a
large sample (n=718) of men and women, each collected at one time point within a relatively
uniform distribution over a full calendar year. Both men and normally-cycling women
displayed seasonal variation in salivary testosterone concentrations, such that testosterone
concentrations are maximal in the fall and minimal in the summer. Notably, normally-cycling
women had testosterone concentrations that were over 100 percent greater at their
maximum in fall compared to their minimum in summer. Women using hormonal
contraceptives not only had consistently lower endogenous testosterone concentrations, but
also showed a flatter seasonal testosterone profile [11577].
Ethnic differences in steroid-related diseases
Differences in circulating steroid hormone levels have been hypothesized to explain ethnic
differences in steroid-related diseases. The aim of one study was to determine the serum
levels of a wide panel of steroid hormones, both androgens and estrogens, in healthy middle-
aged African-Caribbean and European men. Serum steroid hormone levels were determined
in men participating in a systematic public health study funded by the French National Health
Insurance system. Blood was collected in the morning from 304 healthy African-Caribbean
and European men aged between 40 and 69 years. Serum steroids were measured by mass
spectrometry-gas chromatography, except for DHEAS and sex hormone-binding globulin,
which were determined by RIA. Data were analyzed in 10-year age intervals by analysis of
covariance, with adjustment for age, body mass index, waist-to-hip ratio, tobacco and alcohol
consumption, and season of sampling. Compared with Europeans, African-Caribbean men
presented significantly higher serum levels of measured bioavailable testosterone, 4-
androstenedione (4-dione), and estrone (E1) regardless of the age group, of 5-androstene-
diol (5-diol) in those aged 40-49 and 50-59 years, and of testosterone (TT) and dihydrotesto-
sterone in those aged 40-49 years. In contrast, European men aged 40-69 years showed
significantly higher serum levels of DHEA and DHEAS. It was concluded that significant
1277
differences in serum steroid hormone levels were observed in middle-aged African-
Caribbean and European men. Whether such differences could contribute to ethnic
differences in disease risk in adult men remains to be investigated. Some steroids, such as
bioavailable TT, 4-dione, 5-diol, and E1, deserve particular attention [11578].
It is hypothesised that seemingly disparate and unrelated phenomena clustering in persons

of African descent living in the Americas such as outstanding sprinting ability and high
prostate cancer incidence and mortality are in fact related and emerge from enhanced
testosterone responsiveness in descendants of African slaves surviving the transatlantic
trade in Africans. It is postulated that the ability to have survived the middle passage was
positively correlated with greater responsiveness of the androgen receptor to its primary
ligands dihydrotestosterone and testosterone, and that slaves possessing more responsive
androgen receptors experienced a survival advantage engendered by the enhanced anabolic
effects which accrued such as increased red cell mass and therefore greater oxygen carrying
capacity and tissue oxygen delivery enabling these slaves to tolerate stifling conditions in the
hull of the slave ship, increased lean muscle mass and therefore greater surface area to
volume ratio resulting in easier ability to dissipate heat and remain cool, and increased skin
thickness and sebum production resisting the macerating effect of lying in admixed bodily
fluids below deck. These androgen effects as well as others would have produced a survival
advantage under the severe selection pressure created by the inhumane and physiologically
challenging circumstances under which the slaves were transported from the interior of the
African continent and West Africa to the 'New World'. This would result in a population shift
favouring increased androgen receptor responsiveness in descendants of African slaves
populating the Americas and a corresponding geographic and racial distribution of androgen
related phenomena such as sprinting prowess and prostate cancer. African-Americans
having the highest prostate cancer incidence rate and the Caribbean having the highest
prostate cancer mortality rates in the world are consistent with this hypothesis as is the
observation that the 10 fastest men and 9 fastest women of all time are exclusively the
descendants of West African slaves who survived the middle passage. It is predicted that as
yet undiscovered as well as known biological correlates of enhanced androgen receptor
responsiveness such as relatively short CAG-repeats in the poly Q tail of exon 1 of the
androgen receptor gene will be more prevalent among African-Americans and Afro-
Caribbean peoples than among West Africans. It is also predicted that African-Americans
and Afro-Caribbean peoples will have relatively shorter CAG-repeats in the androgen
receptor gene compared to West Africans [11579].
Urinary levels of testosterone and epitestosterone in a Korean male population
Cannabis, or marijuana, the most commonly used illicit drug in the world, has been shown to
be responsible for suppressing the production and secretion of androgens, particularly
testosterone. However, despite such findings in animals, the chronic effects of marijuana use
on human endocrine systems have proved to be inconsistent. Here, it was investigated the
reference ranges of urinary levels of testosterone (T) and epitestosterone (E) as well as their
metabolic ratio of T/E in a Korean male population (n=337), which would enable an
evaluation of abnormal changes in steroid metabolism induced by habitually administered
cannabis. The T/E ratio was significantly decreased in the marijuana group (n=18), while the
urinary testosterone concentrations were also tended to decrease. This study is the first to
provide data for the reference values of two urinary androgens and T/E values among control
Korean males, and, furthermore, suggests that the T/E ratio, though not testosterone levels,
might be used to understand the suppression of human male gonadal function affected by
smoking marijuana [13219].
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Status of lean elite athlets
It was investigated the endocrine profile, body composition, and state of mood in male
Olympic athletes participating in sports that do or do not emphasize leanness. Forty-four
Swedish male Olympic athletes participating in 26 different sport disciplines were studied.
Body composition was determined by dual-energy x-ray absorptiometry, and blood levels of
steroid hormones and biomarkers of nutritional status were analyzed. In addition, states of
mood were assessed employing the profile of mood states (POMS) test. The athletes were
divided into 2 groups on the basis of whether their sporting discipline emphasized leanness
or not. In all subjects, body composition, hormone levels, and POMS scores were within
normal ranges. However, the leanness athletes (n=18) displayed significantly lower
proportion of body fat, higher spinal bone mineral density, lower serum levels of free
testosterone and leptin, and higher serum levels of insulin-like growth factor binding protein 1
than nonleanness athletes (n=26). Leanness athletes also had higher POMS scores for
depression and anger, and a higher global POMS score, the latter being positively correlated
to the frequency of illness before the Olympic Games. It was concluded that although there
were no indications of energy deficiency or endocrine disturbance in the leanness athletes,
their higher POMS scores and frequency of illness may indicate the potential harmfulness of
their pursuit of outstanding athletic performance [13195].
Levels in male Olympics
To investigate the endocrine profile, body composition, and state of mood in male Olympic
athletes participating in sports that do or do not emphasize leanness 44 Swedish male
Olympic athletes participating in 26 different sport discipline were investigated. Body
composition was determined by dual-energy x-ray absorptiometry, and blood levels of steroid
hormones and biomarkers of nutritional status were analyzed. In addition, states of mood
were assessed employing the Profile of Mood States (POMS) test. The athletes were divided
into 2 groups on the basis of whether their sporting discipline emphasized leanness or not. In
all subjects, body composition, hormone levels, and POMS scores were within normal
ranges. However, the leanness athletes (n=18) displayed significantly lower proportion of
body fat higher spinal bone mineral density, lower serum levels of free testosterone and
leptin and higher serum levels of insulin-like growth factor binding protein 1 than
nonleanness athletes (n=26). Leanness athletes also had higher POMS scores for
depression and anger, and a higher global POMS score, the latter being positively correlated
to the frequency of illness before the Olympic Games. CONCLUSION:: Although there were
no indications of energy deficiency or endocrine disturbance in the leanness athletes, their
higher POMS scores and frequency of illness may indicate the potential harmfulness of their
pursuit of outstanding athletic performance [12105].
Associations between dehydration and testosterone at weight loss before competition
In weight class sports, such as judo, taekwondo and wrestling, reducing body weight before
competitions is common. However, it is recommended that weight loss per week should not
exceed 1.5 percent of total body weight otherwise, athletes' metabolism and endocrine
parameters are negatively affected, which will deteriorate their physiology and psychology
and thus decrease their performance. The aim of one study was to determine weight loss
and hydration levels after weight loss before competitions among the elite wrestlers and to
explore the association between hydration levels, and stress and testosterone. An
observational study was undertaken with 56 voluntary athletes who participated in wrestling
championship. With blood samples taken from the wrestlers, glucose, blood urea nitrogen,
1279
sodium (Na), cortisol, prolactin and testosterone hormone analyses were evaluated by a
specialist at a biochemical laboratory. It was found out that according to plasma osmolarity
levels, there were significant differences between those dehydrated and those who
maintained euhydration in terms of cortisol and total testosterone levels. It was detected that
an association was present between plasma osmolarity, and cortisol and total testosterone
levels among the elite wrestlers. It was discovered that elite wrestlers were subjected to
quick and high level of weight losses before competitions in a very short time (1-5 days). It
was seen that their hydration levels differed due to the weight loss, which was explored to be
causing acute dehydration among the wrestlers [150260].
Effect of calorie restriction on testosterone in training
The aim of one study was to evaluate the effect of caloric restriction on the immune and
hormonal responses during exercise in judo athletes. In a randomised order, 11 male
judokas (age: 20.45 ± 0.51; height: 1.71 ± 0.3 m; and body weight: 75.9 ± 3.1 kg) participate
in this study during a period of weight maintenance (baseline) and after 7 days of caloric
restriction (CR). All subjects performed the Special Judo Fitness Test (SJFT) during the two
conditions. Values for nutrient intakes were obtained from a 7 d food record kept during a
period of weight maintenance and after a 7-day food restriction (-5 to 6 MJ/day). The results
showed that CR resulted in significant decreases in body weight and performance. However,
heart rate and SJFT index increase significantly during CR in comparison to baseline.
Moreover, exercise leads to a significant increase in testosterone, cortisol, growth hormone
(GH), leukocytes, neutrophils, TNF-α, and IL-6, in both CR and baseline conditions.
Compared to baseline, TNF-alpha and IL-6 were significantly higher during CR condition.
Additionally, CR leads to an increase in cortisol and GH and a decrease in testosterone
Effects of meal form and composition on plasma testosterone
The purpose of one study was to examine the effects of postexercise feeding on plasma
levels of insulin, testosterone, cortisol, and testosterone:cortisol (T:C). Ten experienced,
resistance trained males (21 years) were given whole food (WF: protein 38 g; carbohydrate
70 g; fat 7 g), a supplemental drink (SD; isocaloric and isonitrogenous to WF), an isocaloric
carbohydrate beverage (C), or a placebo beverage (P) immediately, 2 and 4 hours after a
standardized weight training protocol on 4 days, each separated by 1 week, in a repeated
measures design. Subjects also received a standardized meal at 7 and 12 hours
postexercise. Insulin, testosterone, and cortisol were measured pre-exercise and during 24
hours of recovery (at 0.5, 2.5, 4.5, 8, and 24 hours) using venous blood samples. Significant
(condition 3 time) interactions were found for insulin, testosterone, and T:C, but not for
cortisol. The SD yielded a greater response for insulin than all other conditions. Conversely,
P demonstrated the greatest values for testosterone and T:C at 2.5 and 4.5 hours
postexercise. Cortisol did not vary between conditions and there were no condition effects for
insulin, testosterone, cortisol, and T:C at 8 or 24 hours. In conclusion, the efficacy of
postexercise feeding for optimizing T:C and muscle growth is unclear; however, consumption
of SD appears to maximize circulating insulin for several hours following resistance exercise
[00074].
Effect of sexual activity on testosterone levels
The purpose of one study was to investigate the effect of sexual activity on cycle ergometer
stress test parameters, on plasmatic testosterone levels and on concentration capacity in
high-level male athletes. Experimental design. Analysis of two days of testing accomplished
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in a laboratory setting, comparing a day with to a day without sexual activity (control day).
Participants. Fifteen high-level male athletes, consisting of 8 team players, 5 endurance
athletes and 2 weight-lifters, participated in the study. Measures. Each subject completed the
following on each test day: two maximal graded stress tests on a cycle ergometer and a one-
hour exercise stress test coupled to an arithmetic mental concentration test. Blood samples
of testosterone were obtained and cardiac activity of each athlete was monitored with a 24-
hour ECG tape recording over the two test days. Significantly higher differences were
achieved for post-effort heart rate (HR) values at 5 minutes and at 10 minutes during the
recovery phase of the morning stress test 2 hours after sexual activity. These differences
disappeared during the recovery phase of the afternoon stress test performed approximately
10 hours after sexual intercourse took place. The findings show that sexual activity had no
detrimental influence on the maximal workload achieved and on the athletes' mental
concentration. However, the higher posteffort HR values after the maximal stress test on the
morning of sexual intercourse suggest that the recovery capacity of an athlete could be
affected if he had sexual intercourse approximately 2 hours before a competition event
[00073].
Testosterone levels during recovery
To describe the evolution of cortisol and testosterone levels and testosterone/cortisol (T/C)
ratio in long-distance runners during a relay competition and during the three days following
the competition two teams of four relayers (one male, one female) took part into this six-hour
relay race. Hormonal control during the race was made possible thanks to saliva sampling
during rest periods at each relay. The runners were subelite runners. During the race, cortisol
levels reached approximately 1.5-fold basal levels. These levels remained high till late
evening, (higher than morning values, when normal resting levels are 4 to 6-folds lower).
Surprisingly, wakening levels during the following days were lower than resting levels.
Testosterone did not vary in females; then, male values only are reported. During the race
they decreased gradually and remained low till night. During the following three days,
testosterone levels were higher than resting day levels. The T/C ratio amplifies these
variations: low during the race till retiring, (currently associated with a catabolic tendency)
and reversely high during the following three days (associated with a high anabolic
tendency). As expected, a catabolic tendency occurs during a long distance run (increase in
cortisol level followed by a drop in testosterone level). More surprising is the high anabolic
tendency noted during the recovery period (low cortisol and high testosterone levels)
[00076].
Older men
Little information exists about longitudinal changes in body composition and physical function
in relation to sex hormone levels in older men. The aim of one study was to determine
associations of testosterone, estradiol, and SHBG with changes in body composition and
physical function. It was conducted a prospective cohort study within the Osteoporotic
Fractures in Men (MrOS) study at six US clinical centers. A total of 5994 ambulatory men
aged 65 years or older enrolled in the MrOS. It was examined 1183 men with complete
measures of sex steroid hormones, body composition, and some measure of physical
function. Intervention: There were no interventions. Sex steroids were measured by mass
spectrometry in serum collected at baseline. Measurements of body composition using dual-
energy x-ray absorptiometry and physical performance (grip strength, leg power, timed chair
stands, narrow walk, and 6-m walk) were performed at baseline and repeated 4.5 years later.
Overall, men lost 1.3 kg (± 4.4) weight between study visits. Lean mass, especially
appendicular, declined less at higher baseline testosterone levels. These associations were
1281
most evident in the 40 percent of men who lost more than 2.0 kg during follow-up. In weight
losers, higher testosterone was associated with less decline in timed chair stands. Estradiol
was not related to body composition or physical function changes. Higher SHBG was
associated with less loss of appendicular lean mass and grip strength. Higher endogenous
testosterone is associated with reduced loss of lean mass and lower extremity function in
older men losing weight. Endogenous testosterone may contribute to healthy aging [11580].
Cycling androgens has been reported by athletes to improve physical performance by

enhancing muscle mass and strength, a paradigm that has not been studied, and may have
clinical value in older men being treated with testosterone. It was investigated the efficacy of
a monthly cycled testosterone regimen that uses half the testosterone dose as the current
standard of care continuous therapy on body composition and muscle strength in older men.
Twenty-four community-dwelling older men 70 ± 2 years of age with total testosterone levels
below 500 ng/dL were randomized at the Institute for Translational Sciences-Clinical
Research Center into a 5-month double-blind placebo-controlled trial. Subjects were dosed
weekly for 5 months, receiving continuous testosterone (TE, n=8; 100 mg testosterone
enanthate, im injection), monthly cycled testosterone (MO, n=8; alternating months of
testosterone and placebo), or placebo (PL, n=8). Main outcomes included body composition
by dual-energy x-ray absorptiometry and upper and lower body muscle strength. Secondary
outcomes included body weight, serum hormones, and mixed-muscle protein fractional
synthesis rate (FSR). Total lean body mass was increased and percent fat was reduced after
5 months in TE and MO. Upper body muscle strength increased in TE, and lower body
muscle strength increased in TE and MO. FSR increased in TE and MO but not in PL. It was
concluded that cycled testosterone improved body composition and increased muscle
strength compared with placebo and increased FSR similarly to continuous testosterone
[11581].
Testosterone in Older Men with Mobility Limitations Trial determined the effects of
testosterone on muscle performance and physical function in older men with mobility
limitation. Trial's Data and Safety Monitoring Board recommended enrollment cessation due
to increased frequency of adverse events in testosterone arm. The changes in muscle
performance and physical function were evaluated in relation to participant's perception of
change. Men aged 65 years and older, with mobility limitation, total testosterone 100-350
ng/dL, or free testosterone less than 50 pg/mL, were randomized to placebo or 10 g
testosterone gel daily for 6 months. Primary outcome was leg-press strength. Secondary
outcomes included chest-press strength, stair-climb, 40-m walk, muscle mass, physical
activity, self-reported function, and fatigue. Proportions of participants exceeding minimally
important difference in study arms were compared. Of 209 randomized participants, 165 had
follow-up efficacy measures. Mean (SD) age was 74 (5.4) years and short physical
performance battery score 7.7 (1.4). Testosterone arm exhibited greater improvements in
leg-press strength, chest-press strength and power, and loaded stair-climb than placebo.
Compared with placebo, significantly greater proportion of men receiving testosterone
improved their leg-press and chest-press strengths (43 % vs 18 %) and stair-climbing power
(28 % vs 10 %) more than minimally important difference. Increases in leg-press strength
and stair-climbing power were associated with changes in testosterone levels and muscle
mass. Physical activity, walking speed, self-reported function, and fatigue did not change.
Testosterone administration in older men with mobility limitation was associated with patient-
important improvements in muscle strength and stair-climbing power. Improvements in
muscle strength and only some physical function measures should be weighed against the
risk of adverse events in this population [11582].
Although wide interindividual variations exist, mean total testosterone (TT) and free
testosterone (FT) levels decline with age, whereas DHT and estradiol levels tend to remain
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relatively constant. At age 75 years, the mean TT level in the morning is about two-thirds of
the mean level in men aged 20-30 years, whereas the mean FT and bioactive testosterone
(FT plus albumin- bound testosterone) levels are only 40 percent of the mean levels in
younger men. Furthermore, the circadian rhythm of serum testosterone levels is generally
lost or attenuated in elderly men [12095].
Effects of an oral androgen in older, community-dwelling men

To determine whether oxymetholone increases lean body mass (LBM) and skeletal muscle
strength in older persons, 31 men 65-80 year of age were randomized to placebo (group 1)
or 50 mg (group 2) or 100 mg (group 3) daily for 12 weeks. For the three groups, total LBM
increased by 0.0 + 0.6, 3.3 + 1.2, and 4.2 + 2.4 kg, respectively. Trunk fat decreased by 0.2
+ 0.4, 1.7 + 1.0, and 2.2 + 0.9 kg in groups 1, 2, and 3, respectively. Relative increases in 1-
repetition maximum (1-RM) strength for biaxial chest press of 8.2 + 9.2 and 13.9 + 8.1
percent in the two active treatment groups were significantly different from the change (-0.8 +
4.3 %) for the placebo group. For lat pull-down, 1-RM changed by -0.6 + 8.3, 8.8 + 15.1, and
18.4 + 21.0 percent for the groups, respectively. The pattern of changes among the groups
for LBM and upper-body strength suggested that changes might be related to dose. Alanine
aminotransferase increased and HDL-cholesterol decreased. Thus oxymetholone improved
LBM and maximal voluntary muscle strength and decreased fat mass in older men [02053].
Testosterone therapy in late-life major depression in males

Major depression associated with aging in males may improve with anabolic/androgenic
steroid therapy. The efficacy and safety of testosterone therapy in the treatment of
depression in elderly hypogonadal males is inconclusive. The following study identifies a
subgroup of elderly depressed males who may benefit from testosterone therapy.
Participants included 16 elderly eugonadal males with major depressive disorder (DSM-IV
criteria) and a Hamilton Rating Scale for Depression (HAM-D) score > 18. Following a single-
blind 2-week placebo lead-in, patients were randomly assigned to treatment with either a
physiologic dose of testosterone cypionate (TC), 100 mg/week, or supraphysiologic dose of
200 mg/week IM for 6 weeks. Psychometric testing was carried out at entry into the study, at
the TC injection baseline, and every 2 weeks thereafter. Tests included an objective
measurement, the HAM-D, and the Buss-Durkee Hostility Inventory. One patient meeting
inclusion criteria responded during the placebo lead-in; thus, 15 patients were randomly
assigned to treatment (100 mg/week, n=8; 200 mg/week, n=7). There was a 42 percent
decrease in the mean HAM-D scores from 20.1 to 11.9. However, the majority of the change
was due to improvement in the 10 late-onset (<45 years old) depression patients, whose
mean HAM-D score decreased from 19.8 to 9.3 (53 %), versus the 5 early-onset depression
patients, whose mean HAM-D score decreased from 20.8 to 17.0 (18 %). The TC dose did
not affect the response. Similar HAM-D decreases of 43 and 41 percent occurred for the
respective 100- and 200-mg/week doses. The HAM-D responder analysis found that none of
5 early-onset patients had HAM-D response, whereas 6 (60 %) of 10 late-onset patients
responded. Similarly, none of the early-onset patients experienced a remission whereas 5
(50 %) of the late-onset patients were categorized as remitters. Correlations between the
peak and mean total testosterone concentrations and HAM-D change scores suggested that
only minimal TC doses were required to produce an antidepressant effect. These data
suggest that testosterone therapy would best be limited to men with late-onset depression.
The findings suggest that short-term therapy with TC is safe. Long-term treatment safety is
unknown. Psychiatrists using testosterone therapy should ascertain that patients have been
recently valuated for prostate cancer. If testosterone therapy is initiated, serial serum
prostate-specific antigen sampling should be used for monitoring patients' prostate status
[02054].
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Women
The measurement of serum testosterone in women is challenging due to lack of trueness,

precision, and sensitivity of various available testosterone assays. Accurate assessment of
testosterone in women is crucial especially in conditions associated with alleged over- or
under-production of testosterone, such as in polycystic ovary syndrome (PCOS) or primary
ovarian insufficiency (POI). The aim of this study was to measure and compare androgen
concentrations in women with PCOS, POI, and female controls and to evaluate the
performance of extraction RIA and liquid chromatography-tandem mass spectrometry (LC-
MS/MS) in these women. Carefully phenotyped women with POI (n=208) or PCOS (n=200)
and 45 healthy, regularly cyclic female controls were included. Method comparison analyses
were performed for total testosterone, androstenedione (AD), and DHEA, as measured by
LC-MS/MS and extraction RIA. All androgen levels were significantly elevated in women with
PCOS compared with POI patients and controls. Women with POI presented with similar
androgen concentrations as controls, except for AD. Compared with measurements by
extraction RIA, testosterone, DHEA, and AD concentrations measured by LC-MS/MS were
systematically lower. However, using extraction RIA and LC-MS/MS, testosterone, DHEA,
and AD measurements were shown to have good agreement as assessed by Bland-Altman
analysis and intraclass correlation coefficient: 0.95, 0.83, and 0.96, respectively. It was
concluded that LC-MS/MS, compared with a labor-intensive extraction RIA, shows good
precision, sensitivity, and high accuracy for measuring female testosterone, DHEA, and AD
concentrations under various clinical conditions. LC-MS/MS, therefore, represents a
convenient and reliable assay for both clinical and research purposes, where androgen
measurement in women is required [11439].
To evaluate the role of physiologic levels of androgens and their precursors in the regulation
of body composition, energy and substrate metabolism and aerobic capacity in healthy,
cycling, premenopausal women it was evaluated 30 young (27 ± 1 year) premenopausal,
non-obese (23 ± 0.5 kg/m2), normally-cycling women, without clinical or chemical evidence of
hyperandrogenism or hyperinsulinemia, for parameters of total and regional body
composition, glucose tolerance, aerobic capacity and resting energy expenditure and
substrate oxidation. Serum was assayed for androgens and androgen precursors by
techniques optimized to assess the low androgen levels in this population. Higher serum
testosterone levels correlated with greater fat mass but not abdominal adiposity or other
metabolic/physiologic variables. Additionally, dehydroepiandrosterone (DHEA) was
negatively related to visceral fat content. Other serum androgens did not correlate with total
or regional adiposity, skeletal muscle mass, aerobic capacity, glucose tolerance, or resting
energy and substrate metabolism. It was concluded that this group of non-obese,
premenopausal women with no clinical or chemical evidence of hyperandrogenemia, serum
testosterone levels were positively related with fat mass, but not with abdominal adiposity;
whereas, DHEA was negatively related to visceral adiposity. The data suggest that within the
normal physiologic range, testosterone is a predictor of overall adiposity, but that this effect
does not appear to be associated with concomitant alterations in resting energy or substrate
metabolism that could predispose to weight gain [11440].
Androgen therapy is being increasingly used in the management of postmenopausal women.

The most common indication is to improve sexual function. The aim of one review was to
evaluate current knowledge pertaining to testosterone and sexual function in
postmenopausal women. The change of testosterone levels during the menopause transition
remains controversial. A correlation of endogenous testosterone levels and sexual function is
still inconclusive. A Cochrane Review and recent randomized control trials have, however,
consistently demonstrated that short-term testosterone therapy in combination with traditional
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hormone therapy regimens improves sexual function in postmenopausal women, particularly
surgically menopausal women with hypoactive sexual desire disorder. An adverse effect on
the lipid profile has been identified which appears to be mostly associated with oral
methyltestosterone. Data for other effects of testosterone and long-terms risks are lacking.
Testosterone may act in a variety of ways in different tissues. This is, however, an area that
requires further investigation. Thus, testosterone therapy is a promising option for treating
women with hypoactive sexual desire disorder after surgical menopause. Two remaining
questions need to be answer: who is most likely to benefit from testosterone therapy and
what are the long-term health risks? [06052].
Testosterone after use of chlorinated swimming pools
The goal of one study was to evaluate the associations between testicular hormones at
adolescence and the exposure to chlorination by-products when attending chlorinated
swimming pools. We obtained serum samples from 361 school male adolescents (aged 14-
18years) who had visited swimming pools disinfected with chlorine or by copper-silver
ionization. We analysed serum concentrations of inhibin B (two different assays), total and
free testosterone, sex hormone-binding globulin, luteinizing hormone (LH), follicle stimulating
hormone (FSH) and dehydroepiandrosterone sulphate (DHEAS). There were strong inverse
associations between serum levels of inhibin B (both assays) or of total testosterone,
adjusted or unadjusted for gonadotropins and the time adolescents had spent in indoor
chlorinated pools, especially during their childhood. Adolescents having attended indoor
chlorinated pools for more than 250 h before the age of 10 years or for more than 125 h
before the age of 7 years were about three times more likely to have an abnormally low
serum inhibin B and/or total testosterone (<10th percentile) than their peers who never
visited this type of pool during their childhood (odds ratio 2.83 and 3.67, respectively). Such
associations were not seen with free testosterone, LH, FSH and DHEAS or with the
attendance of outdoor chlorinated pools or of the copper-silver pool. Swimming in indoor
chlorinated pools during childhood is strongly associated with lower levels of serum inhibin B
and total testosterone. The absorption of reprotoxic chlorination by-products across the
highly permeable scrotum might explain these associations [11441].
Testosterone prohormones
Testosterone prohormones such as androstenedione, androstenediol, and

dehydroepiandrosterone (DHEA) have been heavily marketed as testosterone-enhancing
and muscle-building nutritional supplements for the past decade. Concerns over the safety of
prohormone supplement use prompted the United States Food and Drug Administration to
call for a ban on androstenedione sales, and Congress passed the Anabolic Steroid Control
Act of 2004, which classifies androstenedione and 17 other steroids as controlled
substances. As of January 2005, these substances cannot be sold without prescription.
Here, we summarize the current scientific knowledge regarding the efficacy and safety of
prohormone supplementation in humans. We focus primarily on androstenedione, but we
also discuss DHEA, androstenediol, 19-nor androstenedione, and 19-nor androstenediol
supplements. Contrary to marketing claims, research to date indicates that the use of
prohormone nutritional supplements (DHEA, androstenedione, androstenediol, and other
steroid hormone supplements) does not produce either anabolic or ergogenic effects in men.
Moreover, the use of prohormone nutritional supplements may raise the risk for negative
health consequences [06122].
Salivary testosterone (and testosterone to cortisol ratio)
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Salivary testosterone (T) and cortisol (C) concentrations were monitored across a sports
competition. Data were compared using two enzyme-immunoassay (EIA) methods and two
sample preparations to determine their influence on hormone concentrations. A group of
male athletes (n=19) provided a saliva sample the morning before and one day after (24h
post) an international rugby union match. Following an extraction procedure, the samples
were analysed for T and C concentrations using a commercial kit (CME) and an in-house
method (IHE). Raw samples (no extraction procedure) were also tested using the commercial
kit (CMR). There were no significant changes in T and C levels from pre to post competition
with each EIA method and sample preparation, but significant differences in T (IHE>CME>
CMR) and C (CMR)>IHE and CME) concentrations were seen when both samples were
pooled. Bland-Altman analyses confirmed the presence of fixed and proportional bias. Strong
and significant correlations were demonstrated between the IHE and CME measures of
salivary T and C. The T and C values from the raw and extracted samples were also strongly
correlated. The measurement of salivary T and C concentrations across an international
sports event was influenced by different EIA methods and sample preparations, but all
measures were strongly correlated with some bias. Both T and C were unresponsive to the
sports event, but within the group results large individual variation was seen [12106].
To prepare efficiently for competition, wrestlers usually train physically for a period of
approximately 12-20 weeks. Numerous physical qualities must be developed during this
period of preparation: aerobic fitness, maximal strength, muscular endurance, power, and
speed. However, numerous studies have concluded that it is difficult to concurrently develop
strength and aerobic fitness for several reasons, in particular antagonistic endocrine
variations. The study involved 15 elite junior wrestlers who trained at a sports training school
for 15 weeks. To investigate the effects of long-term training and to assess the relationships
between hormonal concentrations (salivary testosterone [T] and cortisol [C]) and
performance changes during simultaneous strength and aerobic fitness training, 6 saliva
samples and 3 physical tests and 2 measures of body composition were made during the
training period. Wrestlers had a significant increase (+1.5 kg) in body weight without changes
in percentage body fat. Apart from the 20-m maximal shuttle speed, all performances
increased significantly during the 15 weeks of training: maximum mechanical power output
(Pmax: +13 %), mean power during 30 seconds (Pmean: +11 %), bench press (+6 %), squat
(+23 %), power clean (+6 %), time to 3,000- and 30-m sprints (-3.6, -1.3 % respectively).
During the period that the C increased, there was no significant variation for the T. The T/C
ratio followed a variation pattern contrary to that of the C. It was found strong correlations
between salivary T, C, and T/C and the variation in explosive strength. The results suggest
that data about subjects' salivary C, T, and T/C may be employed to optimize the training
process for sports people who need to develop strength and aerobic fitness simultaneously
[12107].
It was developed a simple and sensitive method for the simultaneous determination of
testosterone (TES), cortisol (CRT), and dehydroepiandrosterone (DHEA) in saliva by
automated online in-tube solid-phase microextraction (SPME) coupled with liquid
chromatography-tandem mass spectrometry (LC-MS/MS) using a Discovery HS F5 column.
The optimum in-tube SPME conditions were 25 draw/eject cycles of 40 μL of sample at a
flow rate of 200 μL/min using a Supel-Q PLOT capillary column as an extraction device. The
extracted compounds were easily desorbed from the capillary by passage of the mobile
phase, and no carryover was observed. The in-tube SPME LC-MS/MS method showed good
linearity with correlation coefficients r ≥ 0.9998 for TES, CRT, and DHEA using their
respective stable isotope-labeled internal standards. The intra-day and inter-day precisions
(relative standard deviations) were below 4.9 and 8.5 percent (n=5), respectively. This
method was successfully utilized to analyze TES, CRT, and DHEA in saliva samples without
any other pretreatment or interference peaks, and the quantification limits (S/N = 10) of TES,
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CRT and DHEA were about 0.01, 0.03 and 0.29 ng/mL saliva, respectively. The recoveries of
these compounds spiked into saliva samples were each above 94 percent. This method was
applied to analyze changes in salivary TES, CRT, and DHEA levels resulting from stress and
fatigue load [12108].
Circadian rhythm of salivary testosterone

Circadian rhythms of serum testosterone concentrations in men have been shown, in
general, to be highest in the morning and lowest in the evening. Thus, the purpose of this
investigation was to determine the effects of acute resistance exercise upon the waking
circadian rhythm of salivary testosterone over 2 days (with or without resistance exercise).
The subjects included ten resistance-trained men (with at least 1 year of lifting experience). A
matched, randomized, crossover study design was used such that each subject was tested
under both the resistance exercise and control (no exercise) conditions. The resistance
exercise protocol consisted of ten exercises performed for three sets of ten repetitions
maximum with 2 min of rest between sets. Saliva sample 1 was collected at 0615 hours and
resistance exercise began immediately afterwards at approximately 0620 hours, and sample
2 was collected at 0700 hours, which corresponded approximately to a mid-exercise (or
control) time point. Saliva samples were then obtained every hour on the hour from 0800
hours until 2200 hours. No significant differences were observed between the exercise and
resting conditions for salivary testosterone, with the exception of a significant decrease at
0700 hours during the resistance exercise protocol. The results of this investigation indicate
that resistance exercise does not affect the circadian pattern of salivary testosterone
secretion over a 16-h waking period in resistance-trained men [01082].
In women
To compare the baseline free testosterone (T) and cortisol (C) concentrations of elite and
non-elite female athletes 18 females from different sports (track and field, netball, cycling,
swimming, bob skeleton) were monitored over a 12-week period. Baseline measures of
salivary free T and C concentrations were taken weekly prior to any training. The elites (n=9)
and non-elites (n=9) were classified as international and national level competitors,
respectively, with both groups matched by sport. The pooled free T concentrations of the
elites (87 pg/ml) were significantly higher than the non-elites (41 pg/ml) and consistently so
across all weekly time points. Pooled free C concentrations were also greater in the elite
group (2.90 ng/ml) than the non-elites (2.32 ng/ml). The pooled baseline T and C measures
were higher in elite female athletes than non-elites. Higher free T and C concentrations could
indicate a greater capacity for physical performance at higher work rates, which is
commensurate with the demands of elite sport. Speculatively, the T differences observed
could influence female behavior and thereby help to regulate sporting potential [12109].
A sensitive and rapid liquid chromatographic (LC) method for the simultaneous determination
of testosterone (T) and epitestosterone (E) in human urine samples has been developed and
elaborated. The ratio of the both steroids (T/E) in human urine is a widely used as doping
control indicator. A sample pretreatment by solid-phase extraction (SPE) after hydrolysis
using 36 percent hydrochloric acid for determination of total level of T has been applied.
Unconjugated (free) form of the both androgens was determined without hydrolysis steps,
what makes novelty of the method, because simplifies the proposed procedure. In turn, the
measurements of urinary free T and E provided the diagnostic information for excess adrenal
production of steroids. The proposed LC assay was evaluated by analyzing a series of urine
samples containing T, E and methyltestosterone (MT) as internal standard at the range of
concentration 2-300 ng/mL of both analyzed hormones. The proposed method was fully
validated for specificity, linearity, limits of detection and quantitation, precision and trueness
according to the current requirements concerning analytical methods. Interestingly, the
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developed LC method allows to obtain a sensitive enhancement with respect to UV detection
with the quantitation limit for T and E equaled 2 ng/mL. The method was selective and
reliable for identity and enables to detect changes of endogenous levels of T and E in urine
independently of fluctuations characteristic for both analyzed endogenous hormone level in
plasma [12110].
Testosterone deficiency produced by castration
Medical history
China
Castration has been practised for socio-cultural and political purposes since antiquity. Its
major purpose was to generate obedient slaves who were loyal to their masters or rulers
and, being infertile, could not create competing offspring. Set to guarding harems, they also,
and in larger numbers, obtained influential administrative and political positions as in China
and formed elite troops in Islamic countries. In different cultures and over centuries “wealthy
women preferred intercourse (or rather other sexual pleasures) with castrated slaves for a
good reason: there was no risk of pregnancy.”The earliest documentation of creating
eunuchs in China dates back to about 1300 BC. The Chinese eunuch system, with several
thousand's existing at a time, continued until the end of the imperial period in 1912. The last
Chinese eunuch, Sun Yaoting, died at the age of 94 in 1996. Only the fact that imperial
eunuchs could obtain high-ranking positions and considerable power as well as wealth
makes it plausible that adult men underwent this gruesome operation. It was performed by
”licensed surgeons” just outside the imperial court in Beijing by cutting off testes and penis.
About 25 percent of the volunteers did not survive this bloody operation. The severed
genitals were kept in a box, as shown vividly in the film “The Last Emperor”, and were
eventually buried with their owner. During the Ming Dynasty (1368-1644) eunuchs attained
outstanding influence and wealth. The prime example is represented by Liu Jin (1451-1510)
who is counted among the richest persons in history; he accumulated 449 750 kg of gold and
9 000 000 kg of silver, but eventually his criminal intrigues led to his execution. In the
nineteenth century there were still about 2000 eunuchs at the imperial court in Beijing. The
impact of peri- and postpubertal castration on the phenotype of these men was described
extensively by Wagenseil7 who in 1922 established an Anatomical Institute at the Chinese–
German Tung-Chi University in Shanghai where he examined a series of 31 Chinese
eunuchs aged 45–57 years. These eunuchs had no beard growth and sparse body hair and
21 of the 31 had developed kyphosis as a clear sign of osteoporosis [14016].
Egypt
Eunuchs probably already existed in ancient Egypt. From the times of the legendary Queen
Semiramis (about 800 BC) eunuchs were reported from Assyria and the system developed
and continued into the Islamic world in the Middle – East and North Africa. Over centuries,
slaves were deported from Sub-Saharan Africa to the Islamic cities and courts and many of
the slaves who survived the exhausting march through the desert were then castrated to
serve as laborers, guards, administrators and even soldiers. It is astonishing that these tasks
could be fulfilled without the anabolic effects of testosterone [14016].
Greece
In Greek mythology, castration already occurred among the first generation of gods. Gaea,
mother earth, grew out of the chaos and produced Uranos by parthenogenesis with whom
she then generated the titan Chronos. When Uranos prevented Gaea from creating children
with their son Chronos, she induced Chronos to castrate his father. Uranos’ testes, thrown
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into the sea, caused the water to foam and out of these bubbles the foam-born goddess of
love Aphrodite (Venus) was born. Quite extraordinary events in terms of reproductive
physiology! This episode has been depicted beautifully in a fresco by Giorgio Vasari (1511–
1574) in the Palazzo Vecchio in Florence [14016].
Medieval Europe
It has been estimated that the transatlantic deportation of Africans to the Americas between
1450 and 1870 comprised about 11.5 million people while the entire Islamic deportation of
slaves from Africa between 650 and 1920 amounted to 17 million people and several million
of these African slaves were castrated. This constant drain of manpower effectively
prevented economic and cultural development of Sub-Saharan Africa. In medieval times,
slaves were also exported from Europe to the Islamic countries. These slaves were mainly
from Eastern European (Slavic) and Central Asian countries. There were well-established
slave routes through Europe and Verdun in France enjoys the questionable historical fame of
having been the European center for castration of slaves on their way from the East to the
South at those times [14016].
Castration has also been practised as lawful punishment. In medieval Scandinavia,
castration combined with blinding was administered for high treason, especially when the
insurgent was a close relative whom one did not want to kill directly. As told in the Islendinga
Saga, Sturla of the Sturlungar Clan in Iceland castrated and blinded his rebelling relative
Oraekja Snorrason in 1236. When the Normans migrated south, they also introduced this
penal practice in the areas they invaded. When he established his reign in Britain after 1066,
William the Conqueror abolished the Anglo-Saxon death penalty and replaced it by castration
and blinding: “I also forbid that anyone shall be slain or hanged for any fault, but let his eyes
be put out and let him be castrated”. As a further example, when the Normans invaded Sicily,
King William III was castrated and blinded in 1194 after a rebellion against Emperor Henry
VI. This episode forms the historical background for Klingsor's castration in the Parsifal epos.
The Toulouse Law Codex of 1296 describes (and depicts) castration for high treason
[14016].
More recent events in Europe

Throughout the centuries castration was applied to beaten enemies by victorious soldiers for
revenge and as a measure to eliminate the enemies without outright killing. This continues
into recent times. When Italian troops invaded Ethiopia and lost the battle of Aduwa in 1896
supposedly 7000 Italian soldiers were castrated. As reported by Babtschenko, this still
happened on both sides during the Chechen War in the Caucasus in 1996 [14016].
Castration has also been reported as self-mutilation for religious reasons since ancient times
in order to make a life in chastity easier. The early church father Origines (186–254) is one of
the most prominent examples. In the eleventh to fourteenth centuries, the sect of the
Catharers with their strongholds in Southern France promulgated self-castration as part of a
“pure” life. More recently, castration was practised in Southern Russia among members of
the Scoptic sect founded in the eighteenth century and the medical consequences were
documented. The largest contemporary group of castrates is among the hijras in India who
also comprise persons with disorders of sexual development (DSD). They function as
professional well-wishers at birth rites and weddings and receive considerable financial
rewards. Several thousand of them exist [14016].
Castration has also been used as revenge for seduction and adultery through the centuries.
For example, Paris – presumably in the twelfth century BC and preceding the Trojan War –
has been reported to have castrated Peritanos after he had seduced his famous wife Helena.
The case of the great medieval theologian and philosopher Peter Abaelard (1079–1142) has
been celebrated in history and literature. As master of the cathedral school in Paris he
seduced one of his disciples, Heloise (1100–1164), whose uncle in revenge then had
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Abaelard castrated by paid criminals. Despite the lack of testosterone, one of the most
romantic love stories documented by literature developed. This type of revenge continues
into most recent times as demonstrated by an incident in Germany in 2011 when the father
of a 17-year-old girl castrated her 57-year-old lover. These people had migrated to Germany
from Kazakhstan and obviously brought their rules of self-justice with them. The German
court sentenced the father to 6 years imprisonment and Euros 80,000 penalty [14016].
Castration before puberty maintains the high voice of boys so that soprano and alto voices
with the acoustic volume of an adult male result. Such high-pitched voices were considered
desirable among music lovers, especially at times when women were not allowed to sing in
church or in operatic performances. Prepubertal castrates belonged to casts of operas in the
seventeenth and eighteenth centuries; in the Vatican choirs these voices could be heard until
the early twentieth century. Some of these castrates became famous soloists, such as Carlo
Farinelli (1705–1782) or Domenico Annibaldi (1705–1779). The middle Italian cities of Norcia
and Preci were a center for the operation on young boys. In the solitude of this hidden
Apennine valley, a surgical school had already been established in the thirteenth century and
the 30 family dynasties monopolizing the trade there guarantied utmost secrecy concerning
this illegal operation. Strangely enough, while castration was forbidden in the Vatican state,
which extended over most of middle Italy, it was not forbidden to employ castrated singers.
However, most of the thousands of prepubertal castrates lost their virility in vain as they did
not achieve the promised career as a singer, developed only mediocre voices and were
ridiculed by their contemporaries. An impression of the castrato voice, although of very low
recording quality, is preserved from the last Vatican castrato Alessandro Moreschi (1858–
1922) in one of the earliest gramophone recordings, made in 1902 (available today on CD).
Today countertenors applying a trained falsetto sing the castrato roles in, for example,
Händel operas, but their head voices only approximate those of seventeenth century castrati.
Another impression of the castratos’ enormous artistic talents is provided by the recordings
of the mezzo-soprano Cecilia Bartoli who trained her voice to sing the extremely demanding
arias by Nicola Porpora (1686–1768), Georg Friedrich Händel (1685–1759) and others
[14016].
Prepubertal castration provides an involuntary experiment on the influence of testosterone on
longevity. A retrospective comparison of the life expectancy of singers born between 1580
and 1858 and castrated before puberty, in order to preserve their high voices, to intact
singers born at the same time did not reveal a significant difference between the lifespan of
castrated intact singers (66 ± 14 vs 64 ± 14). This would imply that the presence or absence
of normal male testosterone levels at and after the age of puberty has no influence on life
expectancy [14016].
Biological action
The anabolic hormone testosterone induces muscle hypertrophy, but the intracellular
mechanisms involved are poorly known. It was addressed the question whether signal
transduction pathways other than the androgen receptor (AR) are necessary to elicit
hypertrophy in skeletal muscle myotubes. Cultured rat skeletal muscle myotubes were
preincubated with inhibitors for ERK1/2 (PD98059), PI3K/Akt (LY294002 and Akt inhibitor
VIII) or mTOR/S6K1 (rapamycin), and then stimulated with 100 nM testosterone. The
expression of α-actin and the phosphorylation levels of ERK1/2, Akt and S6K1 (a
downstream target for mTOR) were measured by Western blot. mRNA levels were evaluated
by real time RT-PCR. Myotube size and sarcomerization were determined by confocal
microscopy. Inhibition of AR was assessed by bicalutamide. Testosterone-induced myotube
hypertrophy was assessed as increased myotube cross-sectional area (CSA) and increased
alpha-actin mRNA and alpha-actin protein levels, with no changes in mRNA expression of
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atrogenes (MAFbx and MuRF-1). Morphological development of myotube sarcomeres was
evident in testosterone-stimulated myotubes. Known hypertrophy signaling pathways were
studied at short times: ERK1/2 and Akt showed an increase in phosphorylation status after
testosterone stimulus at 5 and 15 min, respectively. S6K1 was phosphorylated at 60 min.
This response was abolished by PI3K/Akt and mTOR inhibition but not by ERK1/2 inhibition.
Similarly, the CSA increase at 12 h was abolished by inhibitors of the PI3K/Akt pathway as
well as by AR inhibition. These results suggest a crosstalk between pathways involving fast
intracellular signaling and the AR to explain testosterone-induced skeletal muscle
hypertrophy [13189].
The mechanisms by which AAS improve athletic performance are not fully understood.
Testosterone administration increases skeletal muscle mass by inducing the hypertrophy of
both type I and 2 fibers; testosterone does not change the absolute number or the relative
proportion of type 1 and 2 fibers. Testosterone administration increases the number of
muscle progenitor cells (satellite cells), which contribute to muscle fiber hypertrophy.
Testosterone promotes myogenic differentiation of muscle progenitor cells.Upon binding to
its cognate androgen receptor (AR), the liganded AR associates with beta-catenin and other
proteins, and the complex translocates into the nucleus where it binds transcription factor-4
and activates a number of Wnt target genes, including follistatin. Follistatin blocks the effects
of a number of transforming growth factor beta family members, including myostatin and
activins, and plays an essential role in mediating testosterone’s effects on myogenic
differentiation. Most of the anabolic effects of testosterone appear to be mediated through
AR-signaling. Testosterone stimulates circulating growth hormone (GH) and IGF-1, although
circulating GH is not essential for mediating testosterone’s effects on muscle mass.
However, IMIGF-1 receptor signaling plays an important role in mediating the effects of
testosterone on myogenesis. The conversion of testosterone to dihydrotestosterone by
steroid 5-alpha reductase is not essential for mediating its effects on the muscle.
Testosterone increases maximal voluntary strength and leg power, but does not increase
specific force. Testosterone also promotes mitochondrial biogenesis and quality control (QC),
and increases net oxygen delivery to the tissue by increasing red cell mass and tissue
capillarity. Testosterone also increases the circulating levels of 2, 3 biphosphoglycerate,
which shifts the oxygen:hemoglobin curve to the left thereby facilitating oxygen unloading
from oxyhemoglobin (HbO2). The observations that testosterone improves neuromuscular
transmission and upregulates acetyl cholinesterase expression in the frog hind limb model,
have led to speculation that testosterone may reduce reaction time, which may contribute to
improved performance in sprint events or in sports requiring high level of hand-eye
coordination such as baseball. Testosterone administration may also affect mood and
motivation, which may indirectly affect athletic performance [14017].
Anabolic steroids are performance enhancers, this being particularly apparent in women,
although there is a high risk of virilization despite the favourable myotrophic-androgenic
dissociation that many xenobiotic steroids confer. Modulation of androgen receptor
expression appears to be key to partial dissociation, with consideration of both intracellular
steroid metabolism and the topology of the bound androgen receptor interacting with co-
activators. An anticatabolic effect, by interfering with glucocorticoid receptor expression,
remains an attractive hypothesis. Behavioural changes by non-genomic and genomic
pathways probably help motivate training. It is important not to exaggerate the medical risks
associated with their administration for sporting or bodybuilding purposes but to emphasize
to users that an attitude of personal invulnerability to their adverse effects is certainly
misguided [08064].
Androgens are modulators of skeletal muscle adaptation and regeneration processes. The
control of satellite cell activity is a key mechanism during this process. In this study, it was
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analyzed the ability of dihydrotestosterone (DHT) and anabolic steroids to induce and
modulate the differentiation of myoblastoma cells toward myotubes. Myoblastoma cells were
dose-dependently treated with DHT and anabolic steroids. The time-dependent effects on
differentiation were measured and correlated with the expression of genes involved in the
regulation of satellite cell activity. The distribution of myoblastoma cells within the cell cycle
was measured by flow cytometry and differentiation by creatine kinase (CK) activity. Gene
expression was analyzed using quantitative real-time PCR and confocal microscopy. The
treatment with DHT and anabolic steroids resulted in a stimulation of myoblastoma cell
proliferation and CK activity. The antiandrogen flutamide was able to antagonize this effect.
The expression of the androgen receptor, SOX8, SOX9, Delta, Notch, myostatin, and paired
box gene7 (Pax7) was modulated by androgens. The treatment with DHT and anabolic
steroids resulted in a strong stimulation of myostatin expression not only in undifferentiated
cells but also in myotubes. The stimulation could be antagonized by flutamide. The
expression of Pax7 was detectable in myoblastoma cells early after treatment with DHT. The
results demonstrate that the key mechanisms of satellite cell differentiation are modulated by
androgens. Androgens stimulate the proliferation of myoblastoma cells, accelerate the
process of differentiation, and increase the expression of myostatin in undifferentiated and
differentiated cells [08065].
Important mechanisms behind the myotrophic effects of testosterone were uncovered both in
athletes using steroids for several years and in short-term controlled studies. Both long-term
and short-term steroid usage accentuates the degree of fibre hypertrophy in human skeletal
muscle by enhancing protein synthesis. A mechanism by which testosterone facilitates the
hypertrophy of muscle fibres is the activation of satellite cells and the promotion of
myonuclear accretion when existing myonuclei become unable to sustain further
enhancement of protein synthesis. Interestingly, long-term steroid usage also enhances the
frequency of fibres with centrally located myonuclei, which implies the occurrence of a high
regenerative activity. Under the action of testosterone, some daughter cells generated by
satellite cell proliferation may escape differentiation and return to quiescence, which help to
replenish the satellite cell reserve pool. However, whether long-term steroid usage induces
adverse effects of satellite cells remains unknown. Testosterone might also favour the
commitment of pluripotent precursor cells into myotubes and inhibit adipogenic
differentiation. The effects of testosterone on skeletal muscle are thought to be mediated via
androgen receptors expressed in myonuclei and satellite cells. Some evidence also suggests
the existence of an androgen-receptor-independent pathway. Clearly, testosterone abuse is
associated with an intense recruitment of multiple myogenic pathways. This provides an
unfair advantage over non-drug users. The long-term consequences on the regenerative
capacity of skeletal muscle are unknown [08066].
There is strong evidence that androgen administration in men increases skeletal muscle
mass, maximal voluntary strength and muscle power. However, there is no good
experimental evidence to support the presumption that androgen administration improves
physical function or athletic performance. Androgens do not increase specific force or whole
body endurance measures. The anabolic effects of testosterone on the skeletal muscle are
mediated through androgen receptor signaling. Testosterone promotes myogenic
differentiation of multipotent mesenchymal stem cells and inhibits their differentiation into the
adipogenic lineage. Testosterone binding to androgen receptor induces a conformational
change in androgen receptor protein, causing it to associate with beta-catenin and TCF-4
and activate downstream Wnt target genes thus promoting myogenic differentiation. The
adverse effects of androgens among athletes and recreational bodybuilders are under
reported and include acne, deleterious changes in the cardiovascular risk factors, including a
marked decrease in plasma high-density lipoproteins (HDL) cholesterol level, suppression of
spermatogenesis resulting in infertility, increase in liver enzymes, hepatic neoplasms, mood
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and behavioral disturbances, and long term suppression of the endogenous hypothalamic-
pituitary-gonadal axis. Androgens are often used in combination with other drugs which may
have serious adverse events of their own [08067].
One study examined the anabolic-hormone response to carbohydrate (CHO)

supplementation at rest and after resistance exercise. Nine recreationally trained men
randomly underwent 4 testing conditions: rest with placebo, rest with CHO, resistance
exercise with placebo, and resistance exercise with CHO. The resistance-exercise protocol
was four sets of Smith machine squats with a 10-repetition-maximum load, with 90-s rests
between sets. Participants then consumed either a placebo or CHO (24 % CHO, 1.5 g/kg)
drink. Blood was taken before exercise, immediately after testing, and then 15, 30, and 60
min after drink ingestion. Blood was analyzed for cortisol, glucose, insulin, and total
testosterone (TTST). Cortisol did not change significantly in any condition. Glucose
concentrations increased significantly. Insulin concentrations increased significantly under
resistant conditions. There were no significant changes in total testosterone concentrations
during rest with or without carbohydrate in kontrast to the exercise groups. It was concluded
that ingesting carbohydrates after resistance exercise resulted in decreased total
testosterone concentrations during recovery, although the mechanism is unclear [00868].
Many hormones (e.g. catecholamines, growth hormone, adrenal steroids, androgens, etc.)
influence health status, exercise/sport performances and the physiological adaptation to
exercise-related stress in athletes. In addition to classic reproductive and sexual effects (e.g.
sexual behaviour, penis growth, erection, secondary sexual characteristics and
spermatogenesis), and also depending on the role of CAG repeat polymorphism on
androgen receptors biological activity, endogenous testosterone exert a wide spectrum of
actions in males. Particularly, testosterone can differently influence body composition (e.g.
muscles growth, fat mass, bone density), central nervous system maturation and functions
(e.g. behavior characteristics, aggression and cognitive processes), endocrine and metabolic
pathways (glucose metabolism, insulin and leptin), muscles physiology and motor behaviour,
erythropoiesis, and adaptation to stress. A biologically normal testosterone secretion appears
therefore fundamental in males to guarantee both a physiological exercise adaptation and
safe sport participation. The reproductive system is highly sensitive to the effects of exercise-
related stress and the reproductive hormones may both increase and decrease after different
acute or chronic exercises. Exercise and sport participation may positively or negatively
influence andrological health status depending on the type, intensity and duration of
performed physical activity and on individual health status. In addition, prohibited substances
administration (e.g. androgenic-anabolic steroids, and so forth) in competitive and non-
competitive athletes represents the main cause of iatrogenic andrological diseases [12094].
Hypothalamic-pituitary-gonadal (HPG) axis
The hypothalamic-pituitary-gonadal (HPG) axis is regulated by a negative feedback

mechanism. Testosterone inhibits the frequency and amplitude of gonadotropin-releasing
hormone (GnRH) release from the hypothalamus and also the secretion of luteinizing
hormone (LH) from the pituitary. The Sertoli cells of the testes, in addition to stimulating
spermatogenesis, also secrete the glycoprotein hormone inhibin, which provides negative
feedback to the pituitary, inhibiting the secretion of follicle stimulating hormone (FSH).
Testosterone is converted to dihydrotestosterone (DHT) by 5-alpha-reductase enzymes or to
estradiol by P450 aromatase in target cells. Testosterone and DHT both bind to the
androgen receptor where they exert their biological effects. Approximately 20 percent of the
DHT in the circulation is produced directly by testicular secretion, with the remaining 80
percent being derived from conversion of testosterone in peripheral tissues. Target cells that
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contain 5-alpha-reductase are concentrated in the prostate, reproductive system, and skin.
Aromatase containing cells predominate in the liver, adipose tissue, and regions of the brain.
Total serum testosterone is composed of 3 components added together. Roughly half of
testosterone is bound to the carrier molecule sex hormone-binding globulin (SHBG), almost
all of the remainder is bound to albumin, and 1-2 percent is unbound or free. Testosterone
binds so tightly to SHBG that it is functionally unavailable to cells. In contrast, albumin-bound
testosterone dissociates readily, meaning that this component and the free component are
available to cells. The term ‘‘bioavailable testosterone’’ refers to a combination of the
albumin-bound and free portions [12095].
Brain
Sex steroids readily pass the blood-brain barrier, and receptors for them are abundant in
brain areas important for the regulation of emotions, cognition and behaviour. The sex
steroid receptors are ligand-activated transcription factors that bind to specific hormone
response elements in their target genes. There are two subtypes of estrogen receptors:
alpha and beta [08069]. In addition, several isoforms of each subtype have been reported
[08070]. The two estrogen receptor subtypes have comparable affinities to estradiol, but
many other ligands show preferential binding to one or the other of them. The two subtypes
also differ with respect to tissue distribution and coregulator interactions [08071, 08072]. The
human estrogen receptor α gene (ESR1) is located on chromosome 6q25.1 [08073] and
composed of 8 exons. A large number of polymorphisms in this gene have been identified,
none of which has as yet been shown beyond doubt to be functional. The human estrogen
receptor β gene (ESR2) is located on chromosome 14q22–24. The gene is composed of 8
exons [08074] and has several polymorphisms.
So far, there seems to be only a single subtype of the androgen receptor and the
progesterone receptor. Alternative splicing of the amino terminal of androgen receptor and
progesterone receptor genes, however, results in different isoforms displaying differences in
both expression and function [08075, 08076].
The human progesterone receptor gene (PGR) is located on chromosome 11q22-23 [077]
and composed of 8 exons. The receptor exists in 2 molecular forms, PR-A and PR-B; these
differ only at the amino terminus, with PR-B containing an additional stretch of amino acids.
This domain plays an important role in identifying target genes that can be activated by the
PR-B protein but not by the PR-A protein. The expression ratio of the 2 PR isoforms in the
brain varies during fetal development and as a result of the estrous cycle and also differs
between males and females. Administration of estrogen and progesterone has been shown
to influence the expression ratio, and some of these variations may therefore be induced by
these hormones. Notably, PR-A has recently been shown to play a key role in both hormone-
dependent and hormone-independent facilitation of female sexual behavior [08078].
The androgen receptor gene (AR) is located on chromosome Xq11-12 and composed of 8
exons [08079, 08080]. Preliminary evidence from several studies also suggests that AR
repeat polymorphisms may be of importance for interindividual differences in personality
traits.
Coregulators of sex steroid receptors play an important role for tissue-specific actions of sex
steroids. Several coregulators of importance for brain function have been identified [08081];
for example, both the steroid receptor coactivator gene and CREB-binding protein have been
shown to be involved in estrogen receptor–mediated effects on sexual behavior [08082].
Moreover, the coactivator estrogen receptor–associated protein 140, which interacts with
both estrogen receptor α and estrogen receptor β, displays its highest expression in the brain
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[083]. Another protein expressed in the brain that, among other tasks, serves as coactivator
for sex steroid receptors, is E6-associated protein (UBE3A) [08084].
The aromatase enzyme converts androgens into estrogens. The human aromatase gene
(CYP19) is located on 15q21.1 and contains several genetic variants [08085, 08086].
Notably, enzymes required for the synthesis of sex steroids, as well as for functionally active
sex steroid metabolites, are expressed locally within the brain; some of the sex steroids
present in the central nervous system are thus probably produced locally [08087]. For
example, within the brain, progesterone is metabolized to allopregnanolone [08088, 08089]
that may influence behaviour by interacting with GABAA receptors. The genes for the 5
alpha-reductase type 1 and type 2 enzymes, which are critical for this conversion, contain
functional polymorphisms [08090] that could be relevant for the study of psychiatric disorders
for which allopregnanolone has been attributed importance, such as premenstrual dysphoric
disorder.
Animal experiments have revealed that sex steroids have both an important early and
permanent influence on brain development and an ongoing influence on brain
neurotransmission in the adult organism. The influence exerted by sex steroids on animal
behaviour, including sexual activity and aggression, is exerted by both these mechanisms
[08091-08094]. That sex steroids also influence behavior in humans is shown by the
reduction in libido that often follows a decrease in serum sex steroids and by conditions such
as premenstrual dysphoric disorder (where the symptoms coincide with sex steroid
fluctuations in serum and can be abolished by means of ovariectomy or treatment with
ovulation inhibitors), postpartum depression, dysphoria induced by oral contraceptives and
changes in behavior induced by anabolic steroids [08094-08097]. The hypothesis that sex
hormones play a role in the regulation of mood and behavior also gains support from the fact
that a large number of psychiatric conditions, including depression, panic disorder,
generalized anxiety disorder, social phobia and eating disorders, are more prevalent in
women than in men. In contrast, alcoholism, attention-deficit hyperactivity disorder and
autism are more common in men. As well, with respect to normal personality traits, there are
subtle but clear differences between women and men at the group level (e.g. with respect to
anxiety-related traits). Similarly, certain aspects of cognitive abilities appear to differ slightly
between the sexes. Autism is a disorder of particular interest in regard to the possible role of
sex steroids; there is evidence suggesting that subjects with autism are characterized by a
brain that, in certain aspects, may be regarded as unusually masculinized [08093, 08094].
Anabolic androgenic steroids at supratherapeutic doses seem to improve physical

appearance and the drug user becomes more bold and courageous. Investigations of the
possible neurochemical effects of AAS have focused partially on the monoaminergic
systems, which are involved in aggressive behaviours and the development of drug
dependence. In one study, it was administered nandrolone decanoate (3 or 15 mg/kg/day
for 14 days) and measured mRNA expression of dopaminergic and serotonergic receptors,
transporters and enzymes in the male rat brain using quantitative real-time polymerase
chain reaction. Expression of the dopamine D1-receptor transcript was elevated in the
amygdala and decreased in the hippocampus while the transcript level of the dopamine D4-
receptor was increased in the nucleus accumbens. No changes in transcriptional levels
were detected among the serotonin-related genes examined in this study. The altered
mRNA expression of the dopamine receptors may contribute to some of the behavioural
changes often reported in abusers of anabolic steroids of increased impulsivity, aggression
and drug-seeking [08098].
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Testosterone and aggressiveness
Aggressiveness is an ancestral behavior common to all animal species. Its
neurophysiological mechanisms are similar in all vertebrates. Males are generally more
aggressive than females. In this review, aggressive behavior in rodents, monkeys, and man
and the role of testosterone and brain serotonin levels have been considered. Interspecific
aggressiveness in rats has been studied considering the mouse-killing behavior; the neonatal
androgenization of females increases adult mouse-killing as does the administration of
testosterone in adults. Intraspecific aggressiveness was studied by putting two or more male
rats (or mice) in the same cage; the condition of subjection or dominance is influenced by
testosterone. In monkeys, testosterone is related to aggressiveness and dominance and,
during the mating season, increases in testosterone levels and aggressive attitude are
observed. In men, higher testosterone levels were obtained in perpetrators of violent crimes,
in men from the army with antisocial behaviors, in subjects with impulsive behaviors,
alcoholics and suicidals, in athletes using steroids, and during competitions. Aggressive and
dominant behavior are distinguished. Testosterone influences both of these, even if man is
usually inclined to affirm his power without causing physical damage. Testosterone receptors
are mainly in some hypothalamic neurons, where it is aromatized into estrogens, which
determine the increase in aggressiveness. A relation between testosterone levels and
diencephalic serotonin has been shown: in fact, the lack of serotonin increases aggressive
behaviors both in animals and man. Testosterone also increases ADH levels in the medial
amygdala, lateral hypothalamus, and preoptical medial area, involved in aggressive
behaviors [05079].
In addition to an action on metabolism, anabolic/androgenic steroids also increase sex drive

and mental acuity. If abused, such steroids can cause irritability, impulsive aggression, and
signs of major depression [Pearson H. Nature 2004; 431, 500-1], but the mechanisms that
produce these symptoms are unknown. One study investigates behavioral and
neurochemical alterations occurring in association with protracted (3-week) administration of
testosterone propionate (TP) to socially isolated (SI) and group-housed male and female
mice. Male but not female SI mice exhibit aggression that correlates with the down-regulation
of brain neurosteroid biosynthesis. However, in female mice, long-term TP administration
induces aggression associated with a decrease of brain allopregnanolone (Allo) content and
a decrease (approximately 40 %) of 5alpha-reductase type I mRNA expression. In spayed
mice treated with TP, restitution experiments with progesterone and estrogen normalize brain
Allo content and prevent aggression. Submicromolar doses of S-norfluoxetine (S-NFLX) that
are insufficient to inhibit serotonin reuptake selectively increase brain Allo content and
abolish TP-induced aggression. The results support the view that TP-induced aggressive
behavior is the result of a TP-mediated neurosteroid biosynthesis down-regulation that can
be reversed by the S-NFLX-induced increase of brain Allo content [05080].
Mood
In the last decade, there has been a surge of new clinical trials studying the impact of
exogenous testosterone on mood. The results of these studies have been inconsistent.
Therefore a meta-analysis of controlled clinical trials using common depression rating scales
was performed. Sixteen trials with a total of 944 subjects met selection criteria. Meta-analysis
of data showed a significant positive impact of testosterone on mood. Subgroup analysis
showed a significant effect size. in the trials with a mean age of <60 years. However, the
effect size was not statistically significant in those trials with a mean age of >60 years. The
effect size in hypogonadal men was 4.19, whereas the result was not statistically significant
in eugonadal men. In addition, the effect size was larger in subthreshold depression
compared with major depression. Oral testosterone compared with oral dehydroepiandro-
sterone, testosterone gel, and intramuscular testosterone did not show a significant result. It
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was concluded that testosterone may be used as a monotherapy in dysthymia and minor
depression or as an augmentation therapy in major depression in middle-aged hypogonadal
men [14062].
Heart
The aim of one study was to investigate the effects of anabolic androgenic steroids on the
cardiac structure and the plasma lipoprotein profile isolated and in combination with
exercise. Transgenic mice with a human lipaemic phenotype (expressing cholesteryl ester
transfer protein on the LDL receptor knockout background) were used in this study.
Sedentary and exercised mice (treadmill running, five times per week for 6 weeks) were
treated with mesterolone (2 microg/g body weight) or vehicle (control-C) in the last 3 weeks.
Four groups were compared: (i) exercise + mesterolone (Ex-M), (ii) exercise + vehicle (Ex-
C), (iii) sedentary + mesterolone (Sed-M) and (iv) sedentary + vehicle (Sed-C). Arterial
blood pressure and body mass increased in all groups along time, but Sed-M reached the
highest values and Ex-C the lowest. Treatment with mesterolone increased total
cholesterol, triglyceride, low-density lipoprotein cholesterol (LDL-c) and very LDL-c (VLDL-
c) plasma levels. However, exercise blunted some of these deleterious effects by increasing
high-density lipoprotein cholesterol and decreasing LDL-c, VLDL-c and triglycerides.
Exercise training induced beneficial effects, such as physiological cardiomyocyte
hypertrophy, increase in myocardial circulation and decrease in cardiac interstitium.
However, mesterolone impaired such physiological gains and in addition increased troponin
T plasma levels both in sedentary and exercised mice. Thus, while mesterolone induced
pro-atherogenic lipoprotein profile and pathogenic cardiac hypertrophy, exercise
counteracted these effects and modified favourably both the lipoprotein profile and the
cardiac remodelling induced by mesterolone [08099].
Tendon
Matrix metallopeptidases (MMPs) are responsible for degradation of the extracellular matrix
components and tissue remodeling. To achieve a better understanding of anabolic steroids
effects in rat tendon, MMP-2 activity in the proximal and distal regions of the calcanear
tendon and proximal, intermediate and distal region of superficial and deep flexor tendons
after mechanical load exercise associated with anabolic androgenic steroids was
investigated. In animals both proximal and distal regions of the calcanear tendon showed
the lowest MMP-2 concentration and the highest proportion in MMP-2 active form. The
intermediate region of the superficial flexor tendons differed significantly from the proximal
and distal regions with higher proportion of active MMP-2 in the sedentary group. The
proportion of active MMP-2 decreased in the proximal region of the calcanear tendon. The
differences in the response to exercise and androgenic anabolic steroid treatment are a
result of distinct metabolism and recruitment of these tendon regions in the exercise
program employed in this study [08100].
Muscle
One presentation discussed investigations into testosterone's effects on muscle protein

metabolism. Protein synthesis is the principal end point, but protein breakdown and the
availability of an amino acid pool are important to the process of net muscle protein
synthesis. The effects of other hormones – including growth hormone, oxoandrolone (a
synthetically derived testosterone), and androstenedione – on muscle protein synthesis also
were discussed. Effects in both normal and elderly men are considered [00077].
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Athletes have long supported the concept that anabolic steroids increase skeletal muscle
mass. However, it was only recently that both testosterone and its synthetic analogue,
oxandrolone, were proven capable of inducing myotrophic effects in postabsorptive human
skeletal muscle. These findings have provided the physiological evidence that anabolic
steroids deserve attention in the clinical arena as a pharmacological intervention against
losses in lean body mass associated with age, disease, trauma and burn injury. However, we
are lacking in vivo molecular evidence that would directly or indirectly link androgens and the
androgen receptor with increases in skeletal muscle mass. Clearly, a need exists to link in
vivo and in vitro studies from both the physiological and molecular arena as they relate to
androgens and the control and regulation of skeletal muscle mass. In one brief review, newly
discovered information and emerging theories relating to the direct, indirect, priming and
antiglucocorticoid action of androgens on skeletal muscle will be presented [00078].
The growing and indiscriminate use of high doses of anabolic androgenic steroid (AAS)
among youth and athletes has raised serious concerns about its hepatotoxic effects. Now the
influence of AAS in the nuclear phenotype of hepatocytes was investigated in sedentary and
trained mice heterozygous for the human CETP (cholesteryl ester transfer protein) transgene
and for LDL-receptor null allele (CETP+/-LDLr+/-) by image analysis. Sedentary blank control
mice showed the lowest chromatin condensation and highest Feulgen-DNA content,
polyploid nuclei frequency, nuclear area and perimeter, suggesting gene activation.
Contrarily, exercised mice showed a highest chromatin condensation, and a significant
decrease of Feulgen-DNA content and decreased frequency of polyploid nuclei, which
suggest gene silencing. Image analysis of the nuclear phenotype offered a coherent
descriptive picture of the changing patterns of chromatin organization, which were shown to
be congruent with the levels of Feulgen-DNA content, geometric nuclear parameters and
hepatocyte activity. In the study, the image analysis permitted the monitoring of the nuclear
response to mesterolone and physical exercise action in liver cells, the molecular mechanism
of which is in prospect [08101].
Use of testosterone enanthate has been shown to significantly increase strength within 6-12
weeks of administration, however, it is unclear if the ergogenic benefits are evident in less
than 6 weeks. The two objectives of this study were to establish if injection of 3.5 mg/kg
testosterone enanthate once per week could increase muscular strength and cycle sprint
performance in 3-6 weeks; and if the WADA-imposed urinary T/E ratio of 4:1 could identify all
subjects being administered 3.5 mg/kg testosterone enanthate. Sixteen healthy young men
were match-paired and were assigned randomly in a double-blind manner to either a
testosterone enanthate or a placebo group. All subjects performed a structured heavy
resistance training program while receiving either testosterone enanthate (3.5 mg/day) or
saline injections once weekly for 6 weeks. One repetition maximum (1RM) strength
measures and 10-second cycle sprint performance were monitored at the pre (week 0), mid
(week 3), and post (week 6) time points. Body mass and the urinary T/E ratio were measured
at the pre (week 0) and post (week 6) time points. When compared with baseline (pre), 1RM
bench press strength and total work during the cycle sprint increased significantly at week 3
and week 6 in the testosterone enanthate group, but not in the placebo group. Body mass at
week 6 was significantly greater than at baseline in the testosterone enanthate group, but not
in the placebo group. Despite the clear ergogenic effects of testosterone enanthate in as little
as 3 weeks, 4 of the 9 subjects in the testosterone enanthate group (approximately 44 %) did
not test positive to testosterone under current WADA urinary T/E ratio criteria [07087].
Testosterone supplementation increases muscle mass primarily by inducing muscle fiber

hypertrophy; however, the mechanisms by which testosterone exerts its anabolic effects on
the muscle are poorly understood. The prevalent view is that testosterone improves net
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muscle protein balance by stimulating muscle protein synthesis, decreasing muscle protein
degradation, and improving the reutilization of amino acids. However, the muscle protein
synthesis hypothesis does not adequately explain testosterone-induced changes in fat mass,
myonuclear number, and satellite cell number. It was postulate that testosterone promotes
the commitment of pluripotent stem cells into the myogenic lineage and inhibits their
differentiation into the adipogenic lineage. The hypothesis that the primary site of androgen
action is the pluripotent stem cell provides a unifying explanation for the observed reciprocal
effects of testosterone on muscle and fat mass [03080].
Testosterone supplementation increases fat-free mass (FFM) and muscle size in healthy,
hypogonadal men, human immunodeficiency virus (HIV)-infected men with low testosterone
levels, and older men with low testosterone levels. However, there are striking qualitative and
quantitative differences in this anabolic response to testosterone administration among the
various studies. Of the six, placebo-controlled, clinical trials evaluating testosterone
administration in HIV-infected men, two reported no significant difference in the change in
FFM between the placebo- and testosterone-treated men. Among the studies that did
demonstrate significant gains in lean body mass (LBM) after androgen administration, the
magnitude of increase varied considerably. In one study in which HIV-infected men with low
testosterone levels were treated with placebo or testosterone patches, the mean gain in LBM
in the testosterone-treated men was 1.4 kg, whereas in another study 100 mg testosterone
enanthate (TE) weekly was associated with a larger (mean 2.9 kg) gain in LBM. Studies of
testosterone supplementation in older men have also demonstrated similar variability in
results. It was reported no significant gains in LBM during testosterone administration, others
found greater gains in older men treated with testosterone than in those treated with placebo.
These data are similar to anecdotal reports that athletes using androgenic steroids differ
significantly in their anabolic response to these agents. It is not known whether these varying
responses in HIV-infected and older men in different studies are because of differences in
testosterone dose, baseline characteristics of the subjects, or methods of body composition
assessment [03081].
Considerable heterogeneity exists in the anabolic response to androgen administration;

however, the factors that contribute to variation in an individual's anabolic response to
androgens remain unknown. We investigated whether testosterone dose and/or any
combination of baseline variables, including concentrations of hormones, age, body
composition, muscle function, and morphometry or polymorphisms in androgen receptor
could explain the variability in anabolic response to testosterone. Fifty-four young men were
treated with a long-acting gonadotropin-releasing hormone (GnRH) agonist and one of five
doses (25, 50, 125, 300, or 600 mg/wk) of testosterone enanthate (TE) for 20 wk. Anabolic
response was defined as a change in whole body fat-free mass (FFM) by dual-energy X-ray
absorptiometry (DEXA), appendicular FFM (by DEXA), and thigh muscle volume (by
magnetic resonance imaging) during TE treatment. We used univariate and multivariate
analysis to identify the subset of baseline measures that best explained the variability in
anabolic response to testosterone supplementation. The three-variable model of TE dose,
age, and baseline prostate-specific antigen (PSA) level explained 67% of the variance in
change in whole body FFM. Change in appendicular FFM was best explained (64% of the
variance) by the linear combination of TE dose, baseline PSA, and leg press strength,
whereas TE dose, log of the ratio of luteinizing hormone to testosterone concentration, and
age explained 66 percent of the variation in change in thigh muscle volume. The models
were further validated by using Ridge analysis and cross-validation in data subsets. Only the
model using testosterone dose, age, and PSA was a consistent predictor of change in FFM
in subset analyses. The length of CAG tract was only a weak predictor of change in thigh
muscle volume and lean body mass. Hence, the anabolic response of healthy, young men to
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exogenous testosterone administration can largely be predicted by the testosterone dose
[03081].
Dose-dependently increase of maximal voluntary strength and leg power

Testosterone supplementation in men increases fat-free mass, but whether measures of
muscle performance, such as maximal voluntary strength, power, fatigability, or specific
tension, are improved has not been determined. Furthermore, the extent to which these
measures of muscle performance are related to testosterone dose or circulating
concentration is unknown. To examine the relationship between testosterone dose and
muscle performance, 61 healthy, eugonadal young men (aged 18-35 years) were
randomized to 1 of 5 groups, each receiving a long-acting GnRH agonist to suppress
endogenous testosterone production plus weekly injections of 25, 50, 125, 300, or 600 mg
testosterone enanthate for 20 wk. These doses produced mean nadir testosterone
concentrations of 253, 306, 542, 1345, and 2370 ng/dl, respectively. Maximal voluntary
muscle strength and fatigability were determined by a seated leg press exercise. Leg power
was measured using a validated leg power instrument. Specific tension was estimated by the
ratio of one repetition maximum muscle strength to thigh muscle volume determined by
magnetic resonance imaging. Testosterone administration was associated with a dose-
dependent increase in leg press strength and leg power, but muscle fatigability did not
change significantly during treatment. Changes in leg press strength were significantly
correlated with total and free testosterone as was leg power, but not muscle fatigability.
Serum IGF-I concentrations were not significantly correlated with leg strength, power, or
fatigability. Specific tension did not change significantly at any dose. It was concluded that
the effects of testosterone on muscle performance are specific; it increases maximal
voluntary strength and leg power, but does not affect fatigability or specific tension. The
changes in leg strength and power are dependent on testosterone dose and circulating
testosterone concentrations and exhibit a log-linear relationship with serum total and free
testosterone. Failure to observe a significant testosterone dose relationship with fatigability
suggests that testosterone does not affect this component of muscle performance and that
different components of muscle performance are regulated by different mechanisms [03082].
Testosterone-induced muscle hypertrophy and satellite cells

Testosterone supplementation increases muscle mass in healthy hypogonadal men, older
men with low testosterone levels, and men with chronic illnesses and low testosterone levels.
The anabolic effects of testosterone on muscle mass are dose and concentration dependent.
Testosterone-induced increase in muscle mass is associated with a dose-dependent
increase in cross-sectional areas of both type I and type II muscle fibers but is not attended
by a change in muscle fiber number. However, the mechanisms by which testosterone
increases muscle mass are not well understood. The prevalent dogma for the past 50 years
has been that testosterone increases muscle mass by stimulating fractional muscle protein
synthesis. Indeed, lowering of circulating testosterone concentrations by administration of a
gonadotropin-releasing hormone (GnRH) agonist is associated with a reduction in fractional
muscle protein synthesis; conversely, testosterone replacement in healthy hypogonadal men
increases muscle protein synthesis. However, in a study it was noted that testosterone-
induced muscle fiber hypertrophy is associated with a dose-dependent increase in
myonuclear number. It would be difficult to explain this increase in myonuclear number if the
sole effect of testosterone were on muscle protein synthesis. Muscle growth during postnatal
development or hypertrophy is dependent on the addition of myonuclei to muscle fibers.
Because the nuclei within the muscle fibers are postmitotic, new myonuclei must be
contributed by the satellite cells that are outside the muscle fiber. Inhibition of satellite cell
proliferation by gamma irradiation at doses that do not produce overt cellular damage
prevents the muscle growth and increase in myonuclear number that follow muscle atrophy
due to hindlimb suspension. Muscle remodeling and repair following injury often involve
1300
satellite cell replication and recruitment of new stem cells into the myogenic cell lineage.
Similarly, the hypertrophy of levator ani muscle in the female rat induced by exogenous
testosterone administration is associated with satellite cell entry into the cell cycle and
proliferation. Taken together, these animal data suggest that an increase in satellite cell
number is an important antecedent of an increase in myonuclear number and muscle fiber
adaptation leading to hypertrophy. However, we do not know whether similar changes in
satellite cell number are also observed in testosterone-treated humans. Furthermore,
previous reports studied satellite cells in the levator ani muscle of rodents, which differs
significantly from skeletal muscle in the magnitude of response to castration and testosterone
supplementation. Therefore, in this study, we tested the hypothesis that testosterone-induced
muscle fiber hypertrophy would be associated with an increase in satellite cell number in the
skeletal muscle of testosterone-treated men. Because the gains in muscle mass during
testosterone supplementation are correlated with testosterone dose and concentrations, it
was hypothesized that the change in satellite cell number would also be correlated with
testosterone dose and concentrations [03083].
It was hypothesized that testosterone (T)-induced increase in muscle fiber size is associated
with a dose-dependent increase in satellite cell number. It was quantitated satellite cell and
myonuclear number by using direct counting and spatial orientation methods in biopsies of
vastus lateralis obtained at baseline and after 20 weeks of treatment with a gonadotropin-
releasing hormone agonist and a 125-, 300-, or 600-mg weekly dose of T enanthate. T
administration was associated with a significant increase in myonuclear number in men
receiving 300- and 600-mg doses. The posttreatment percent satellite cell number, obtained
by direct counting, differed significantly among the three groups: the mean posttreatment
values (5.0 and 15.0 %) in men treated with 300- and 600-mg doses were greater than
baseline (2.5 and 2.5 %, respectively). The absolute satellite cell number measured by
spatial orientation at 20 week (1.5 and 4.0/mm) was significantly greater than baseline (0.3
and 0.6/mm) in men receiving the 300- and 600-mg doses. The change in percent satellite
cell number correlated with changes in total and free T concentrations. Satellite cell and
mitochondrial areas were significantly higher and the nuclear-to-cytoplasmic ratio lower after
treatment with 300- and 600-mg doses. It was concluded that T-induced muscle fiber
hypertrophy is associated with an increase in satellite cell number, a proportionate increase
in myonuclear number, and changes in satellite cell ultrastructure [03083].
In myocytes
In myocytes, testosterone binds to the intracellular androgen receptor (AR), initiating an
activation cascade with conformational changes and nuclear translocation of the AR-steroid
complex. Binding of the complex to androgen responsive elements (ARE) in the DNA results
in specific activation or repression of the transcription in target genes [12096].
Breast
Gynecomastia is a common finding in adolescent men. While gynecomastia has long been
attributed to an imbalance between estrogen and androgen concentrations, recent literature
has begun to illuminate other potential mechanisms for breast development in adolescent
men. Increased leptin levels, as well as human chorionic gonadotropin and luteinizing
hormone receptors on male breast tissue, may play a role. Newer treatment strategies, such
as the antiestrogen raloxifene, have shown promising results; however, further studies are
needed to determine long-term efficacy. As a result of the limited pharmaceutical treatment
options, many more adolescents are seeking surgical intervention. Careful attention should
be paid to both the breast and testicular examination. A detailed history should include an
inquiry regarding the use of illicit substances, anabolic-androgenic steroids, herbal products,
and medications. The impact of gynecomastia on the adolescent's mental health should be
1301
assessed. Reassurance remains the standard of care for physiologic gynecomastia [08102].
Liver
Anabolic steroid abuse is associated with a number of medical complications. Reported

hepatic complications include cholestasis, elevation of aminotransferases, jaundice, benign
hepatic adenomas, and rare cases of hepatocellular carcinoma. Histologic findings include
peliosis hepatis, a lesion characterized by hepatic sinusoidal dilatation that is often cystic.
Rupture of these cysts can cause fatal internal hemorrhage. The risk of androgen-associated
liver tumors appears to correlate with the cumulative androgen dose and the potency of the
steroid used [08103-08110].
It was described a case of a 27-year-old male bodybuilder with multiple hepatic adenomas
induced by anabolic steroids. He initially presented with tumor hemorrhage and was treated
with left lateral hepatic segmentectomy. Regression of the remaining tumors was observed
with cessation of steroid use. However, 3 years and a half after his initial hepatic
segmentectomy, he presented with recurrent tumor enlargement and intraperitoneal
hemorrhage in the setting of steroid abuse relapse. This was the first reported case of
hepatic adenoma re-growth with recidivistic steroid abuse, complicated by life-threatening
hemorrhage [08111].
Fat and fat metabolism
Many, but not all, studies of testosterone replacement in healthy, young, hypogonadal men
have reported a decrease in fat mass after testosterone supplementation. Studies that
recruited lean, young, hypogonadal men have not demonstrated significant changes in fat
mass. It was also reported that testosterone supplementation in middle-aged men with low to
low-normal testosterone concentrations is associated with a significant reduction in the
visceral fat cross-sectional area, plasma insulin levels, and blood glucose concentrations and
an improvement in insulin sensitivity. Long-term, placebo-controlled, randomized trials of
testosterone replacement in older men have also been in agreement that testosterone
replacement in older men with low-normal testosterone concentrations is associated with
gains in fat-free mass and a loss of fat mass. To determine the effects of graded doses of
testosterone on regional adipose tissue distribution, we randomized 61 healthy men, aged
18–35 years, to receive 20 weeks of treatment with a long-acting GnRH agonist (to suppress
endogenous T secretion) plus 1 of 5 weekly intramuscular doses (25, 50, 125, 300, or 600
mg) of testosterone enanthate. Energy and protein intake were standardized. Fat mass was
measured by underwater weighing, dual energy x-ray absorptiometry (DEXA), and MRI of
the abdomen and thigh. The combined administration of a GnRH agonist plus graded TE
doses resulted in serum nadir testosterone levels of (mean ± SE) 221 ± 51, 295 ± 32, 597 ±
89, 1405 ± 230, and 2205 ± 241 ng/dL, respectively. The change in whole-body fat mass was
inversely correlated with testosterone dose and concentration. Whole-body fat mass,
measured by underwater weighing and DXA, increased significantly in men who received 25
and 50 mg/week of TE and decreased in those who received 300 and 600 mg/week. There
was a significant reduction in both appendicular and truncal fat at the 300 and 600 mg/week
doses but a significant increase at the 25 and 50 mg/week doses. The absolute change in fat
mass was not significantly different between the trunk and the appendices. The
subcutaneous as well as the intermuscular fat mass decreased significantly in men who
received the 300- and 600-mg doses. The absolute changes in subcutaneous and
intermuscular fat mass were not significantly different. There was a significant decrease in
intra-abdominal fat volume at the 125-mg (18 ± 41 cm3 decrease) and 600-mg (46 ± 39 cm3
decrease) doses. The changes in both intra-abdominal and intermuscular fat mass were
negatively correlated with testosterone dose. The changes in visceral fat mass were also
1302
negatively correlated with serum testosterone concentration. Therefore, changes in
testosterone concentration among healthy young men are associated with dose-dependent
changes in whole-body fat mass that reflect changes in both the truncal and appendicular fat
depots [03078].
It was studied the uptake and turnover of triglycerides from the peripheral and abdominal
subcutaneous fat depots after the administration of labeled oleic acid. In contrast to
dihydrotestosterone (DHT) and placebo, testosterone inhibited the uptake of label into the
abdominal subcutaneous fat, increased its turnover, and was associated with lower
lipoprotein lipase (LPL) activity only in the abdominal subcutaneous fat but not in the femoral
subcutaneous fat. Androgens increase insulin-independent glucose uptake and modulate
LPL activity [03078].
Testosterone effects on plasma lipids

Testosterone effects on plasma lipids depend on the dose, route of administration, and type
of androgen (whether it is aromatizable). Supraphysiological doses of androgens, particularly
those that are administered orally (nonaromatizable androgens), undoubtedly lower plasma
HDL and increase low-density lipoprotein (LDL) cholesterol concentrations. A meta-analysis
of 20 studies has shown that healthy hypogonadal men have higher HDL cholesterol and that
physiological testosterone replacement lowers HDL modestly, by 4 mg/dL, on average, and
total cholesterol by 5 mg/dL and has no effect on plasma LDL or triglycerides. Testosterone
supplementation also lowers lipoprotein a and plasminogen activator inhibitor 1 (PAI-1).
Lower testosterone levels in men are associated with higher levels of dense LDL particles
and prothrombotic factors. A number of studies have been in agreement that the
physiological testosterone replacement of HIV-infected men with weight loss does not
significantly affect plasma lipid levels. Similarly, placebo-controlled, randomized controlled
trials of testosterone replacement in older men have not demonstrated significant changes in
plasma HDL cholesterol concentrations [03078].
Effect of testosterone on muscle protein synthesis
Muscle mass is dependent on the ratio of muscle protein synthesis (anabolism) to muscle
protein breakdown (catabolism). Trauma from surgery and limited movement of the knee
result in atrophy and breakdown due to a catabolic state and reduced anabolism of skeletal
muscle proteins. Testosterone increases myofibrillar protein synthesis and promotes
anabolism of muscle tissue. It also modulates the activity of immune, fibroblast, and
myogenic precursor cells, which are all involved in muscle regeneration. Furthermore,
testosterone administration increases lean tissue and maximal voluntary strength in a dose-
dependent manner. Animal models have shown that exogenous testosterone aids in muscle
regeneration following several types of injury, such as crush injuries, venom-induced muscle
injury, muscle disuse atrophy, and injury following muscle graft surgery. With testosterone,
successful regeneration of healthy mouse muscle can occur within 2–3 weeks of injury where
muscle strength can return to pre-injured values. Testosterone may also induce muscle
growth via growth hormone (GH) and insulin-like growth factor 1 (IGF-1). IGF-1 can stimulate
muscle protein synthesis and satellite cell activity and anabolic steroids increase both
circulating IGF-1 and muscle mRNA expression of IGF-1. Akt (protein kinase B) is a signaling
mediator of IGF-1 that can regulate muscle mass. However, it is also known that
testosterone can work outside of the Akt signaling pathway in order to maintain skeletal
muscle hypertrophy. It was studied key regulatory proteins such as Akt1, mTOR, and
FOXO3a, which have not been studied in concurrent states of muscle trauma and
testosterone administration. There is evidence that the Akt pathway regulates gene
transcription through the inactivation of a group of transcription factors (FOXOs) located in
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the nucleus. FOXO3a increases the transcription of atrophy related genes (atrogens),
decreases protein degradation, and results in muscle atrophy. Phosphorylation of FOXO3a
by phosphorylated Akt1 inactivates the transcription factor, releasing it from DNA and
resulting in translocation of the inactive FOXO3a to the cytosol. Phosphorylated Akt1 will also
activate the mTOR pathway to stimulate protein synthesis and result in hypertrophy.
Therefore, the Akt pathway may regulate both skeletal muscle protein synthesis and
degradation by different but complimentary mechanisms in response to testosterone. By
analyzing muscle tissue following an ACL rupture with or without testosterone administration,
we can learn more about the mechanism through which muscle adapts to trauma and how
testosterone affects this process. Therefore, we may learn more about testosterone’s
mechanism of action in skeletal muscle during the catabolic stimulus of bedrest and surgery.
In addition to understanding the possible mechanisms of testosterone, there may be
improved recovery time as measured by questionnaires and function tests [14763]
Low levels of testosterone

To investigate how suppression of endogenous testosterone during an 8-week strength
training period influences the activity of satellite cells and myonuclei 24 moderately trained
young men participated in this randomized, placebo-controlled, and double-blinded
intervention study. The participants were randomized to treatment with a GnRH analogue,
goserelin (n=12), which suppresses testosterone or placebo (n=10) for 12 weeks. The
strength training period of 8 weeks started after 4 weeks of treatment and included exercises
for all major muscles. Biopsies were obtained from the mid-portion of the vastus lateralis
muscle. Testosterone resting level in goserelin was 10-20 times lower compared with
placebo, and the training-induced increase in the level of testosterone was abolished in
goserelin. Training increased satellite cells number in type II fibres by 20 percent in placebo
and by 52 percent in goserelin, whereas the myonuclear number significantly increased by
12 percent in type II fibres in placebo and remained unchanged in goserelin. No changes in
satellite cells and myonuclei were seen in type I fibres in either group. Data from the
microarray analysis indicated that low testosterone affects the bone morphogenetic proteins
signalling, which might regulate proliferation vs. differentiation of satellite cells. It was
concluded that eight weeks of strength training enhances the myonuclear number in type II
fibres, and this is largely blocked by the suppression of testosterone. The data indicate that
low testosterone levels could reduce the differentiation of satellite cells to myonuclei via the
bone morphogenetic proteins signalling pathway, resulting in reduced increases in lean leg
mass [14764].
Effect of different doses of testosterone on lipid profile
To study the effect and time profile of different doses of testosterone enanthate on the blood
lipid profile and gonadotropins 25 healthy male volunteers aged 27-43 years were given 500
mg, 250 mg, and 125 mg of testosterone enanthate as single intramuscular doses of
Testoviron(®) Depot. Luteinizing hormone (LH), follicle-stimulating hormone (FSH), blood
lipid profile (total cholesterol, plasma [p-] low-density lipoprotein, p-high-density lipoprotein
[HDL], p-apolipoprotein A1 [ApoA1], p-apolipoprotein B, p-triglycerides, p-lipoprotein(a),
serum [s-] testosterone, and 25-hydroxyvitamin D3) were analyzed prior to, and 4 and 14
days after dosing. Testosterone and epitestosterone in urine (testosterone/epitestosterone
ratio) were analyzed prior to each dose after a washout period of 6-8 weeks. All doses
investigated suppressed the LH and FSH concentrations in serum. LH remained suppressed
6 weeks after the 500 mg dose. These results indicate that testosterone has a more profound
endocrine effect on the hypothalamic-pituitary-gonadal axis than was previously thought.
There was no alteration in 25-hydroxyvitamin D3 levels after testosterone administration
compared to baseline levels. The 250 and 500 mg doses induced decreased concentrations
1304
of ApoA1 and HDL, whereas the lowest dose (125 mg) did not have any effect on the lipid
profile. The single doses of testosterone produced a dose-dependent increase in serum
testosterone concentrations together with suppression of s-LH and s-FSH. Alterations in
ApoA1 and HDL were observed after the two highest single doses. It is possible that long-
time abuse of anabolic androgenic steroids will lead to alteration in vitamin D status.
Knowledge and understanding of the side effects of anabolic androgenic steroids are
important to the treatment and care of abusers of testosterone [14765].
Testosterone and insulin sensitivity
Cross-sectional epidemiological studies have reported a direct correlation between serum

testosterone concentrations and insulin sensitivity. Low testosterone levels are associated
with an increased risk of type 2 diabetes mellitus in men. However, the data from intervention
studies on the effects of testosterone replacement on insulin sensitivity are conflicting. It has
been reported that testosterone administration in replacement doses to middle-aged men
with low-normal testosterone levels improves insulin sensitivity and lowers serum insulin
levels. However, in a recent dose-response study in which we administered a range of
testosterone doses to healthy men, the insulin sensitivity index, measured by a frequently
sampled intravenous glucose tolerance test using the Bergman minimal model, did not
significantly change across a range of testosterone doses and concentrations that were
tested. However, the participants in that study were young and very lean; it is possible that,
in middle-aged and older HIV-infected men with higher fat mass, testosterone replacement
might improve insulin sensitivity by reducing fat mass. Testosterone effects on insulin
sensitivity depend on the dose. For instance, in one study the effects of surgical castration
and testosterone administration on insulin sensitivity were studied in adult male rats using
the insulin clamp method. Adult male rats became severely insulin resistant when they were
castrated. Physiological testosterone replacement in these castrated rats restored their
insulin sensitivity to levels seen in intact rats. Supraphysiological doses of testosterone made
the rats insulin resistant again. These beneficial effects of testosterone replacement on
insulin sensitivity were independent of free fatty acid concentrations [03078].
Effects of testosterone supplementation on inflammation markers
Testosterone replacement in young men does not affect inflammation markers. Thus, C-
reactive protein levels in healthy young men were unaffected by testosterone administration
across a range of testosterone doses. In another prospective study of older men, DHT or
human chorionic gonadotropin administration did not significantly affect high-sensitivity C-
reactive protein, soluble vascular cell adhesion molecule–1, or soluble intracellular adhesion
molecule levels [03078].
Testosterone effects on coagulation and fibrinolytic factors
In cross-sectional epidemiological studies, serum testosterone levels have been positively

correlated with tissue plasminogen activator and inversely correlated with PAI-1, fibrinogen,
alpha-2 anti-plasmin, and factor VIIc levels. Men with hypogonadism have low baseline
fibrinolytic activity, which is accounted for by an increased synthesis of PAI-1. There have
been isolated case reports of stroke and deep-vein thrombosis in young men who were
taking large amounts of androgenic steroids. However, the results of the intervention studies
with testosterone replacement have been less clear; testosterone administration increases
the circulating levels of both pro- and anticoagulant factors [03078].
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Serum testosterone and physiological effects
The earlier mentioned SHBG is the most important carrier protein for androgens. The dimeric
protein consists of two identical peptide chains of 370 amino acids. SHBG synthesis is
stimulated by oestrogen in the liver and decreased by androgens and anabolic steroids.
Together with serum albumin (binding 40–50 % of T), SHBG (binding ≈50–60 % of T) forms
circulating reservoir of T, balances the concentration of free fraction and decreases the rate
of metabolism in the liver. With respect to genetic variation, studies have revealed SNP
which alters SHBG binding affinity for T. Parallel to carrier proteins, there are transporter
proteins which are involved in the absorption, distribution and elimination of drugs by
participating to permeation of the drugs into cells and access of the drugs to their targets.
Genetic polymorphism has also been shown to occur at this phase of bioprocesses, of which
an example is the organic ion transporter OATP1B3 (encoded by SLCO1B3 gene) and its
two polymorphic variants which transport T with varying efficiencies [14450].
The actions of anabolic androgenic steroids are executed via various mechanisms. At
androgen receptor (AR) level, these mechanisms include indirect modulation of expression
by intracellular metabolism and direct effect on the AR topology, which leads to subsequent
interaction with coactivators and transcriptional activity. Human AR is a nuclear transcription
factor, belongs to the nuclear receptor superfamily and mediates male sexual differentiation
as well as the development and maintenance of sexual characteristics. The molecular
structure of AR is well characterised and comprises polymorphic N-terminal domain, a
central well-conserved DNA-binding domain and a C-terminal ligand-binding domain.
According to the literature, more than 300 mutations in the X linked AR gene result in
androgen-insensitivity syndrome, and most of the mutations in the ligand-binding domain
disrupt binding of the natural ligands dihydrotestosterone (DHT) and T [14450].
Testosterone deficiency
Testosterone deficiency (TD) afflicts approximately 30 percent of men aged 40-79 years, with
an increase in prevalence strongly associated with aging and common medical conditions
including obesity, diabetes, and hypertension. Clinical symptoms of TD include fatigue,
decreased libido, ED, and negative mood states. TD also is associated with changes in body
composition, including decreased lean body mass, increased fat mass, and decreased bone
mineral density. A significant increased risk of TD is noted in association with common
medical conditions such as obesity, type 2 diabetes mellitus (T2DM), and hypertension. In
addition, a strong relationship was observed between TD and the metabolic syndrom.
Further, recent studies in women with complete androgen insensitivity syndrome showed
increased body fat, abnormal values of cholesterol, and homeostasis model assessment of
insulin resistance (HOMA-IR), suggesting that disruption of androgen signaling in women
also is associated with metabolic disorders. Repletion of testosterone (T) in T-deficient men
with these co-morbidities may indeed reverse or delay their progression. Studies of
testosterone replacement therapy (TRT) on sexual function and performance vary in quality,
although findings are generally consistent. Most studies show that TRT increased sexual
awareness and arousal, erectile function, and the frequency of spontaneous erections, but is
less consistent in enhancing sexual behavior and performance. A number of TRT
preparations are currently available. Intramuscular injections of short-acting testosterone
derivatives achieve good serum concentrations within 2-3 days, with levels returning to
baseline in most men by 2 weeks, resulting in an injection schedule of 1-2 weeks. Topical
gels or patches provide a more stable serum-testosterone concentration over time than
injections. Patches currently available are associated with a high rate of skin reaction, and
1306
their use has been largely supplanted by testosterone gels. The main disadvantages of
testosterone gels are cost and a black box warning concerning transfer potential to women
and children. A long-acting injection formulation (testosterone undecanoate) is dosed every
10-12 weeks, and is available internationally. Testosterone pellets provide 3-6 months of
normal serum testosterone, and are placed subcutaneously in the gluteal region via an in-
office surgical implantation procedure under local anesthesia; this formulation also has some
disadvantages such as extrusion of pellets post surgical procedure. In addition, the genetic
background relating to the patient responsiveness to androgens, hence, androgen receptor
polymorphisms, are likely to play an inter-individual role. Adverse events that have been
definitively associated with treatment are reversible with cessation of treatment. These
include acne, gynecomastia, erythrocytosis, and edema. A number of additional risks have
appeared in the literature, but their relationship to TRT is less well established. These include
sleep apnea, worsening of urinary voiding symptoms, and prostate cancer. Standard forms of
TRT do not appear to adversely affect lipid profiles and does not appear to cause liver
toxicity, with the exception of oral alkylated testosterone preparations (e.g,
methyltestosterone), which should not be used for testosterone replacement therapy for this
reason. A key area of controversy relates to the biochemical determination of TD. There is no
defined serum threshold for testosterone. Yet all published guidelines recommend one
arbitrary threshold or another, generally ranging from 200 to 350 ng/dL (6.94-12.15 nmol/L)].
Variation in sex hormone-binding globulin levels also confounds the interpretation of
bioavailable testosterone levels. There is general agreement that free or bioavailable
tesosterone provides a better estimation of testosterone status, but there is uncertainty about
the reliability of those assays. In addition, genetic variation may influence response to
circulating testosterone [11328].
Opioid-induced androgen deficiency (OPIAD)
Morphine sulfate, through its binding to opioid receptors, is known to act in several body
regions from the gut to the brain. Previous studies have demonstrated that morphine induces
a dramatic long-lasting decrease in testosterone, which persists during opioid therapy even if
the treatment lasts for months or years, in both males and females. The effect can occur
after a few hours, with testosterone concentrations reaching castration levels (< 1 ng/mL).
Aloisi and colleagues have also shown that once opioid treatment is interrupted, testosterone
levels recover in a few hours/days. Furthermore, spinal (intrathecal or epidural)
administration of morphine resulted in a similar reduction in testosterone in both males and
females. Opioid therapy is one of the most effective forms of analgesia currently in use. In
the past few decades, the use of opioids as a long-term treatment for chronic pain has
increased dramatically. Accompanying this upsurge in the use of long-term opioid therapy
has been an increase in the occurrence of opioid associated endocrinopathy, most
commonly manifested as an androgen deficiency and therefore referred to as opioid
associated androgen deficiency (OPIAD). This syndrome is characterized by the presence of
inappropriately low levels of gonadotropins (follicle stimulating hormone and luteinizing
hormone) leading to inadequate production of sex hormones, particularly testosterone.
Symptoms that may manifest in patients with OPIAD include reduced libido, erectile
dysfunction, fatigue, hot flashes, and depression. Physical findings may include reduced
facial and body hair, anemia, decreased muscle mass, weight gain, and osteopenia or
osteoporosis. Additionally, both men and women with OPIAD may suffer from infertility. While
the literature regarding OPIAD remains limited, it is apparent that OPIAD is becoming
increasingly prevalent among chronic opioid consumers but often goes unrecognized. OPIAD
can have a significant negative impact on the the quality of life of opioid users, and clinicians
should anticipate the potential for its occurrence whenever long-term opioid prescribing is
undertaken. Once diagnosed, treatment for OPIAD may be offered utilizing a number of
androgen replacement therapy options including a variety of testosterone preparations and,
1307
for female patients with OPIAD, dehydroepiandrosterone (DHEA) supplementation. Follow-
up evaluation of patients receiving androgen replacement therapy should include a review of
any unresolved symptoms of hypogonadism, laboratory evaluation, and surveillance for
potential adverse effects of androgen replacement therapy including prostate disease in
males [12095].
Testosterone and motivation to compete
It is possible that high-testosterone individuals have increased motivation to compete in

sports. High-testosterone individuals may select into sports as a function of testosterone's
positive influence on dominance striving, also known as power motivation. Basal
testosterone is positively correlated with power motivation in men, whereas basal estradiol is
positively correlated with power motivation in women. High concentrations of testosterone
are also positively associated with selection into power-laden careers, e.g. trial law and
acting. Knowing that power-motivated individuals are motivated to pursue dominance and
find dominance experiences rewarding, the positive association between testosterone and
power motivation suggests that high testosterone individuals may be the individuals most
motivated to pursue athletic competition. Testosterone is also associated with reduced
empathy, reduced perception of negative emotions enhanced attention to social threat, and
enhanced amygdala responses to social threat. Additionally, testosterone has been linked to
increased risk-taking in economic domains and social domains. Lastly, testosterone is
associated with enhanced visuospatial ability, which may provide greater abilities in the
perceiving critical targets and navigating the physical sports environment, i.e., field, rink, or
court. Thus, high endogenous concentrations of testosterone may confer both psychological
and physiological advantage in sports [12100].
It was tested the effects of different post-match recovery interventions on the subsequent
hormonal responses to a physical stress-test and game performance in professional rugby
union players. On four occasions, participants (n=12) completed a video session (1 h each)
with accompanying coach feedback the day after a rugby union match. The interventions
showed either video footage of player mistakes with negative coach feedback (NCF1) or
player successes with positive feedback (PCF1). Both approaches were repeated (NCF2 and
PCF2). In the following week, participants were assessed for their free testosterone (T) and
cortisol (C) responses to a physical stress-test, pre-game T and game-ranked performance.
The PFC1 and PCF2 approaches were both associated with significantly greater free T (36
% to 42 %) responses to the stress-test when compared to NCF1 and NCF2 (16 % to -3 %),
respectively. The PCF interventions were also associated with higher (28 % to 51 %) pre-
game T concentrations and superior game-ranked performances than the NCF approaches.
In conclusion, the post-game presentation of specific video footage combined with different
coach feedbacks appeared to influence the free hormonal state of rugby players and game
performance several days later. Therefore, within the sporting context, future behaviour and
performance might be modified through the use of simple psychological strategies. These
data are applicable to generalised human stress responses and their modifiability by prior
exposure to a stressor [12104].
Long-term effects of testosterone
A study published in the New England Journal of Medicine in 1996 confirmed, in a

randomized and controlled trial, numerous anecdotal reports that had long suggested that
anabolic steroids promoted hypertrophy in human skeletal muscle and thus were
1308
performance enhancing. By acting on androgen receptors expressed by myonuclei and
muscle stem (satellite) cells, or through a rapid intracellular androgen receptor-independent
mode, testosterone stimulates muscle protein synthesis to increase muscle mass.
Subsequent work from the same group showed that there was a dose-response effect of
testosterone enanthate not only on human muscle fiber size also on the number of muscle
satellite cells and myonuclei. The positive relationship between muscle cell size and the
number of myonuclei was clearly demonstrated in 1999 in a study with a wide range of
muscle fiber sizes, including extremely large fibres obtained from self-reported anabolic
steroid users. This and other work provided the basis for the myonuclear domain and ceiling
effect hypotheses. Simply put, these suggest that each nucleus is responsible for managing
a certain volume of cytoplasm and this has a maximum limit. If the myonuclear domain is
below this “ceiling,” then an increase in nuclear transcription and protein synthesis can drive
hypertrophy in the muscle cell. However, once this ceiling is approached, additional nuclei
are required to facilitate further growth, these nuclei being donated by the satellite cells. It
has also been published a report from studies on mice which demonstrated an increase in
myonuclear number in response to overload, which was shown to be maintained after
overload. Interestingly, this was shown to be maintained after overload had ended and in the
face of a declining muscle fiber cross-sectional area. This important observation prompted
discussion about whether muscles might remain “primed” and more amenable to further
hypertrophy at a later date. Given that anabolic steroids have also been shown to increase
the myonuclear number in humans would an athlete who had tested positive for anabolic
steroid abuse and had served a 2-year competitive ban still be in position to reap the
physiological benefits of the initial doping offence at a later date? In other words, having
returned “clean” and no longer taking the prohibited substance, would athletes still have an
unfair advantage? A second and more recently published study shows that this is a real
possibility. In a study when mice received testosterone propionate for 14 days myonuclear
number was markedly increased. However, the unique part of the study was when the
animals undertook a muscle loading regimen 3 weeks after drug treatment had been
removed (i.e. when “clean”). The results showed that in the testosterone treated group mean
muscle fibre area from the extensor digitorum longus increased by 31 percent, whilst there
was no significant change in the sham treated animals. Indeed, this positive effect on
adaptation was still shown to be present even when a delay of 3 months (equivalent to
approximately 10 years in humans) was given between treatment withdrawal and the onset
of the loading intervention. Thus, it seems that there is now convincing evidence to show that
the administration of anabolic steroids can result in giving skeletal muscles “memory” in the
form of more myonuclei, which results in a more adaptive response to training long after the
initial effects of the drug have worn off. These results provide a challenge to WADA in regard
to the legitimacy of the length of competitive bans imposed on athletes, as it is now clear that
there are serious concerns surrounding the reversibility, or permanence, of the drug-
mediated performance-enhancing effects of anabolic steroids. However, given that muscle
protein turnover rates are markedly higher in mice than in human beings, it is important that
these data are confirmed in human studies [14708].
Effect of magnesium on testosterone levels
One study was performed to assess how 4 weeks of magnesium supplementation and
exercise affect the free and total plasma testosterone levels of sportsmen practicing tae
kwon do and sedentary controls at rest and after exhaustion. The testosterone levels were
determined at four different periods: resting before supplementation, exhaustion before
supplementation, resting after supplementation, and exhaustion after supplementation in
three study groups, which are as follows: group 1-sedentary controls supplemented with 10
mg magnesium per kilogram body weight; group 2-tae kwon do athletes practicing 90-120
1309
min/day supplemented with 10 mg magnesium per kilogram body weight; and group 3-tae
kwon do athletes practicing 90-120 min/day receiving no magnesium supplements. The free
plasma testosterone levels increased at exhaustion before and after supplementation
compared to resting levels. Exercise also increased testosterone levels relative to sedentary
subjects. Similar increases were observed for total testosterone. The results show that
supplementation with magnesium increases free and total testosterone values in sedentary
and in athletes. The increases are higher in those who exercise than in sedentary individuals
[10080].
Psychological influence on testosterone levels
Previous research indicates that testosterone concentrations are highly responsive to human
competitive interactions and that winners have elevated testosterone concentrations relative
to losers. Also, there is some evidence that simply observing others compete can have a
similar effect on the endocrine system. Here, in two studies, it was examined the extent to
which elite male hockey players would demonstrate an increase in testosterone
concentrations after watching themselves engaged in a previous successful competitive
interaction. Results indicated that watching a previous victory produced a significant increase
in testosterone concentrations (42-44 % increase), whereas watching a previous defeat or a
neutral video did not produce a significant change in testosterone (17 % and 6 %,
respectively). Given that natural fluctuations in testosterone have been shown to influence
future competitive and aggressive behaviours, the current studies may have important
practical implications for individuals involved in competitive sports [09086].
Influence on testosterone levels by sports environment
One study examined the social environment effects during a post-match video presentation
on the hormonal responses and match performance in professional male rugby union
players. The study participants (n=12) watched a 1-hour video of mixed content (player
mistakes and successes) from a match played 1 day earlier in the presence of; (1) strangers
who were bigger (SB), (2) strangers who were smaller (SS), (3) friends who were bigger (FB)
and (4) friends who were smaller (FS). The salivary testosterone (T) and cortisol (C)
responses to a physical stress test were assessed 3 days later, along with pre-match T
levels and match-ranked performance 6-7 days later. All treatments were associated with
elevated T responses (% change from baseline) to the stress test with SS>SB and FB>FS.
The C stress responses after the SS and SB interventions were both greater than FS and
FB. On match-day, the FB approach was linked to higher T concentrations than SB and
better ranked performance than FS and SS. The subsequent testing of a population sub-
group (n=8) across a video (V) and a non-video (NV) presentation in a neutral social
environment produced similar stress-test and performance outcomes, but pre-match T
concentrations differed (V>NV). In conclusion, the presence of other males during a post-
match video assessment had some influence on the hormonal responses of male athletes
and match performance in the week that followed. Thus, the social environment during a
post-match assessment could moderate performance and recovery in elite sport and, in a
broader context, could be a possible modulator of human stress responses [14261].
Influence of psychosocial environment
The aim of one survey was to provide a literary review of current knowledge of the possible
association between the psychosocial working environment and relevant physiological
parameters measured in blood and urine. Literature databases (PubMed, Toxline, Biosis and
1310
Embase) were screened using the key words job, work-related and stress in combination
with selected physiological parameters. In total, 51 work place studies investigated the
associations between the psychosocial working environment and physiological changes, of
which 20 were longitudinal studies and 12 population-based studies. The studied exposures
in work place/population-based studies included: job demands (26/8 studies), job control
(24/10 studies), social support and/or leadership behaviour (12/3 studies), effort-reward
imbalance (three/one studies), occupational changes (four studies), shift work (eight studies),
traumatic events (one study) and other (five studies). The physiological responses were
catecholamines (adrenaline, noradrenaline) (14 studies), cortisol (28 studies), cholesterol (23
studies), glycated haemoglobinA(1c) (six studies), testosterone (nine studies), oestrogens
(three studies), dehydroepiandrosterone (six studies), prolactin (14 studies), melatonin (one
study), thyroxin (one study), immunoglobulin (Ig) A (five studies), IgG (four studies), IgM (one
study) and fibrinogen (eight studies). In general, fibrinogen and catabolic indicators, defined
as energy releasing, were increased, whereas the anabolic indicators defined as constructive
building up energy resources were decreased when the psychosocial working environment
was perceived as poor. In conclusion, in this review the association between an adverse
psychosocial working environment and HbA(1c), testosterone and fibrinogen in serum was
found to be a robust and potential candidate for a physiological effect of the psychosocial
working environment. Further, urinary catecholamines appear to reflect the effects of shift
work and monotonous work [09087].
Testosterone concentrations and playing position in professional basketballers
The effects of basketball on basal concentrations of testosterone and cortisol and its
associations to body composition and physical performance remain to be determined. The
main aim of one study was to determine the effects of playing position on physical fitness,
percentage of body fat and hormonal profile in professional basketball players. Jump
performance (SJ, CMJ and ABK), 30 m running speed and treadmill VO2max tests were
conducted in 12 males (24 years) from the first division league of Spain (ACB). The
percentage of body fat was determined from anthropometry, and hemoglobin, glucose,
testosterone and cortisol concentrations were measured from fasting blood samples. The
players were divided into 3 groups depending on playing positions: guards (GU), forwards
(FW) and centers (CE) (n=4 in each group). GU had significantly greater percentage of body
fat (%BF) than CE. CE developed greater positive mechanical impulse than GU in all jump
types and achieved higher maximal instantaneous power than GU and FW in the SJ and
ABK. Centers had more plasma testosterone than guards. All groups a similar relative
VO2max. It was concluded that center position was associated to lower adiposity and higher
jumping performance than playing as guards. All playing positions induced a similar effect on
aerobic power [150232].
Differences between winners and losers
The aim of one study was to investigate the hormonal, physiological and physical responses
of simulated kickboxing competition and evaluate if there was a difference between winners
and losers. Twenty athletes of regional and national level participated in the study
(mean±SD; age: 21.3 ± 2.7 years; height: 170.0 ± 5.0cm). Hormones [cortisol, testosterone,
growth hormone (GH)], blood lactate [La] and glucose concentrations, as well as upper-body
Wingate test, countermovement jump (CMJ) performances were measured before and after
combats. Heart rate (HR) was measured throughout rounds (R) R1, R2 and R3 and rating of
perceived exertion (RPE) was taken after each one. All combats were recorded and
analysed to determine the length of different activity phases (high-intensity, low-intensity and
referee pause) and the frequency of techniques. Hormones, glucose, [La], HR, and RPE
1311
increased pre-to-post combat, while a decrease was observed for CMJ, Wingate test
performance, body mass and time of high-intensity activities. There was no difference
between winners and losers for hormonal, physiological and physical variables. However,
winners executed more jab-cross, total punches, roundhouse kicks, total kicks and total
attacking techniques compared to losers. Kickboxing is an intermittent physically demanding
sport inducing changes in the stress-related hormones soliciting the anaerobic lactic system.
Training should be orientated to enhance kickboxers' anaerobic lactic fitness and their ability
to strike at a sufficient rate. Further investigation is needed to identify possible differences in
tactical and mental abilities that offer some insight into what makes winners "winners"
[150233].
In one study, it was reported evidence from sport competition that is consistent with the
biosocial model of status and dominance. Results show that testosterone levels rise and
drop following victory and defeat in badminton players of both sexes, although at lower
circulating levels in women. After losing the match, peak cortisol levels are observed in both
sexes and correlational analyses indicate that defeat leads to rises in cortisol as well as to
drops in testosterone, the percent change in hormone levels being almost identical in both
sexes. In conclusion, results show the same pattern of hormonal responses to victory and
defeat in men and women [12097].
Home versus away competition: effect on psychophysiological variables
One study evaluated the effect of game venue and starting status on precompetitive
psychophysiological measures in elite rugby union. Saliva samples were taken from players
(starting XV, n=15, and nonstarters, n=9) on a control day and 90 min before 4 games played
consecutively at home and away venues against local rivals and league leaders.
Precompetition psychological states were assessed using the Competitive State Anxiety
Inventory-2. The squad recorded 2 wins (home) and 2 losses (away) over the study period.
Calculated effect sizes (ESs) showed higher pregame cortisol- (C) and testosterone- (T)
difference values before all games than on a baseline control day (ES 0.7-1.5). Similar
findings were observed for cognitive and somatic anxiety. Small between-venues C
differences were observed in starting XV players (ES 0.2-0.25). Conversely, lower home T-
(ES 0.95) and higher away C- (ES 0.6) difference values were observed in nonstarters.
Lower T-difference values were apparent in nonstarters (vs starting XV) before home games,
providing evidence of a between-groups effect (ES 0.92). Findings show an anticipatory rise
in psychophysiological variables before competition. Knowledge of starting status appears a
moderating factor in the magnitude of player endocrine response between home and away
games [150234].
The authors examined the extent to which changes in testosterone concentrations before
competition would be associated with performance among elite male hockey players. Saliva
samples were collected on two noncompetition days (baseline) and before two playoff games
(1 home game, 1 away game). Individual performance was assessed by the coaching staff
after each game. Results indicated that changes in testosterone before competition predicted
performance, but this effect was influenced by game location. Unexpectedly, the authors
found a significant negative relationship between a rise in testosterone and performance for
the away game and a nonsignificant positive relationship for the home game. These findings
indicate that game location should be considered in studies examining the neuroendocrine
correlates of athletic competition [12098].
Spectators
1312
One field study investigated the release of testosterone and cortisol of a vicarious winning
experience in Spanish fans watching the finals between Spain and the Netherlands in the
2010 FIFA World Cup Soccer. Spanish fans (n=50) watched the match with friends or family
in a public place or at home and also participated in a control condition. Consistent with
hypotheses, results revealed that testosterone and cortisol levels were higher when watching
the match than on a control day. However, neither testosterone nor cortisol levels increased
after the victory of the Spanish team. Moreover, the increase in testosterone secretion was
not related to participants' sex, age or soccer fandom, but the increase in total cortisol
secretion during the match was higher among men than among women and among fans that
were younger. Also, increases in cortisol secretion were greater to the degree that people
were a stronger fan of soccer. Level of fandom further appeared to account for the sex effect,
but not for the age effect. Generally, the testosterone data from this study are in line with the
challenge hypothesis, as testosterone levels of watchers increased to prepare their organism
to defend or enhance their social status. The cortisol data from this study are in line with
social self-preservation theory, as higher cortisol secretion among young and greater soccer
fans suggests that especially they perceived that a negative outcome of the match would
threaten their own social esteem [12099].
No effect of red color
One study examined the testosterone responses of men to an exercise bout simulating a
competitive sporting effort in order to determine if the wearing of red-colored apparel
influenced the hormonal response. Male subjects (n=10) were placed into sets of matched-
pairs and performed VO2max cycle ergometry exercise test to exhaustion to simulate the
competitive effort. Each member of a pairing was randomly assigned to one of two treatment
groups-the wearing of red-colored clothing, or the wearing of black-colored clothing. Blood
samples were collected before exercise (REST), an immediate postexercise sample was
collected at exhaustion (EXH), and a final sample was taken at 15 min into recovery (REC)
from exercise. Blood was biochemically analyzed for total testosterone. In response to the
exercise, performance characteristics (i.e. VO2max and maximal workload) of treatment groups
did not differ significantly. A significant increase in the testosterone was observed in both
treatment groups postexercise at EXH and at REC as compared to REST. However, no
differences were observed between treatment groups in the before or postexercise hormonal
concentrations. These findings suggest that the wearing of red-colored apparel had no
affects on the testosterone responses to an exercise bout simulating a competition [05071].
Red coloration is a sexually selected, testosterone-dependent signal of male quality in a

variety of animals, and in some non-human species a male's dominance can be
experimentally increased by attaching artificial red stimuli. Here it was shown that a similar
effect can influence the outcome of physical contests in humans--across a range of sports,
we find that wearing red is consistently associated with a higher probability of winning. These
results indicate not only that sexual selection may have influenced the evolution of human
response to colours, but also that the colour of sportswear needs to be taken into account to
ensure a level playing field in sport [05072].
Influence of stress on testosterone (and other anabolics) levels
To determine whether cycling has an effect on serum PSA, gonadotropins, and uroflowmetric
parameters a total of 34 healthy male athletes from the National Cycling Team and 24
healthy male student volunteers from University and medical staff were prospectively
enrolled in a study. Blood samples for serum total prostate-specific antigen (tPSA), free PSA
1313
(fPSA, fPSA/tPSA, follicle-stimulating hormone (FSH), luteinizing hormone (LH), and
testosterone determinations were obtained before and after cyclists completed 300 km
bicycle ride and with each cyclist seated without changing posture and with minimal
movement for 10 minutes before blood collection. The athletes and the control group were
well matched by age. There was no significant difference between the 2 groups in terms of
serum tPSA, fPSA, f/t PSA values, FSH, LH, and testosterone levels and uroflowmetric
parameters. The postcycling serum testosterone level was significantly lower than precycling
levels (mean, 604 ng/dL vs 425 ng/dL). There was no correlation between body mass index
values, postcycling serum FSH, LH levels, age, and testosterone levels [09088].
Hormone profile in men
Steroid profiling provides valuable information to detect doping with endogenous steroids.
Apart from the traditionally monitored steroids, minor metabolites can play an important role
to increase the specificity and efficiency of current detection methods. The applicability of
several minor steroid metabolites was tested on administration studies with low doses of oral
testosterone (T), T gel, dihydrotestosterone (DHT) gel and oral dehydroepiandrosterone
(DHEA). The collected data for all monitored parameters were evaluated with the respective
population based reference ranges. Besides the traditional markers T/E, T and DHT, minor
metabolites 4-OH-Adion and 6alpha-OH-Adion were found as most sensitive metabolites to
detect oral T administration. The most sensitive metabolites for the detection of DHEA were
identified as 16alpha-OH-DHEA and 7beta-OH-DHEA but longest detection up to three days
(after oral administration of 50 mg) was obtained with non-specific 5β-steroids and its ratios.
Steroids applied as a gel had longer effects on the metabolism but were generally not
detectable with universal decision criteria. It can be concluded that population based
reference ranges show limited overall performance in detecting misuse of small doses of
natural androgens. Although some minor metabolites provide additional information for the
oral testosterone and DHEA formulations, the topical administered steroids could not be
detected for all volunteers using universal reference limits. Application of other population
based threshold limits did not lead to longer detection times [10455].
Hormone profile in women
It is unclear whether hormone profiles obtained in two consecutive months are consistent
within females. It was prospectively examined month to month consistency in daily, nadir,
peak and mean hormone concentrations during the early follicular and luteal phases in
recreationally active, young eumenorrheic females. Sixty healthy, non-smoking females who
reported normal and consistent menstrual cycles lasting 26-32 days for the past 6 months
were followed prospectively to obtain serum samples for the first 6 days of menses, and for 8
days following a positive ovulation test over two consecutive months. Month to month
consistency of daily concentrations of estradiol (pg/mL), progesterone (ng/mL), testosterone
(ng/dL), SHBG (nmol/L) and FAI were determined using linear mixed models. Month to
month consistency in nadir, peak and mean concentrations were then assessed using
intraclass correlation coefficients and standard error of the measurement to more precisely
examine intra-individual consistency. Linear mixed models revealed stable hormone
concentrations across cycles and cycles by day. Reliability estimates for nadir, peak, mean
menses and mean postovulatory concentrations range from 0.56-0.86 for estradiol, 0.44-0.91
for progesterone, 0.60-0.86 for testosterone, 0.88.0.97 for SHBG, and 0.78-0.91 for FAI. It
was concluded that hormone profiles were reproducible over two consecutive months. In
order to reduce month to month intra-individual variations and improve measurement
consistency, it was recommended that multiple samples be taken over consecutive days as
1314
opposed to a single sample [09102].
Plasma testosterone and work-related neck and shoulder disorders
The aims of one study were to study the association between anabolic hormone testosterone
in plasma and the presence of musculoskeletal disorders among female workers and to
study the association between changes in testosterone and changes in musculoskeletal
complaints. In a cross-sectional design 145 women from 2 different industries filled out
questionnaires about current musculoskeletal complaints, participated in a clinical
examination of the neck and upper extremities, and gave a blood sample for the analysis of
free testosterone in plasma. Individual characteristics, psychosocial job factors, and stress
reactions were evaluated by questionnaires. In a follow-up study a subgroup of 73 sewing
machine operators from the cross-sectional study was reexamined after 1 year. The group of
women with clinically verified neck or shoulder disorders had significantly lower plasma
testosterone than the women with no disorders. Furthermore, the testosterone level showed
a negative association with age and a positive association with smoking and body mass
index. Changes in pain status or clinically diagnosed musculoskeletal disorders were not
associated with changes in testosterone levels. However, this finding may well be due to a
strong plant influence in that marked changes in testosterone levels were observed for 2 of
the 3 participating plants. There is thus some indication of an association between
musculoskeletal disorders in the neck and shoulders and a low level of free plasma
testosterone. The study failed to clarify the associations found between changes in
testosterone and changes in musculoskeletal complaints over time [00080].
Influence of fasting (Ramadan)
The Ramadan fasting period is associated with changes in sleep habits and increased
sleepiness, which may affect physical performance in athletes, and may induce metabolic,
hormonal, and inflammatory disturbances. In 8 middle-distance athletes (25 + 1 years), a
maximal aerobic velocity (MAV) test was performed 5 days before Ramadan fasting period
(day -5), and on days 7 and 21 of Ramadan fasting period. The same days, saliva samples
were collected to determine cortisol and testosterone concentrations before and after the
MAV test. During Ramadan fasting period, mean body mass and body fat did not statistically
change. Compared with day -5, MAV values decreased significantly at days 7 and 21, while
testosterone/cortisol ratio values did not change significantly. Nocturnal sleep time and
energy intake were significantly lower at day 21 than before Ramadan fasting period. At the
end of Ramadan fasting period (day 31), the fatigue score on the Profile of Mood States
questionnaire was significantly increased. In conclusion, Ramadan fasting period is
accompanied by significant metabolic, hormonal, and inflammatory changes. Sleep
disturbances, energy deficiency, and fatigue during Ramadan fasting period may decrease
physical performance in Muslim athletes who maintain training. Reduction of work load and
(or) daytime napping may represent adequate strategies to counteract Ramadan fasting
period effects for Muslim athletes [09089].
Circadian rhythm
The study investigated the effects of circadian rhythm of cortisol (C) and testosterone (T) on
maximal force production (Fpeak) and power output (Ppeak). Twenty male university students
(mean age 24 years) performed 4 time-of-day testing sessions consisting of
countermovement jumps (CMJs), squat jumps (SJ), isometric midthigh pulls (IMTPs), and a
1315
1-repetition maximum (1RM) squat. Saliva samples were collected at 0800, 1200, 1600, and
2000 hours to assess T and C levels on each testing day. Session rate-of-perceived exertion
(RPE) scores were collected after each session. The results showed that Fpeak and Ppeak
presented a clear circadian rhythm in CMJ and IMTP but not in SJ. One repetition maximum
squat did not display a clear circadian rhythm. Session RPE scores collected at 0800 and
2000 hours were significantly higher than those obtained at 1200 and 1600 hours. Salivary T
and C displayed a clear circadian rhythm with highest values at 0800 hours and lowest at
2000 hours; however, no significant correlation was found between T and C with Fpeak and
Ppeak. A very strong correlation was found between Taural with Fpeak of CMJ and IMTP and
Ppeak of CMJ. Thus, the study showed the existence of a circadian rhythm in F peak and Ppeak in
CMJ and IMTP. The evidence suggests that strength and power training or testing should be
scheduled later during the day. The use of Taural seemed to be a more effective indicator of
physical performance than hormonal measures, and the use of session RPE should also be
closely monitored because it may present a circadian rhythm [11095].
Genetic influence (polymorphism)
Doping with anabolic agents is regulated within a number of sports. Testosterone and its
functional analogs are popular compounds for increasing muscle mass, physical
performance, recovery, and reducing body fat. While routine tests for anabolic drugs exist
(e.g. hair, urine, and blood analysis), the aim of the present study is to determine specific
gene expression profiles (induced by testosterone and exercise) which may be used as
effective biomarkers to determine the use of anabolic drugs. In one study, whole blood
samples of 19 male volunteers were analyzed by semi-quantitative real-time polymerase
chain reaction (RT-PCR) for gene expression profiles in the context of exercise and
transdermal testosterone application (1.5 mg/kg body weight). The hormone application was
monitored by urine and saliva analysis for testosterone. Both urinary and saliva levels
indicate that transdermal testosterone application leads to an increase of testosterone,
especially after exercise. RT-PCR results showed a clear variation in the expression of target
genes as well as established housekeeping genes. Only one of the nine common
housekeeping genes, cyclophilin b (PPIB), appears to be independent of both exercise and
testosterone. Out of 14 candidate genes, five are unregulated; all others were more or less
influenced by the mentioned variables. Only interleukin-6 appeared to be exclusively
dependent on long-term testosterone application. This study indicates that many genes are
not influenced by testosterone alone while exercise modulates gene expression in whole
blood samples. As such, exercise must be considered when validating gene expression
techniques for doping analysis [11442].
The ability to identify anabolic steroid use continues to improve. A sudden elevation of the
testosterone–epitestosterone ratio typically indicates illegitimate use of an anabolic steroid.
However, it is well known that some individuals may have naturally elevated ratios, and it has
recently been recognized that 40 percent of those with a certain genotype found in two-thirds
of people of Asian ancestry may not reach the threshold elevation of the ratio after
administration of testosterone [08112].
The heritability of most behavioural traits, including personality, cognitive abilities and
susceptibility to psychiatric illness, is considerable, but as yet, only few genes of definite
importance in this context have been identified. Given the important role of sex steroids for
brain function, it is unfortunate that relatively few studies so far have addressed the possible
influence of sex steroid-related genes on interindividual differences with respect to
personality, cognition and susceptibility to psychiatric disorders [08113].
1316
Testosterone is excreted mainly as glucuronide conjugates after metabolism by uridine
diphospho (UDP)-glucuronosyl transferases (UGT). It is well established that UGT2B7,
UGT2B15 and UGT2B17 are the principal catalysts of the glucuronidation of androgens and
their metabolites in the human [08114]. Testosterone is mainly conjugated by UGT2B17 and,
to a minor extent, by UGT2B15. The main androgen substrate of UGT2B15 is androstane
3α,17β-diol [115]. UGT2B17 shares 96 percent homology with UGT2B15 [08116], but its
substrate specificity is broader [08115]. UGT2B7 has been shown to have the capacity to
conjugate epitestosterone [08117] while testosterone is a poor substrate for this enzyme
[08115].
Testosterone abuse is conventionally assessed by the urinary testosterone/epitestosterone

(T/E) ratio, levels above 4.0 being considered suspicious. An alternative is determination of
the 13C/12C ratio of selected steroids (IRMS analysis) provides the possibility to distinguish
between pharmaceutical and natural testosterone because exogenous compounds contain
less 13C than their endogenous homologues [08118].
The large variation in testosterone glucuronide excretion and its strong association with a
deletion polymorphism in the UGT2B17 gene challenge the accuracy of the T/E ratio test.
Therefore, it was investigated in an open 3-armed comparative study whether genotype
based cut-off values will improve the sensitivity and specificity of the test. Fifty-five healthy
male volunteers with either two, one or no allele of the UGT2B17 gene were investigated
after a single intramuscular dose of 500 mg testosterone enanthate. It was found that the
degree and rate of increase in testosterone glucuronide excretion rate was highly dependent
on the UGT2B17 genotype with a 20-fold average maximum difference. Forty percent of the
subjects with deletions never reached the T/E ratio of 4.0 on any of the 15 days after the
dose. This means that consideration of the genetic variation in disposition of androgens will
improve the sensitivity and specificity of the testosterone doping test [203]. The
polymorphism was considerably more common in a Korean Asian than in a Swedish
Caucasian population, with 67 and 9 percent deletion/deletion homozygotes respectively
[286, 287]. Continued experience of testing for anabolic steroids indicated that Asian
individuals excrete lower amounts of testosterone glucuronide and hence have lower T/E
ratios, thus increasing the risk of false-negative doping test results [08119]. This means that
there are possible genetic differences between groups of individuals.
Testosterone is excreted in urine as water soluble glucuronidated and sulphatated

conjugates. The ability to glucuronidate testosterone and other steroids depends on a
number of different glucuronidases (UGT) of which UGT2B17 is essential. A clinical study of
116 healthy boys aged 8 to 19 years had UGT2B17 genotyping performed using quantitative
PCR. Serum FSH, LH, T, estradiol (E2) and SHBG were analysed by immunoassays, and
urinary levels of androgen metabolites were quantitated by gas chromatography/mass
spectrometry in all subjects. Ten out of 116 subjects (9 %) presented with a homozygote
deletion of the UGT2B17 gene (del/del), while 52 and 54 boys were hetero- or homozygous
carriers of the UGT2B17 gene (del/ins and ins/ins), respectively. None of the reproductive
hormones were affected by UGT2B17 genotype. In all subjects, mean urinary T/E ratio was
1.56 + 1.14 and unaffected by age or pubertal stage. Subjects with homozygous deletions of
UGT2B17 had significantly lower urinary levels of T, and 5alpha- and 5beta-androstanediol.
Mean urinary T/E was significantly reduced in del/del subjects (0.29 + 0.30). It was
concluded that in pubertal boys, a common homozygous deletion in the UGT2B17 gene
strongly affected urinary excretion pattern of androgen metabolites, but did not influence
circulating androgen levels [08120].
It has been known for more than a decade that urinary T/E ratios are significantly lower in
certain ethnic groups. This observation has limited the effectiveness of a population-based
1317
T/E ratio as a screening test for testosterone use. Recent evidence has demonstrated that a
deletion polymorphism in the UGT2B17 gene is responsible for reduced urinary testosterone
levels. UGT2B17 deletion polymorphism testing would be difficult to perform on every athlete
at this time, and GC/C/IRMS is not a practical screening test for testosterone use [08016].
T/E ratio testing provided a solution for detecting synthetic testosterone use until Asian men
were found to have a lower urinary T/E ratio (compared with kaukasians) more than a decade
ago. Circulating concentrations of steroid hormones are controlled by the UDP-glucuronosyl
transferase 2B (UGT2B) subfamily of uridine diphospho-glucuronosyl transferases, which
facilitate urinary excretion by glucuronidation reactions that make steroid molecules more
hydrophilic. UGT2B17 is the major enzyme in the UGT2B subfamily that conjugates
glucuronide to testosterone, dihydrotestosterone, and androsterone in the liver and tissues. A
common deletion polymorphism in the UGT2B17 gene was recently shown to differ among
ethnic groups, being more common in whites than in African Americans. Further studies
revealed that large differences in urinary testosterone concentrations are associated with a
deletion polymorphism in the UGT2B17 gene. Men homozygous for the UGT2B17 deletion
polymorphism have extremely low or undetectable urinary testosterone concentrations, and
this genotype is 7 times more common in Korean men (67 %) than Swedish men (9 %).
Epitestosterone concentrations are similar in the 2 ethnic groups, regardless of whether they
have low or high urinary testosterone concentrations [08016].
The strong association of the UGT2B17 deletion polymorphism with testosterone excretion
brings into question the ability of a population-based T/E ratio to detect testosterone use.
Unfortunately, it would be difficult at this time for laboratories to incorporate genetic testing
into their routine test menu and screen each athlete for UGT2B17 deletions. An alternative
would be to use a low urinary T/E ratio (<0.2) as evidence for the del/del polymorphism.
However, this would be problematic since it would incorrectly classify athletes that are doping
with a combination of testosterone and epitestosterone (to lower their T/E ratio) as having the
del/del polymorphism. In these cases, either genotyping or other tests to detect doping with
epitestosterone would be required, such as the epitestosterone to 5-androstene-3β,17 -diol
ratio or 13C/12C ratio of epitestosterone [08016].
Testosterone abuse is conventionally disclosed by urinary assay of the

testosterone/epitestosterone (T/E) glucuronide ratio, which should not exceed 4. A
noteworthy number of athletes, however, have higher natural ratios than 4, most likely
because of decreased excretion of epitestosterone glucuronide. Urine from different study
populations was analysed for androgen glucuronides by gas chromatography-mass
spectrometry. All men were genotyped for the uridine diphospho-glucuronosyltransferase
(UGT) 2B17 deletion polymorphism and single nucleotide polymorphisms in the cytochrome
P-450c17alpha (CYP17), UGT2B15 and UGT2B7 genes. Expression of UGT2B15 mRNA in
human liver samples was analysed using real-time PCR. A T>C (A1>A2) promoter
polymorphism in the CYP17 gene was associated with the urinary glucuronide levels of
epitestosterone and its putative precursor androstene-3beta, 17alpha-diol, resulting in 64
percent higher T/E ratios in A1/A1 homozygotes. Individuals devoid of UGT2B17 had
significantly higher UGT2B15 mRNA levels in liver than individuals carrying two functional
UGT2B17 alleles. The CYP17 promoter polymorphism may partly explain high natural (>4)
T/E ratios. The data indicate that 5-androstene-3beta, 17alpha-diol is an important precursor
of epitestosterone and that CYP17 is involved in its production. In addition, it was found that
lack of the UGT2B17 enzyme may be compensated for by increase in UGT2B15
transcription [08121].
Testosterone and epitestosterone are endogenous steroids that differ in the configuration of
the hydroxyl-bearing carbon at the C-17. Testosterone is the predominant male sex hormone
1318
while the role of epitestosterone is largely unclear. In humans, both androgens are mainly
excreted as glucuronide conjugates and the urinary ratio of testosterone to epitestosterone
(T/E), used to expose illicit testosterone abuse by male athletes, indicates the relative
concentrations of the respective glucuronides. Some male athletes have T/E above the
accepted threshold value, 4.0, even without testosterone abuse. It was therefore analyzed
athletes urine samples and found that the main reason for such "false positives" in doping
tests was low epitestosterone glucuronide concentration, not high level of testosterone
glucuronide. Sulfate conjugates of both testosterone and epitestosterone were also detected
in the different urine samples. Glucuronidation assays with the 19 human UDP-
glucuronosyltransferases (UGTs) of subfamilies UGT1A, UGT2A and UGT2B revealed that
UGT2B17 is the most active enzyme in testosterone glucuronidation. UGT2B17 does not
glucuronidate epitestosterone, but inhibition studies revealed that it binds epitestosterone
with similar affinity as testosterone. Epitestosterone glucuronidation is mainly catalyzed by
UGT2B7 and the Km of this reaction is significantly lower than the Km of UGT2B17 for
testosterone. While UGT2B7 and UGT2B17 exhibited high, although converse,
stereoselectivity in testosterone and epitestosterone glucuronidation, UGT2A1, an
extrahepatic enzyme that is mainly expressed in the nasal epithelium, catalyzed the
glucuronidation of both steroids at considerable rates and similar kinetics [08122].
To study the disposition of serum testosterone and seven of its metabolites before and after
2 days of an intramuscular dose (500 mg) of testosterone enanthate in relation to the
phosphodiesterase (PDE7B) and the uridine 5'-diphospho-glucuronosyltransferase
(UGT2B17) genotypes patients were genotyped for UGT2B17 deletion polymorphism and
single nucleotide polymorphisms in the PDE7B gene. The involvement of PDE7B in
hydrolysis of enanthate was assessed in human liver homogenates. Genetic variation in the
PDE7B gene was found to be associated with the serum level of testosterone. Individuals
homozygous for PDE7B rs7774640 G allele had a smaller increase (2.5-fold) in the serum
testosterone levels compared with carriers of the A allele (3.9-fold). In addition, genetic
variation in the PDE7B gene significantly influences the testosterone/epitestosterone ratio, a
biomarker of testosterone doping. An in-vitro incubation studies confirmed that PDE7B
serves as a catalyst of the hydrolysis of testosterone enanthate. The UGT2B17 deletion
polymorphism did not show any significant association with serum testosterone levels or the
other androgen metabolites investigated. It was concluded that it was found that PDE7B is
involved in the hydrolysis of testosterone enanthate and that genetic variation in the PDE7B
gene is a determinant of the systemic levels of testosterone after administration of
testosterone enanthate. It is reasonable to believe that the genetic variation in testosterone
bioavailability may be correlated to varying effects of this androgen, whether it is used for
replacement therapy or abused in doping. Thus the results may be important to consider in
doping test programmes and in therapeutics with androgens and other esterified drugs
[11091].
The deletion polymorphism of the enzyme UGT2B17 is known to correlate with the level of
the testosterone to epitestosterone (T/E) ratio in urine specimen. Due to the importance of
the T/E ratio to detect testosterone abuse in doping analysis, a PCR-ELISA system was
established to identify the UGT2B17 phenotype in urine samples. Epidemiological
investigations in a set of 674 routine doping controls (in- and out-of-competition) resulted in
23 percent homozygote gene-deleted and 75 percent UGT2B17-positive athletes. The
validated test system has shown to be robust and sensitive: in only 18 cases (3 %) isolation
of cell material from urine failed. Following hydrolysis of glucuronidated conjugates, steroids
were analyzed as bis-TMS derivatives by gas chromatography-mass spectrometry (GC-MS),
for example, testosterone (T) and epitestosterone (E). Additionally, isotope ration mass
spectrometry (IRMS) analysis and luteinizing hormone (LH) measurement were applied.
Mean T/E ratios significantly correlated with the UGT2B17 phenotype (del: T/E 0.9; pos: 1.7),
1319
however the values did not differ as distinctive as reported in previous studies. Additionally,
the T/E ratios in the gene-deleted group did not show a normal curve of distribution (median
of T/E 0.5). Obviously, beside the UGT2B17 deletion further influences have to be taken into
account, for example, polymorphisms or induction of other metabolizing enzymes. The
results indicate that the UGT2B17 polymorphism might be insufficient when utilized solely as
a crucial parameter for individual interpretation of T/E in urine. Nevertheless, the detection of
the UGT2B17-gene deletion in urine samples would provide additional information important
for gathering evidence in analysis of steroids in doping control [11444].
The conspicuous interindividual differences in metabolism and urinary excretion of

testosterone and its metabolites make it challenging to reveal testosterone doping. The
variation in testosterone glucuronide excretion is strongly associated with a deletion
polymorphism in the uridine diphosphate-glucuronosyltranferase (UGT) 2B17 gene. The
objective of one study was to identify additional biomarkers to detect testosterone abuse and
to elucidate alternative pathways for testosterone elimination in individuals devoid of the
UGT2B17 enzyme. For this purpose a new ultraperformance liquid chromatographic tandem
mass spectrometric method for simultaneous determination of 10 different sulfo- and
glucuronide-conjugated steroids was developed. Fifty-four healthy male volunteers with two,
one, or no allele (ins/ins, ins/del, or del/del) of the UGT2B17 gene participated in the study.
Intervention included a single im dose of 500 mg testosterone enanthate. Urinary sulfo- and
glucuronide-conjugated steroids were measured. Testosterone sulfate levels decreased in all
individuals after the dose. The individual differences in the excretion of all sulfated
metabolites were large. Thus, these metabolites will not serve as appropriate biomarkers for
testosterone abuse. However, androsterone glucuronide excretion increased in all of our
study subjects after the testosterone dose. Etiocholanolone sulfate was excreted at
significantly higher levels in UGT2B17 del/del individuals. It was proposed that the
androsterone glucuronide to epitestosterone glucuronide ratio may serve as a
complementary biomarker to reveal testosterone abuse [11445].
Influence of exercise on testosterone levels
Sex steroid hormones are secreted mainly by the ovary and testis and regulate diverse
physiological processes in target tissues. Recent studies have shown that sex
steroidogenesis-related mRNA and protein expressions, such as for 17β-hydroxysteroid
dehydrogenase (HSD), 3beta-HSD, 5alpha-reductase and aromatase cytochrome P-450
(P450arom) enzymes, are detected in the skeletal muscle, while testosterone, estradiol, and
5α-dihydrotestosterone (DHT) were locally synthesized in skeletal muscle from
dehydroepiandrosterone (DHEA). Moreover, in animal and human studies, the sex
steroidogenesis enzymes and sex steroid hormone levels in skeletal muscle are upregulated
by acute and chronic exercise stimulation. The enhanced muscle sex steroidgenesis is
associated with glycemic control via upregulation of muscle glucose transporter-4 (GLUT-4)
signaling in obese and diabetic rats and with muscle mass and strength in older men. Thus,
an exercise-induced increase of sex steroid hormone in muscle may positively impact age-
related concerns such as life-related diseases and sarcopenia [150235].
The purpose of one study was to explore the mechanisms for increased exercise
performance in conditions of competition. Endurance trained subjects (n=14) performed
incremental treadmill running to exhaustion in control laboratory conditions (non-competition)
and in conditions of simulated competition to assess performance (running duration). Heart
rate and respiration gases were monitored continuously through each exercise condition.
Blood lactate, cortisol, growth hormone and testosterone concentrations were also
1320
determined at pre- (rest) and postexercise in each condition. Results indicated competition
exercise performance was significantly increased as was peak VO2 response versus non-
competition. No significant differences were found in peak measurements of minute
ventilation, respiratory exchange ratio, ventilation threshold, post-exercise lactate, heart rate,
or the ventilation equivalent for O2 between the exercise conditions. In both conditions growth
hormone and testosterone concentrations increased significantly in response to exercise,
whereas cortisol responses post-exercise were significantly elevated in the competition but
not in the control condition. These findings support that in competitive situations the affective
state (motivation) experienced by athletes can enhance performance in exercise events, and
lead to an increased peak oxygen uptake. The magnitude of the improvement is of a
substantial nature and of a level seen with some training programs. Competitive conditions
also augment the cortisol response to exercise, suggesting that enhanced sympatho-adrenal
system activation occur in such situations which may be one of the key "driving forces" to
performance improvement [10081].
Although adaptations to water-based resistance exercise and conventional water-based

exercise have been investigated, little is known regarding acute anabolic and catabolic
hormonal responses to these two types of exercise. The purpose of this study was to
investigate the acute responses of salivary testosterone and cortisol to two water-based
exercise protocols in which the different intensities were determined using Borg's perceived
exertion scale. Ten young (24 + 3 yr) and 7 elderly men (65 + 6 yr) who were familiar with
exercise in water were subjects of the study. Salivary samples were collected at rest and 5
minutes after the 2 water-based exercise protocols. One session involved intermittent water
resistance training at a Borg-scale intensity of 19 (W19), whereas the other involved
continuous water aerobic training at an intensity of 13 (W13). The samples were used to
determine salivary levels of free testosterone and cortisol. There was a significant increase
on salivary testosterone in both groups after the W19 protocol, but no such alteration was
observed after W13. The testosterone response to the W19 protocol was significantly higher
in young than in elderly men. Although no modification on salivary cortisol was observed
after either protocol, in young men, the cortisol response to W19 was higher than in elderly
men. Water-based exercise with emphasis on strength development was found to stimulate a
more acute increase on salivary testosterone than water-based aerobic exercise, probably as
a result of the higher intensity used in that training protocol. Given the known relationship
between acute hormonal responses and chronic neuromuscular adaptations, the
testosterone response after W19 should be considered when prescribing water-based
exercise, especially to older populations [09090].
It was investigated whether the myosin heavy chain (MyHC) proportion and androgen
receptor (AR) concentration in skeletal muscle differ following 21 weeks of strength,
endurance and combined training in untrained older men. Strength (S) and endurance (E)
groups trained twice per week and combined (S+E) group trained four times per week (two
strength and two endurance). Muscle biopsies were obtained before and after the training
period from m. vastus lateralis (VL) and AR mRNA and protein concentration and MyHC
proportion were determined. 1RM increased during the training period in S, S+E and E but
the changes were greater in S and S+E than in E. Statistically significant increases were
observed only in S and S+E in maximal isometric force as well as in VL thickness. VO 2max
increased significantly only in E. MyHCIIa proportion increased in S, while MyHCIIa
proportion decreased and MyHCI increased significantly intheendurance group. No
statistically significant changes were observed in serum testosterone and in AR mRNA or
protein concentrations. The present results indicate that 21 weeks of strength, endurance or
combined training changed MyHC proportion according to the training method but did not
have an effect on androgen mRNA or protein expression in skeletal muscle at rest [09091].
1321
One study compared the neuromuscular performance (speed, power, strength) of elite rugby
union players, by position, and examined the relationship between player performance and
salivary hormones, by squad and position. Thirty-four professional male rugby players were
assessed for running speed (10-m, 20-m or 30-m sprints), concentric mean and peak power
during a 70-kg squat jump and 50-kg bench press throw, and estimated 1 repetition
maximum (1RM) strength for a box squat and bench press. Tests were performed on
separate days with absolute and normalized (power and strength only) values computed.
Saliva was collected before each test and assayed for testosterone and cortisol. The
testosterone and/or cortisol concentrations of players correlated significantly to speed,
power, and strength, especially for the backs, thereby confirming relationships between
neuromuscular performance and hormone secretion patterns. Based on these findings, it
was suggested that training to increase whole-body and muscle mass might facilitate general
performance improvements. Training prescription might also benefit from acute and chronic
hormone monitoring to identify those individuals likely to respond more to hormonal change
[09092].
The effect of a single exercise as well as exercise training on the growth hormone (GH)-
insulin-like growth factor (IGF-I) axis and inflammatory cytokines was studied mainly in adults
participating in individualized endurance-type sports. The gender-specific effect of exercise
on these systems in adolescents is unknown. Therefore, the purpose of one study was to
evaluate the effect of a typical volleyball practice on anabolic (GH, IGF-I, and testosterone)
and catabolic hormones (cortisol) and inflammatory mediators (interleukin-6) in elite, national
team level, male (n=14) and female (n=13) adolescent volleyball players (13-18 years,
Tanner stage 4-5). Exercise consisted of a typical 1-hour volleyball practice. Blood samples
were collected before and immediately after the practice. Exercise led to significant increases
in GH in men and women, testosterone (6.1 + 0.9 to 7.3 + 1.0 and 2.4 + 0.6 to 3.3 + 0.7 ng x
mL, in men and women, respectively), and interleukin-6. There were no gender differences in
the hormonal response to training. Changes in GH and testosterone after the volleyball
practice suggest exercise-related anabolic adaptations [09093].
Effect of training and recovery on testosterone levels
The psychobiologic status of cyclists after 4 days of training and the kinetics of recovery were
assessed by measuring the sympatho-adrenal level, the central noradrenergic activity and
the cortisol/testosterone status by non-invasive methods. For this purpose, urinary excretion
of methoxyamines (metanephrine [MN], normetanephrine [NMN]), which are metabolites of
circulating catecholamines, 3-methoxy-4-hydroxyphenyl glycol sulfate (MHPG-S), a
metabolite of brain norepinephrine, and salivary output of cortisol and testosterone were
measured in twelve national cyclists (aged 20 years), just before (T 1 ) and at the end of the
training (T 2), and during the three following recovery days (R 1, R 2, R 3). Urinary and
salivary samples were also collected during a period of relative rest, in order to get reference
values (T 0). At T 0, T 1 and T 2, mood states, as measured by the Profile of Mood States,
and rating of perceived muscle soreness were assessed. The overall mood and muscle
soreness levels were not affected by the training. The load increased by 187 % as an
average between the first and the fourth day of training. A significant increase in NMN levels
and a decrease in T:F ratio were observed at T 2, while MHPG-S excretion remained
unchanged. Persistent high urinary output of NMN and MN were observed during the post-
training recovery period for 24 h (R 1) and 48 h (R 2), respectively. After 72 h of recovery (R
3), MN levels had returned to baseline while NMN output was lower than the control level.
T:F values returned to their control levels within 48 h of recovery. The strenuous training
seems to induce an alteration in peripheral neuro-endocrine parameters without
modifications of central factors. The hormonal status remained altered for at least 1 day of
post-training recovery and seemed to be achieved within 3 days [02056].
1322
The aim of one study was to compare hormonal changes in plasma total testosterone (T),
cortisol (C), luteinizing hormone (LH), follicle-stimulating hormone (FSH) and prolactin (P) in
two world-class teams, both consisting of 9 top male pro-cyclists, during a real sports
situation (the 1998 "Vuelta a España", a 3-week multi-stage international cycling
competition). Venous blood samples were taken the day before the race (S0), after 1 week
(S1), after 2 weeks (S2) and at the end of the race (S3). The S0 T level was significantly
lower in the team with more racing days in the previous month. There was a significant basal
T decrease during the race in comparison with the initial value, in spite of the difference in S0
T level between teams. However, there was no difference between teams in the mean
decrease in T level. C decreased significantly between S0 and S1 and between S1 and S2,
but not between S2 and S3. There were no differences in P concentration between teams or
throughout the study, except for a significant increase between S2 and S3. There were no
initial differences in LH nor FSH concentration between the teams, nor at any of the study
follow-up times. It was concluded that in professional top-level athletes S0 T values depend
on the number of competition days of the previous month. It was observed a similar decrease
in the T levels in both teams, independent of the absolute S0 values. In both teams C
decreased during long-lasting cycling competition [02057].
One study represents the first time that anaerobic power performance was examined during
an actual intercollegiate American football game. In addition, biochemical and endocrine
responses also were examined to assess the physiological stress imposed by this
competitive contest. Twenty-one members of a NCAA Division III football team were divided
into two groups. Group one (ST) were starters (n=11). The second group (RS) consisted of
red-shirt players (n=10). Blood samples were obtained 24 h (Pre1) and 2.5 h (Pre2) before
the game and within 15 min of game conclusion (IP). Anaerobic power measures were
recorded approximately 10 min before kickoff (pre) and following the first (Q1), second (Q2),
third (Q3), and fourth (Q4) quarters. Peak force (PF) and power (PP) in both squat and
countermovement jumps decreased from pre to Q2 in both ST and RS; however, all
variables returned to baseline levels by Q4. When averaged across trials, PF and PP in both
jumps were greater in ST than RS. No significant changes in testosterone concentrations
with respect to time or between groups were seen. Cortisol concentrations were significantly
higher for ST at IP than RS. No significant changes in creatine kinase, alanine
aminotransferase, urea, or uric acid were observed in either group from Pre2 to IP. In
addition, no between group differences were seen in these variables. Myoglobin and
aspartate aminotransferase significantly increased from Pre2 to IP in ST, and a significant
difference in myoglobin concentrations was seen between the groups at IP. It was concluded
that performance, biochemical, and endocrine changes in these NCAA Division III football
players reflected the stress and muscle damage that occurs as a result of a competitive
American football game [02058].
Maintenance of testosterone status after increased training mileage

The primary objective of one study was to evaluate the early effect of increased training
mileage on testosterone (T) status in recreational joggers. Serum total (T(tot)) and free
(T(free)) concentrations at rest, overnight urinary T(tot) excretion, and the T(tot) and T(free)
responses to maximal exercise were used as indicators of T status. A group of 13 male
(mean age 25 years) fitness joggers (maximal oxygen consumption, VO2max, 53 mL/kg/min)
qualified as subjects. The training intervention consisted of a 100 percent increase in the
habitual distance run (12 miles/week) for 2 consecutive weeks, while maintaining the
customary training intensity. Blood samples were obtained at rest and after maximal exercise
tests, at the beginning and end of a control week of habitual jogging (baseline) and also
following the 1st and 2nd weeks of the intervention. The treadmill exercise endurance time
1323
were unchanged across sampling times. Serum T(tot) and T(free) concentrations did not
change significantly. Urinary T(tot) excretion also remained unchanged during the
intervention. Relative increases in T(tot) (23 %) and T(free) (22%) were observed following
maximal exercise compared to rest. However, the exercise-related increases in serum T(tot)
and T(free) were not evident after adjustment for the change in plasma volume. It was
concluded, that the training intervention did not alter T status in these fitness joggers [02059].
Resistance training on testosterone metabolism in younger and older men

One study investigated the effects of resistance training (RT) on the metabolism of
testosterone (T) in younger (n=5, 28 ± 3 years) and older (n=8, 70 ± 2 years) men.
Experimental heavy resistance exercises (5×10 RM leg presses) were performed before and
after a 12-month of RT. No age differences were found in the production or metabolic
clearance rate of T (determined by stable isotope dilution method), skeletal muscle androgen
receptor content or serum LH concentrations due to acute or chronic RT. The T production
capacity response to gonadotropin stimulation and the concentrations of the urinary T
metabolites (androsterone and etiocholanolone) were lower in the older compared to
younger men. This study further showed that RT may have acute effect on T production and
clearance rates, while the exercise-induced increases in serum T appeared to be induced by
decreased metabolic clearance rate of T. Attenuated T production capacity and urinary
excretion of T metabolites in older men may reflect the known reduction in testicular
steroidogenesis upon aging. No changes were observed in T metabolism due to RT
indicating a homeostatic stability for this hormone in men of different ages [150237].
In trained and in not-trained
It is a common view that strength and sprint trained athletes are characterized by high
plasma/serum testosterone (T) concentration, which is believed to be partly responsible for
their performance level. This opinion, however, has poor scientific background. The aim of
one study was to give evidence-based information on this issue. It was examined gonadal
hormone status at rest after overnight fasting in high and top-class track and field sprinters
(n=16) and in untrained men (n=15). It was shown that basal T, free testosterone (fT),
bioavailable testosterone (bio-T), and sex hormone-binding globulin concentrations were not
significantly different in sprinters versus untrained subjects. Further comparison of the results
of the basal serum T concentration in 8 sprinters showed its significant changes during an
annual training period. Significantly higher T concentration during a low-intensity training
period (beginning of December) than during heavy sprint specific training period (end of
March) was observed in these athletes. It was concluded that basal gonadal hormone
concentration in high and top-class athletes (sprinters and jumpers) did not appear to be
significantly different when compared with untrained subjects. Moreover, basal T
concentration in sprinters can differ significantly during an annual training period. This fact
should be taken into consideration when interpreting the results of gonadal hormone status in
athletes at varied training stages [11082].
Effects of hard exercise
Resistance exercise has been shown to elicit a significant acute hormonal response. It
appears that this acute response is more critical to tissue growth and remodelling than
chronic changes in resting hormonal concentrations, as many studies have not shown a
significant change during resistance training despite increases in muscle strength and
hypertrophy. Anabolic hormones such as testosterone and the superfamily of growth
hormones (GH) have been shown to be elevated during 15-30 minutes of post-resistance
exercise providing an adequate stimulus is present. Protocols high in volume, moderate to
1324
high in intensity, using short rest intervals and stressing a large muscle mass, tend to
produce the greatest acute hormonal elevations (e.g. testosterone, GH and the catabolic
hormone cortisol) compared with low-volume, high-intensity protocols using long rest
intervals. Other anabolic hormones such as insulin and insulin-like growth factor-1 (IGF-1)
are critical to skeletal muscle growth. Insulin is regulated by blood glucose and amino acid
levels. However, circulating IGF-1 elevations have been reported following resistance
exercise presumably in response to GH-stimulated hepatic secretion. Recent evidence
indicates that muscle isoforms of IGF-1 may play a substantial role in tissue remodelling via
up-regulation by mechanical signalling (i.e. increased gene expression resulting from stretch
and tension to the muscle cytoskeleton leading to greater protein synthesis rates). Acute
elevations in catecholamines are critical to optimal force production and energy liberation
during resistance exercise. More recent research has shown the importance of acute
hormonal elevations and mechanical stimuli for subsequent up- and down-regulation of
cytoplasmic steroid receptors needed to mediate the hormonal effects. Other factors such as
nutrition, overtraining, detraining and circadian patterns of hormone secretion are critical to
examining the hormonal responses and adaptations to resistance training [05076].
In the last years, mainly 2 high-intensity-training (HIT) protocols became common: First, a
Wingate-based "all-out" protocol and second, a 4×4 min protocol. However, no direct
comparison between these protocols exists, and also a comparison with high-volume-training
(HVT) is missing. Therefore, the aim of the present study was to compare these 3 endurance
training protocols on metabolic, hormonal, and psychological responses. Twelve subjects
performed: 1) HVT [130 min at 55 % peak power output (PPO)]; 2) 4×4 min at 95 % PPO; 3)
4×30 s all-out. Human growth hormone (hGH), testosterone, and cortisol were determined
before (pre) and 0', 30', 60', 180' after each intervention. Metabolic stimuli and perturbations
were characterized by lactate, blood gas (pH, BE, HCO3 -, pO2, PCO2), and spirometric
analysis. Furthermore, changes of the person's perceived physical state were determined.
The 4×30 s training caused the highest increases in cortisol and hGH, followed by 4 × 4 min
and HVT. Testosterone levels were significantly increased by all 3 exercise protocols.
Metabolic stress was highest during and after 4×30 s, followed by 4×4 min and HVT. The
4×30 s training was also the most demanding intervention from an athlete's point of view. In
conclusion, the results suggest that 4×30 s and 4×4 min promote anabolic processes more
than HVT, due to higher increases of hGH, testosterone, and the T/C ratio. It can be
speculated that the acute hormonal increase and the metabolic perturbations might play a
positive role in optimizing training adaptation and in eliciting health benefits as it has been
shown by previous long term training studies using similar exercise protocols [13197].
Intramuscular anabolism following high volume and high intensity resistance exercise
Resistance exercise paradigms are often divided into high volume (HV) or high intensity (HI)
protocols, however, it is unknown whether these protocols differentially stimulate mTORC1
signaling. The purpose of this study was to examine mTORC1 signaling in conjunction with
circulating hormone concentrations following a typical HV and HI lower-body resistance
exercise protocol. Ten resistance-trained men (24.7 ± 3.4 years; 90.1 ± 11.3 kg;
176.0 ± 4.9 cm) performed each resistance exercise protocol in a random, counterbalanced
order. Blood samples were obtained at baseline (BL), immediately (IP), 30 min (30P), 1 h
(1H), 2 h (2H), and 5 h (5H) postexercise. Fine needle muscle biopsies were completed at
BL, 1H, and 5H. Electromyography of the vastus lateralis was also recorded during each
protocol. HV and HI produced a similar magnitude of muscle activation across sets.
Myoglobin and lactate dehydrogenase concentrations were significantly greater following HI
compared to HV, whereas the lactate response was significantly higher following HV
compared to HI. The growth hormone, cortisol, and insulin responses were significantly
greater following HV compared to HI. No significant differences between protocols were
observed for the IGF-1 or testosterone response. Intramuscular anabolic signaling analysis
1325
revealed a significantly greater phosphorylation of IGF-1 receptor at 1H following HV
compared to HI. Phosphorylation status of all other signaling proteins including mTOR,
p70S6k, and RPS6 were not significantly different between trials. Despite significant
differences in markers of muscle damage and the endocrine response following HV and HI,
both protocols appeared to elicit similar mTORC1 activation in resistance-trained men
[150238].
Adaptations to high-intensity interval training in professional male canoe polo athletes

One study compared the effects of two different high-intensity interval training (HIIT)
programs in professional male canoe polo athletes. Responses of peak oxygen uptake
(VO2peak), ventilatory threshold (VT), peak and mean anaerobic power (PPO & MPO), blood
volume, and hormonal adaptations to HIIT were examined. Male athletes (n=21, age: 24 ± 3
years; height : 181 ± 4 cm; mass: 85 ± 6 kg and Body fat: 12.9 ± 2.7 %) were randomly
assigned to one of three groups (n=7): 1) (G1) interval paddling with variable volume (6, 7, 8,
9, 9, 9, 8, 7, 6 repetitions/session from 1 to 9 session respectively) × 60-second at lowest
velocity that elicited VO2peak (vVO2peak), 1:3 work to recovery ratio); 2) (G2) interval paddling
with variable intensity (6 × 60-second at 100, 110, 120, 130, 130, 130, 120, 110, 100 percent
vVO2peak from 1 to 9 session respectively, 1:3 work to recovery); and 3) (GCON) the control
group performed three 60 min paddling sessions (75% vVO2peak) per week for 3 weeks. High-
intensity interval training resulted in significant (except as shown) increases compared with
pretest, in: VO2peak (G1=+8.8 %, G2=+8.5 %), heart rate at VT (b/min) (G1=+9.7 %, G2=+5.9
%) and (%maximum) (G1=+6.9 %, G2=+6.5 %), PPO (G1=+9.7 %, G2=+12.2 %), MPO
(G1=+11.1 %; G2=+16.2 %), total testosterone (G1=+29.4 %, G2=+16.7 %), total
testosterone/cortisol ratio (G1=+40.9 %, G2=+28.1 %), and mean corpuscular hemoglobin
(G1=+1.7 %, G2=+1.3 %). No significant changes were found in GCON. High-intensity
interval paddling may improve both aerobic and anaerobic performances in professional
male canoe polo athletes under the conditions of this study [150239].
Intensive exercise training suppressing testosterone during bed rest

Spaceflight and prolonged bed rest (BR) alter plasma hormone levels inconsistently. This
may be due, in part, to prescription of heavy exercise as a countermeasure for ameliorating
the adverse effects of disuse. The initial project was to assess exercise programs to maintain
aerobic performance and leg strength during BR. One study evaluated the effect of BR and
the performance of the prescribed exercise countermeasures on plasma steroid levels. In a
30-day BR study of male subjects, the efficacy of isotonic (ITE, n=7) or isokinetic exercise
(IKE, n=7) training was evaluated in contrast to no exercise (n=5). These exercise
countermeasures protected aerobic performance and leg strength successfully. BR alone
(no-exercise group) did not change steroidogenesis, as assessed by the plasma
concentrations of cortisol, progesterone, aldosterone, and free (FT) and total testosterone
(TT). In the exercise groups, both FT and TT were decreased: FT during IKE from 24 + 1.7 to
18 + 2.0 pg/mL and during ITE from 21 + 1.5 to 18 + 1 pg/ml, and TT during IKE from 748 +
68 to 534 + 46 ng/dL and during ITE from 565 + 36 to 496 + 38 ng/dL. The effect of intensive
exercise countermeasures on plasma testosterone was not associated with indexes of
overtraining. The reduction in plasma testosterone associated with both the IKE and ITE
countermeasures during BR supports our hypothesis that intensive exercise
countermeasures may, in part, contribute to changes in plasma steroid concentrations during
spaceflight [05077].
After continuous and intermittent training in male rats

To gain more information on the effects of training types on testosterone secretion, the
present study investigated the relationship between serum testosterone (ST) and the
activities of oxydoreductive enzymes in Leydig cells to continuous and intermittent training
regimes. Male rats swam with a load of 3.5 percent body weight for 90 min in the continuous
1326
training group, and 15 min separated by a 7-min rest interval x 6 times in the intermittent
training group, 6 days per week for 5 weeks. ST were measured immediately and 24 h after
exercise, and the activities of SDH, LDH and G6PDH in Leydig cells were measured 24 h
after exercise, following 5 weeks of training. It was found that ST declined following
continuous (0.54 + 0.32 nmol/L) and intermittent (1.64 + 1.80 nmol/L) exercise compared to
sedentary group (9.55 + 5.17 nmol/L). This diminishing effect on ST was still significant 24 h
after continuous exercise (5.96 + 2.79 nmol/l), not after intermittent exercise (7.41 + 4.77
nmol/L). The activities of SDH and LDH increased, whereas G6PDH decreased in Leydig
cells, after both continuous and intermittent training. SDH and G6PDH showed the high
activities in the intermittent training group relative to continuous training group. These
differences in the activities of SDH and G6PDH might be considered as the possible causes
for ST responses to training types [04071].
The aim of one study was to compare the levels of serum immunoglobulin (IgA, IgM, IgG),
testosterone and cortisol in semi-endurance elite runners during general preparation and
competition phase of training. Thirteen semi-endurance elite male runners with an average
age of 19 years volunteered to take part in this study. The runners participated in the
selected training for a period of 14 weeks and 12 sessions per week (in the morning and
afternoon). Blood samples were collected during the three phases of training (before-
preparation phase, after-preparation phase and before-competition phase). The levels of
serum IgM in semi-endurance elite runners after preparation phase reduced significantly,
while these levels during the competition phase increased even though significantly. The
levels of serum IgG and IgA also reduced, however not significantly, during both phases.
Moreover, after preparation phase, there was no significant change in serum IgA levels;
though, these levels reduced, however not significantly, before competition phase. Cortisol
levels significantly decrease after preparation phase; although, it increased before
competition phase. Testosterone/cortisol ratio increases significantly after preparation phase,
and it decreased before competition phase. Testosterone levels intangibility increased and
decreased respectively after preparation and before competition phases. Findings indicated
that long and intensive exercises weaken the immune system, while moderate and short
drills strengthened this system [12101].
Acute exercise, depending on its characteristics, demands a physiological increase in

testosterone. In fact, the majority of investigations showed that total and/or free testosterone
acutely increased immediately after acute strenuous and/or prolonged sub-maximal
endurance and resistance exercises. Unfortunately, the mechanisms responsible for
testosterone increase after acute exercise are still unknown. Probably due to exercise
standardization and/or to individual variability gonadotropins levels have been reported
unchanged, increased or, rarely, decreased after both sub-maximal and maximal acute
exercise. Consequently, other mechanisms, such as a possible adaptation of secretory
capacity of the Leydig cells, adrenergic and/or lactate stimulation, modifications of clearance
rate, plasma volume reductions and changes in testicular blood flow should be investigated.
Studies on the effects of chronic exercise (e.g. training) frequently showed a reduction of free
and total testosterone concentrations in endurance-trained men, and the few prospective
studies showed contradictory results probably due to the features of the training period, the
magnitude of training stimulus and the volume of training load employed. In addition,
modifications of androgen receptors status have also been described [12094].
Generally, cardiovascular exercise and resistance training transiently increase testosterone

concentrations in men although a few studies report null effects. Testosterone concentrations
also vary both before and after competition in a systematic and consistent manner. Wingfield
1327
et al. in 1990)proposed the “challenge hypothesis”, which posits that during mating seasons
and times of resource scarcity testosterone concentrations rise to facilitate competition,
particularly amongst males. The challenge hypothesis is relevant to human competition in the
world of sports. As predicted by the challenge hypothesis, pre-competition concentrations of
testosterone rise in male and female athletes in anticipation of the impending competition. In
men, testosterone commonly increases following victory and decreases following loss.
However, this main effect of winning or losing on changes in men's testosterone is not
always observed. Several studies have shown that other factors like context, individual
differences, e.g. power motivation, social anxiety, and motivation to win (Suay et al., 1999),
as well as cognitive appraisal can play an important role in predicting post-competition
testosterone changes. Dominance-motivated individuals, who positively value interpersonal
dominance and dislike submission, are those most likely to experience outcome-dependent
changes in testosterone. Competitors' level of engagement is also relevant to testosterone
changes, such that men's testosterone increases are greatest when one's opponents feel
more confident. An elite athlete in an international competition is likely to bemore engaged
and to value victory and defeat much more significantly than a participant in laboratory
manipulations with cognitive games. Accordingly, testosterone changes in situations of high
value and importance are likely to be of greater magnitude. A winner will likely benefit from
continued victory and increased access to resources, whereas a loser, who may be injured
or still in the presence of the victory-primed winner, will likely benefit from disengagement.
Several animal studies have elucidated the mediating effects of testosterone in the winner
and loser effect, which have been subsequently studied in humans. A single sample
collected at the peak of endogenous testosterone production has potential to produce a false
positive result, when compared against a population-based average. There is a notable
sexual dimorphism in testosterone responses to competition in humans. Testosterone
responses to winning and losing appear to principally apply to men. Only a single study has
reported an effect winning/losing on differential testosterone changes in women, whereas
many more studies have failed to find an effect. This is likely a function of the different source
glands for testosterone between the sexes, which include the testes and adrenals in men
and ovaries and adrenals in women [12100].
One study examined the predictive relationships between the salivary free testosterone (T)
concentrations of elite athletes and the expression of force and power. A group of elite male
rugby players (n=64) were assessed for peak force (PF), peak rate of force development
(PRFD), force at 100 milliseconds (F100 ms) and 250 milliseconds (F250 ms) during an
isometric mid-thigh pull (IMTP), and/or peak power (PP) and height during a counter-
movement jump (CMJ). Saliva samples were collected before testing and assayed for free T.
Relationships between individual T concentrations and performance were assessed as a
pooled group and 4 sub-groups of equal size. As pooled data sets, none of the IMTP and
CMJ performance variables were significantly correlated with free T in either the PF or PP
groups. The PF and PP abilities of the 4 sub-groups were significantly different, so that
PF1>PF2>PF3>PF4 and PP1>PP2>PP3>PP4. When the 4 sub-groups were analysed, the T
concentrations of the PF4 group were significantly correlated to PRFD and F100 ms during
the IMTP, as was F100 ms in the PF1 group. In the PP1 group, free T also correlated to CMJ
height. The key conclusion is that the expression of force and power in an elite athletic group
may be dependent, to some extent, on individual variation in salivary free T concentrations
and existing strength or power levels. The current results also confirm that the grouping of
elite athletes of mixed strength or power ability may bias predictive results in a manner not
reflective of sub-groups within this population [12102].
Previous pharmacological and pathological studies have reported negative relationships

between circulating testosterone and certain stress hormones (i.e. cortisol and prolactin) in
humans. These relationships have subsequently been used in hypotheses explaining the
1328
subclinical resting testosterone levels often found in some endurance-trained males, but as
of yet no one has specifically examined these relationships as they relate to exercise. Thus,
we examined the relationship between total and free testosterone levels and cortisol, and
between total and free testosterone and prolactin following prolonged endurance exercise in
trained males. Twenty-two endurance-trained males volunteered to run at 100 percent of
their ventilatory threshold (VT) on a treadmill until volitional fatigue. Blood samples were
taken at pre-exercise baseline (B0); volitional fatigue (F0); 30 min (F30), 60 min (F60), and
90 min (F90) into recovery; and at 24 h post-baseline (P24 h). At F0 (mean running time 85
min), exercise induced significant changes from B0 in total testosterone, cortisol and
prolactin. All three of these hormones were still significantly elevated at F30; but at F60 only
cortisol and prolactin were greater than their respective B0 values. Free testosterone
displayed no significant changes from B0 at F0, F30, or the F60 time point. At F90, neither
cortisol nor prolactin was significantly different from their B0 values, but total and free
testosterone were reduced significantly from B0. Cortisol, total testosterone and free
testosterone at P24 h were significantly lower than their respective B0 levels. Negative
relationships existed between peak cortisol response (at time F30) versus total testosterone.
There were no significant relationships between prolactin and total or free testosterone. In
conclusion, the present findings give credence to the hypothesis suggesting a linkage
between the low resting testosterone found in endurance-trained runners and stress
hormones, with respect to cortisol [04072].
Testosterone after a four month endurance training

To investigate the effects of a four month endurance training programme on body
composition and reproductive hormone levels in a PRE and POST training comparative
study 77 male recruit volunteers participating in the Zimbabwe Defence Forces cadet training
programme had percent body fat, fat free mass, body mass index, total serum testosterone,
luteinising hormone (LH) and follicle stimulating hormone (FSH) compared using the paired t-
test. There was a significant decrease in all parameters measured after four months of
endurance training. Decreases in body composition parameters were 54 percent in percent
body fat, 6 percent in fat free mass, and 13 percent in body mass index. There was a
dramatic 58 percent drop in testosterone, 60 percent drop in LH and 15 percent drop in FSH
after four months of endurance training. It was concluded that the Zimbabwe Defence Forces
cadet training programme, an endurance training programme induces a state of negative
energy balance in trainees. This results in a decrease in percent body fat and body mass
index due to utilisation of fat stores as a source of energy and a decrease in fat free mass
due to gluconeogenic utilisation of muscle protein as energy source for muscle activity. There
is impaired hypothalamic-pituitary-testicular axis function as evidenced by the state of
hypogonadal-hypogonadism (low testosterone, LH and FSH). This may be attributed to
gonadotrophin releasing hormone pattern generator malfunction due to the stress of intense
physical activity and withdrawal of energy expenditure from reproductive machinery as a way
of conserving energy for more vital processes in the prevailing state of energy starvation
[00075].
Responses to intensive interval versus steady-state endurance exercise

Free testosterone (FT) hormonal responses were compared between high-intensity interval
exercise (IE) and steadystate endurance exercise (SSE) in endurance trained males (n=15).
IE session was repeated periods of 90-sec treadmill running at 100-110 percent maximal
oxygen uptake (VO2max) and 90-sec active recovery at 40 percent VO2max for 42-47 min. The
SSE session consisted of a continuous 45-min run at 60-65 percent VO2max. Total work
output was equal for each exercise session. A 45-min supine rest control session (CON) was
also performed. All three sessions were on separate days. Pre-session (PRE), immediate
post-session (POST), and 12-h post-session (12POST) blood samples were collected and
used to determine FT, SHBG, LH, 3- alpha-androstanediol glucuronide (3-alphaDiol G) and
1329
cortisol. Analysis of variance compared IE and SSE biomarker responses to the reference
CON session. IE and SSE each caused an increase in FT, but IE more so than SSE. The
5alpha-reductase marker 3-alpha Diol G response at 12POST IE was elevated while FT was
reduced; no such change occurred following SSE. These findings suggest IE might produce
a more pronounced turnover of FT by androgen sensitive tissue than the SSE form of
exercise [12103].
Effect of resistance training
One study assessed the effect of different resistance exercise scheme (RES) designs of
similar total of load lifted on the responses of testosterone, cortisol, and creatine kinase (CK).
Twenty-seven healthy males performed 1 of 4 bench press workouts described by the 1
repetition maximum (1RM) load: 4 sets of maximum repetitions at 50 percent-1RM (50%-
1RM RES), 5 sets of maximum repetitions at 75 percent-1RM (75%-1RM RES), 10 sets of
maximum repetitions at 90 percent-1RM (90%-1RM RES), or 8 sets of maximum repetitions
at 110 percent-1RM (110%-1RM RES). Each RES was equated by the total volume of load
lifted (repetitions x sets x load). Blood samples, collected pre-exercise (Pre) and post-
exercise (Post) at 1 and 24 hours (24 h), were analyzed for total and free testosterone, total
cortisol, and CK. In general, testosterone and cortisol showed little change within or between
the different RES, possibly because of the relatively low volume lifted and/or the small
muscle mass activated by the bench press exercise. Cortisol was elevated after the 75%-
1RM RES at the Post sample, with this response also significantly exceeding the other RES.
The 24 h CK response was also elevated after the 75%-1RM RES, thereby suggesting
greater training strain for the same volume of load. These results confirm previous
recommendations regarding the prescription of resistance exercise and the importance of
total volume as a stimulus for activating the endocrine system and achieving long-term
adaptation [09094].
One study examined the effects of heavy resistance training on physiological acute exercise-
induced fatigue (5 x 10 RM leg press) changes after two loading protocols with the same
relative intensity (%) (5 x 10 RM) and the same absolute load (kg) (5 x 10 RM) as in
pretraining in men (n=12). Exercise-induced neuromuscular (maximal strength and muscle
power output), acute cytokine and hormonal adaptations (i.e. total and free testosterone,
cortisol, growth hormone (GH), insulin-like growth factor-1 (IGF-1), IGF binding protein-3
(IGFBP-3), interleukin-1 receptor antagonist (IL-1ra), IL-1beta, IL-6, and IL-10 and metabolic
responses (i.e. blood lactate) were measured before and after exercise. The resistance
training induced similar acute responses in serum cortisol concentration but increased
responses in anabolic hormones of free testosterone and growth hormone. This enhanced
hormonal and cytokine response to strength exercise at a given relative exercise intensity
after strength training occurred with greater accumulated fatigue and metabolic demand (i.e.
blood lactate accumulation). The magnitude of metabolic demand or the fatigue experienced
during the resistance exercise session influences the hormonal and cytokine response
patterns. Similar relative intensities may elicit not only higher exercise-induced fatigue but
also an increased acute hormonal and cytokine response during the initial phase of a
resistance training period [09095].
One study examined the effects of heavy resistance training on physiological acute exercise-
induced fatigue (5 x 10 RM leg press) changes after two loading protocols with the same
relative intensity (%) (5 x 10 RM) and the same absolute load (kg) (5 x 10 RM) as in
pretraining in men (n=12). Exercise-induced neuromuscular (maximal strength and muscle
power output), acute cytokine and hormonal adaptations (i.e.,total and free testosterone,
cortisol, growth hormone, insulin-like growth factor-1, IGF binding protein-3 (IGFBP-3),
interleukin-1 receptor antagonist (IL-1ra), IL-1beta, IL-6, and IL-10 and metabolic responses
1330
(i.e., blood lactate) were measured before and after exercise. The resistance training induced
similar acute responses in serum cortisol concentration but increased responses in anabolic
hormones of free testosterone and GH, as well as inflammation-responsive cytokine IL-6 and
the anti-inflammatory cytokine IL-10, when the same relative load was used. This response
was balanced by a higher release of pro-inflammatory cytokines IL-1beta and cytokine
inhibitors (IL-1ra) when both the same relative and absolute load was used after training.
This enhanced hormonal and cytokine response to strength exercise at a given relative
exercise intensity after strength training occurred with greater accumulated fatigue and
metabolic demand (i.e. blood lactate accumulation). The magnitude of metabolic demand or
the fatigue experienced during the resistance exercise session influences the hormonal and
cytokine response patterns. Similar relative intensities may elicit not only higher exercise-
induced fatigue but also an increased acute hormonal and cytokine response during the
initial phase of a resistance training period [09096].
The purpose of one study was to determine the acute anabolic and catabolic hormone
response to endurance and resistance exercise bouts of equal volume in subjects with
differing training status. Twenty-two healthy men were recruited who were either resistance
trained (n=7), endurance trained (n=8), or sedentary (n=7). Three sessions were completed:
a resting session, a 40-min run at 50-55 percent maximal oxygen consumption, and a
resistance exercise session. Expired gases were monitored continuously during exercise,
and the endurance and resistance exercise sessions were individually matched for caloric
expenditure. Blood samples were drawn before exercise and 1, 2, 3, and 4 h after the start of
the exercise. Plasma was analyzed for luteinizing hormone, dehydroepiandrosterone sulfate,
cortisol, and free and total testosterone. Androgens increased in response to exercise,
particularly resistance exercise, whereas cortisol only increased after resistance exercise.
Dehydroepiandrosterone sulfate levels increased during the resistance exercise session and
remained elevated during recovery in the resistance-trained subjects. Endurance-trained
subjects displayed less pronounced changes in hormone concentrations in response to
exercise than resistance-trained subjects. After an initial postexercise increase, there was a
significant decline in free and total testosterone during recovery from resistance exercise,
particularly in resistance-trained subjects. On the basis of the results of this study, it appears
that the endogenous hormone profile of men is more dependent on exercise mode or
intensity than exercise volume as measured by caloric expenditure. The relatively catabolic
environment observed during the resistance session may indicate an intensity-rather than a
mode-dependent response [04073].
High-intensity interval resistance training organization on testosterone production

The use of high-intensity interval training (HIIT) is widely diffused as strategy to enhance
aerobic fitness and body composition. In order to offer a more complete training, resistance
exercises have been added to HIIT (HIIRT). Aims of one study were to characterize both
heart rate and hormonal responses elicited by three different protocols of HIIRT having the
same exercises, the same load and number of repetitions for each exercise. Eight healthy
trained men (29 years) performed three different workouts: exercise order, recovery and
speed of execution were differently organized according to workout. Salivary samples were
collected before and after each workout, at 11:00 p.m. and at 7:00 a.m. of the following day.
Salive was also collected during a non-training day. Before and after the workout, plasma
lactate was measured while a beat-to-beat heart rate recording was executed during each
workout. Cortisol (C) and testosterone (T) were measured in salivary samples. Workouts
elicited the same heart rate response while random organization seems to elicit the highest
lactate, C and T increases. Also when we studied the effects of workouts on prolonged
hormones production we observed that workout organization influenced post-exercise
hormonal production until the following morning modifying their physiological trend. Thus,
even if exercises, load and number of repetitions were maintained fixed, exercise order,
1331
structured recovery and speed of execution determined different acute and prolonged
effects. The knowledge of these responses is very important because may positively or
negatively influence performance and health [14762].
Effect of diet on response resistance training

Relationship between dietary intake and serum anabolic hormone concentrations of
testosterone (T), free testosterone (FT), and growth hormone were examined at rest as well
as after the heavy-resistance exercise (HRE) in 8 strength athletes (SA) and 10 physically
active non-athletes (NA). In the first part of the study serum basal anabolic hormone
concentrations and dietary intake were examined in the total group of subjects. In the second
part of the study a subgroup of 5 SA and 5 NA performed the high volume and high intensity
HRE. Dietary intake was registered by dietary diaries for 4 days preceding the loading day.
Significant correlations were observed between serum basal T and fat and protein intake in
the total group of subjects. However, when the two groups were examined separately the
significant relationships between serum basal T and dietary fat and protein could be noticed
in SA only. Both serum T and FT responses to HRE were correlated with fat and protein. The
results suggest the possible role of diet leading to alterations in serum T and FT during
prolonged strength training, and that diets with insufficient fat and/or excessive protein may
compromise the anabolic hormonal environment over a training program [04074].
Short-term effects on resistance training
The aim of one review is to highlight two emerging concepts for the elite athlete using the
resistance-training model with short-term effects of testosterone (T) and cortisol (C) on the
neuromuscular system; and the dose-response training role of these endogenous hormones.
Exogenous evidence confirms that T and C can regulate long-term changes in muscle
growth and performance, especially with resistance training. This evidence also confirms that
changes in T or C concentrations can moderate or support neuromuscular performance
through various short-term mechanisms (e.g. second messengers, lipid/protein pathways,
neuronal activity, behaviour, cognition, motor-system function, muscle properties and energy
metabolism). The possibility of dual T and C effects on the neuromuscular system offers a
new paradigm for understanding resistance-training performance and adaptations.
Endogenous evidence supports the short-term T and C effects on human performance.
Several factors (e.g. workout design, nutrition, genetics, training status and type) can acutely
modify T and/or C concentrations and thereby potentially influence resistance-training
performance and the adaptive outcomes. This novel short-term pathway appears to be more
prominent in athletes (vs non-athletes), possibly due to the training of the neuromuscular and
endocrine systems. However, the exact contribution of these endogenous hormones to the
training process is still unclear. Research also confirms a dose-response training role for
basal changes in endogenous T and C, again, especially for elite athletes. Although full proof
within the physiological range is lacking, this athlete model reconciles a proposed permissive
role for endogenous hormones in untrained individuals. It is also clear that the steroid
receptors (cell bound) mediate target tissue effects by adapting to exercise and training, but
the response patterns of the membrane-bound receptors remain highly speculative. This
information provides a new perspective for examining, interpreting and utilizing T and C
within the elite sporting environment. For example, individual hormonal data may be used to
better prescribe resistance exercise and training programmes or to assess the trainability of
elite athletes. Possible strategies for acutely modifying the hormonal milieu and, thereafter,
the performance/training outcomes were also identified. The limitations and challenges
associated with the analysis and interpretation of hormonal research in sport (e.g. procedural
issues, analytical methods, research design) were another discussion point [11083].
1332
One study examined the effects of short-cycle sprints on power, strength, and salivary
hormones in elite rugby players. Thirty male rugby players performed an upper-body power
and lower-body strength (UPLS) and/or a lower-body power and upper-body strength (LPUS)
workout using a crossover design (sprint vs. control). A 40-second upper-body or lower-body
cycle sprint was performed before the UPLS and LPUS workouts, respectively, with the
control sessions performed without the sprints. Bench throw (BT) power and box squat (BS)
1 repetition maximum (1RM) strength were assessed in the UPLS workout, and squat jump
(SJ) power and bench press (BP) 1RM strength were assessed in the LPUS workout. Saliva
was collected across each workout and assayed for testosterone (Sal-T) and cortisol (Sal-C).
The cycle sprints improved BS (2.6 ± 1.2 %) and BP (2.8 ± 1.0 %) 1RM but did not affect BT
and SJ power. The lower-body cycle sprint produced a favorable environment for the BS by
elevating Sal-T concentrations. The upper-body cycle sprint had no hormonal effect, but the
workout differences (%) in testosterone and cortisol concentrations correlated to the BP,
along with the testosterone/cortisol. In conclusion, the cycle sprints improved the BP and BS
1RM strength of elite rugby players but not power output in the current format. The
improvements noted may be explained, in part, by the changes in absolute or relative
hormone concentrations. These findings have practical implications for prescribing warm-up
and training exercises [11084].
Effect of testosterone administration and weight training on muscle architecture

The purpose of one study was to assess muscle architecture changes in subjects who were
administered supraphysiologic doses of testosterone enanthate (TE) and concurrently
performed heavy resistance training. Ten subjects were randomly selected from the 21
subjects who participated in a previously published study. Subjects were allocated to one of
two groups as per Giorgi et al and received either a saline-based placebo (nonTE) or a 3.5
mg/kg body weight dose of TE by deep intramuscular injection once a week for 12 wk.
Subjects also performed heavy resistance training using exercises that targeted the triceps
brachii muscle. Before and after the training period, free-weight one-repetition-maximum (1-
RM) bench press strength was tested, muscle thickness and pennation of the triceps brachii
lateralis were measured using ultrasound imaging, and fascicle length was estimated from
ultrasound photographs. There were no significant between-group differences in muscle
thickness changes despite a trend toward increased thickness in TE subjects (TE, 23.5 %, vs
nonTE, 13.8 %). However, 1-RM bench press performance and muscle pennation increased
significantly in TE subjects compared with nonTE subjects. There was also a trend toward
longer fascicle lengths in the muscles of nonTE subjects. The results of the present study
suggest that the use of TE in conjunction with heavy resistance training is associated with
muscle architecture changes that are commonly associated with high-force production. Since
there was little difference between the groups in muscle thickness, changes in pennation and
possibly fascicle length may have contributed to strength gains seen in TE subjects [01084].
Effect of different types of training
In the last years, mainly 2 high-intensity-training (HIT) protocols became common: first, a
Wingate-based "all-out" protocol and second, a 4×4 min protocol. However, no direct
comparison between these protocols exists, and also a comparison with high-volume-training
(HVT) is missing. Therefore, the aim of one study was to compare these 3 endurance
training protocols on metabolic, hormonal, and psychological responses. Twelve subjects
performed: 1) HVT (130 min at 55 % peak power output, PPO); 2) 4×4 min at 95 percent
PPO; 3) 4×30 s all-out. Human growth hormone (hGH), testosterone, and cortisol were
determined before (pre) and 0', 30', 60', 180' after each intervention. Metabolic stimuli and
perturbations were characterized by lactate, blood gas (pH, BE, HCO₃⁻, pO₂, PCO₂), and
spirometric analysis. Furthermore, changes of the person's perceived physical state were
1333
determined. The 4×30 s training caused the highest increases in cortisol and hGH, followed
by 4 × 4 min and HVT. Testosterone levels were significantly increased by all 3 exercise
protocols. Metabolic stress was highest during and after 4×30 s, followed by 4×4 min and
HVT. The 4×30 s training was also the most demanding intervention from an athlete's point
of view. In conclusion, the results suggest that 4×30 s and 4×4 min promote anabolic
processes more than HVT, due to higher increases of hGH, testosterone, and the T/C ratio. It
can be speculated that the acute hormonal increase and the metabolic perturbations might
play a positive role in optimizing training adaptation and in eliciting health benefits as it has
been shown by previous long term training studies using similar exercise protocols [13199].
Two different orders of concurrent training

Concurrent training has been widely used in fitness centers in order to simultaneously
optimize cardiovascular and neuromuscular fitness, and induce a high-energy expenditure.
Therefore, the aim of this study was to compare the acute effects of two different orders of
concurrent training on hormonal responses in concurrent trained men. Fourteen men were
randomly divided into 2 groups: endurance training followed by strength (ES, n=7) and
strength training followed by endurance (SE, n=7). Serum concentrations of testosterone,
cortisol, growth hormone and IGFBP3 were measured before and after both training orders.
A significant interaction between exercise order and time was only found in the IGFBP-3
levels. The testosterone and IGFBP-3 concentrations significantly increased in the ES group
after the exercise trainings but did not change significantly in the SE group. Conversely,
cortisol and growth hormone concentrations significantly increased in both ES and SE
groups compared with baseline values. No significant correlations were found between the
changes in the hormonal concentrations. In conclusion, these results suggest that
immediately post-exercise testosterone and IGFPB-3 responses are significantly increased
only after the endurance plus strength training order. Therefore, an ES training order should
be prescribed if the main focus of the training intervention is to induce an acute post-exercise
anabolic environment [14459].
Testosterone responses to interval and tempo runs

The aim of one study was to examine the acute response to plasma and salivary cortisol and
testosterone to three training protocols. Ten trained endurance athletes participated in three
experimental trials, such as interval training (INT), tempo run (TEMP) and bodyweight-only
circuit training (CIR), on separate days. Blood and saliva samples were collected pre- and 0,
15, 30 and 60 min post-exercise. Peak post-exercise salivary cortisol was higher than pre-
exercise in all trials. After INT, salivary cortisol remained elevated above pre-exercise than
60 min post-exercise. Salivary testosterone also increased post-exercise in all trials. Plasma
and salivary cortisol were correlated between individuals and within individuals. Plasma and
salivary testosterone was also correlated between and within individuals. Peak cortisol and
testosterone levels occurred simultaneously in plasma and saliva, but timing of post-exercise
hormone peaks differed between trials and individuals. Further investigation is required to
identify the mechanisms eliciting an increase in hormones in response to CIR. Furthermore,
saliva is a valid alternative sampling technique for measurement of cortisol, although the
complex, individual and situation dependent nature of the hormone response to acute
exercise should be considered [14043].
Effect of different short-time types of exercise
Hormonal responses to exercise could be used as a marker of overreaching. A short

exercise protocol that induces robust hormonal elevations in a normal trained state should be
able to highlight hormonal changes during overreaching. One study compared plasma and
salivary cortisol and testosterone responses to 4 exercise trials at continuous cycle to fatigue
1334
at 75 percent of peak power output (W max) (FAT), 30-minute cycle alternating 1-minute 60
percent and 1 minute 90 percent W max (60/90), 30-minute cycle alternating 1-minute 55
percent and 4-minute 80 percent W max (55/80), and squatting 8 sets of 10 repetitions at 10
repetition maximum (RESIST). Blood and saliva samples were collected pre-exercise and at
0, 10, 20, 30, 40, 50, and 60 minute postexercise. Pre- to postexercise plasma cortisol
increased in all exercise trials, except 60/90. Increases in 55/80 remained above pre-
exercise levels for the entire postexercise period. Salivary cortisol increased from pre- to
postexercise in FAT and 55/80 trials only. Once elevated after 55/80, it remained so for the
postexercise period. Plasma testosterone increased from pre- to postexercise in all trials
except 55/80. Saliva testosterone increased from pre- to postexercise in all trials with the
longest elevation occurring after 55/80. Area under the curve analysis indicated that the
exercise response of salivary hormones was greater in all cycle trials (cortisol) and in the
60/90 and 55/80 trials (testosterone) compared with the other trials. The study indicates that
the 55/80 cycle protocol induces a prolonged salivary and plasma cortisol and salivary
testosterone response compared with the other trials and so may be a useful diagnostic tool
of overreaching [11085].
Effect of standardized short-term sub-maximal and maximal endurance exercises

Few and conflicting data on the acute adaptive role of the hypothalamic-pituitary-testicular
(HPT) axis to sub-maximal endurance exercise exist.To investigate the acute HPT axis
responses to standardized endurance exercises in a laboratory setting and the correlations
between testosterone and classic adaptive hormones variations 12 healthy male volunteers
were recruited for this experimental study. Serum PRL, GH, ACTH, LH, cortisol, DHEAS,
testosterone [total (TT), calculated free (cFT) and bioavailable (cBioT)], SHBG, and
respective ratios, were evaluated before and after a 30-min sub-maximal exercise on cycle
ergometer at individual anaerobic threshold (IAT) and a maximal exercise until exhaustion.
Blood samples were collected before exercise (30, 15 min and immediately before),
immediately after and at different time points during recovery (+15, +30 and +60 min) for
hormones assays. Oxygen consumption and lactate concentration were evaluated.
Testosterone (TT, cFT and cBioT) acutely increased in all volunteers after both exercises.
Testosterone increased in parallel to GH after both exercises and to cortisol only after
maximal exercise. Differently from other increased hormones, testosterone increases were
not correlated to exercise-intensity-related variables. The anabolic/catabolic steroids ratios
were higher after sub-maximal exercise, compared to maximal. It was concluded that a 30-
min sub-maximal endurance exercise acutely increased serum testosterone similarly to
maximal exercise, but without cortisol increases. Exercise-related testosterone peaks should
be considered adaptive phenomena, but few data on their short- and long-term effects exist.
Investigations on the mechanisms of adaptation to exercise in active individuals with
physiological or pathological hypo-testosteronemia are warranted [14061].
Order effects of combined strength and endurance training on testosterone
Concurrent training (CT) has been widely used in fitness centers to simultaneously optimize
cardiovascular and neuromuscular fitness, and induce a high-energy expenditure. Therefore,
the aim of this study was to compare the acute effects of 2 different orders of CT on
hormonal responses in concurrently trained men. Fourteen men (mean ± SD: 24.7 ± 5.1
years) were randomly divided into 2 groups: endurance training followed by strength (ES,
n=7) and strength training followed by endurance (SE, n=7). Serum concentrations of
testosterone, cortisol, growth hormone, and IGF-1 binding protein 3 (IGFBP-3) were
measured before and after both training orders. A significant interaction between exercise
order and time was only found in the IGFBP-3 levels. The testosterone and IGFBP-3
concentrations significantly increased in the ES group after the exercise trainings (57.7 ±
35.1 %, and 17.0 ± 15.5 %, respectively) but did not change significantly in the SE group
1335
(15.5 ± 36.6 %, and -4.2 ± 13.9 %, respectively). Conversely, cortisol and growth hormone
concentrations significantly increased in both ES and SE groups compared with baseline
values. No significant correlations were found between the changes in the hormonal
concentrations. In conclusion, these results suggest that immediately postexercise
testosterone and IGFPB-3 responses are significantly increased only after the ES order.
Therefore, an ES training order should be prescribed if the main focus of the training
intervention is to induce an acute postexercise anabolic environment [150240].
Explosive performances
The primary objective of one study was to analyze the relationship between testosterone
levels and vertical jumping performance in elite men and women athletes. The secondary
objective was to verify whether testosterone levels and vertical jumping performance were
different in men and women athletes and if those measurements were different between
different athletic groups. Seventy (22 women and 48 men) elite athletes in track and field
(sprinters), handball, volleyball, and soccer competing at national and international levels
participated in the study. After 10 hours of fasting and 1 day of rest, blood samples were
drawn from the antecubital vein for determining testosterone levels. Vertical jumping tests
consisted of countermovement jumps conducted on a resistive platform connected to a
digital timer. Resting testosterone levels in women were 10 percent of those of the men.
Countermovement jump performance was significantly different between women and men
athletes, with women's jumping ability 86 percent of that of men. A significant positive
relationship was identified between testosterone levels and vertical jump performance when
all data where considered [06053].
Testosterone response to acute resistance exercise in obese versus lean
Resistance exercise induces a host of endocrine responses that potentiate its effects on
body composition and metabolism. Excess adiposity negatively affects some hormonal
responses to exercise in sedentary men. One study compared the resistance exercise (RE)-
associated growth hormone (GH), insulin-like growth factor-1 (IGF-1), and testosterone
responses in lean versus obese physically active men. Ten healthy physically active obese
males (body fat % 36.2 ± 4.03, mass 104.5 ± 15.5 kg) were compared to ten lean
counterparts (body fat % 12.7 ± 2.9, mass 77.1 ± 6.4 kg). The muscular endurance RE
protocol consisted of six sets of ten repetitions per leg of stepping onto an elevated platform
(20 % of participant's height) while wearing a weighted-vest (50 % of participant's lean
mass). Pre-, immediately post-exercise (IP), and three more blood samples were collected
during the one-hour recovery. When accounting for baseline differences there were no group
by time interactions for GH; or LH. Lean presented a trend towards significance for higher
IGF-1 IP than obese. Testosterone IP was similar in obese and lean, but lower in obese than
lean at 30 min into recovery. AUC were lower in obese than lean for all hormones. These
findings suggest that excess adiposity does not appear to negatively affect the immediate
GH and T responses to RE in active males; but possibly negatively affects IGF-1. However,
the baseline and integrated concentrations during recovery appear negatively affected by
excess adiposity [150236].
Active recovery versus passive recovery
The aim of one study was to compare the effects of active (A) versus passive (P) recovery
during high-intensity interval training on the acute hormonal and metabolic response. Twelve
triathletes/cyclists performed four 4 min intervals on a cycle ergometer, either with A- or P-
recovery between each bout. Testosterone, hGH, cortisol, VEGF, HGF and MIF were
1336
determined pre, 0', 30', 60' and 180' after both interventions. Metabolic perturbations were
characterized by lactate, blood gas and spirometric analysis. A-recovery caused significant
increases in circulating levels of cortisol, testosterone, T/C ratio, hGH, VEGF and HGF.
Transient higher levels were found for cortisol, testosterone, hGH, VEGF, HGF and MIF after
A-recovery compared to P-recovery, despite no differences in metabolic perturbations. A-
recovery was more demanding from an athlete's point of view. Based on the data of
testosterone, hGH and the T/C-ratio, as well as on the data of VEGF and HGF it appears that
this kind of exercise protocol with A-recovery phases between the intervals may promote
anabolic processes and may lead to pro-angiogenic conditions more than with P-recovery.
These data support the findings that also the long term effects of both recovery modes seem
to differ, and that both can induce specific adaptations [13200].
The exercise-induced metabolic stress can be influenced by the mode of recovery and is
associated with acute hormonal responses. Therefore, it is hypothesized that active recovery
between high intensity intervals reduces the metabolic stimulus and therefore the hormonal
response compared to passive recovery. Twelve male cyclist/triathletes performed four 30s
all-out intervals, either with active (A) or passive (P) recovery between each bout. Human
growth hormone (hGH), testosterone and cortisol, vascular endothelial growth factor (VEGF),
hepatocyte growth factor (HGF) and macrophage migration inhibitory factor (MIF) were
determined pre, 0', 30', 60' and 180' after both interventions. Metabolic stimuli and
perturbations were characterized by lactate, blood gas (pH, BE, HCO3-, PO2, PCO2), and
spirometric analysis. Both interventions caused a transient increase in circulating levels of
cortisol, testosterone, testosterone/cortisol-ratio, hGH, VEGF and HGF. Transient differences
between A- and P-recovery were found only for testosterone and HGF directly after exercise,
despite significant differences in metabolic disturbances (lactate, acid base status). Based on
the data of testosterone, hGH and the testosterone/cortisol-ratio, as well as on the data of
VEGF and HGF it appears that this kind of exercise protocol may promote anabolic
processes and may lead to pro-angiogenic conditions independent of the mode of recovery.
However transient differences between A- and P-recovery were shown for testosterone and
HGF. In contrast, cortisol and hGH, which are known to be sensitive for metabolic
perturbations (e.g. pH) showed no differences. Therefore, it is proposed that if a certain
threshold for metabolic perturbations is exceeded, a hormonal response is induced, which
does not differ between A- and P-recovery [13201].
Effect of mood changes
Endogenous hormones are essential on the control of physiological reactions and

adaptations during sport performance. One study aims to compare the mood state and the
salivary levels of cortisol and testosterone during an official female association football
tournament. Twenty female football players (22.85 ± 4.2 years) from the Portuguese
women's national team were included in the study. Mood, salivary cortisol and testosterone
levels were examined in five moments over the championship (M1, neutral measures; M2-
M5, on every match day). Saliva samples were collected before breakfast and immediately
after each match. Mood was measured by the profile of mood states questionnaire (POMS);
hormone levels were measure by immunoassay methods. Iceberg Profiles of POMS were
observed during all the moments of evaluation (M2-M5), showing a decrease in vigor and an
increase in tension and depression in both team defeats (M2 and M5). There is no
relationship between the hormones levels and the outcome of the competition, once cortisol
and testosterone decrease from pre-match to post-match in both wins (M2 and M5) and
defeats (M3 and M4). For testosterone the observed decrease is significantly different before
and after all matches. The results show a pattern in mood states behavior. Cortisol and
testosterone decrease after match and throughout the tournament, independently of the
1337
match outcome. The absence of hormone flutuations related to competiton performance
points out that top-level professional football players training systematically and regularly
seem to be very well adapted to competition stress effect [150245].
Influence of red color
One study examined the testosterone responses of men to an exercise bout simulating a
competitive sporting effort in order to determine if the wearing of red-colored apparel
influenced the hormonal response. Male subjects (n = 10) were placed into sets of matched-
pairs and performed VO2max cycle ergometry exercise test to exhaustion to simulate the
competitive effort. Each member of a pairing was randomly assigned to one of two treatment
groups-the wearing of red-colored clothing, or the wearing of black-colored clothing. Blood
samples were collected before exercise (REST), an immediate postexercise sample was
collected at exhaustion (EXH), and a final sample was taken at 15 min into recovery (REC)
from exercise. Blood was biochemically analyzed for total testosterone. In response to the
exercise, performance characteristics (i.e. VO2max and maximal workload) of treatment
groups did not differ significantly. A significant increase in the testosterone was observed in
both treatment groups postexercise at EXH and at REC as compared to REST. However, no
differences were observed between treatment groups in the before or postexercise hormonal
concentrations. These findings suggest that the wearing of red-colored apparel had no
affects on the testosterone responses to an exercise bout simulating a competition [06054].
Long rest interval promotes durable testosterone responses
The purpose of this study was to examine the influence of rest period duration (1 vs. 3-
minute between sets) on acute hormone responses to a high intensity and equal volume
bench press workout. Ten resistance trained men (25.2 ± 5.6 years; 78.2 ± 5.7 kg; 176.7 ±
5.4 cm; bench press relative strength: 1.3 ± 0.1 kg/kg of body mass) performed 2 bench
press workouts separated by 1 week. Each workout consisted of 5 sets of 3 repetitions
performed at 85 percent of 1-repetition maximum, with either 1or 3-minute rest between sets.
Circulating concentrations of total testosterone (TT), free testosterone (FT), cortisol (C),
testosterone/cortisol ratio (TT/C) and growth hormone (GH) were measured at pre-workout
(PRE), and immediately (T0), 15 minutes (T15) and 30 minutes (T30) post-workout. Rating of
perceived exertion (RPE) was recorded before and after each set. For TT, both rest lengths
enhanced all post-exercise verifications (T0, T15 and T30) compared to PRE, with 1-minute
showing decreases on T15 and T30 compared to T0. For FT, both 1 and 3-minute rest
protocols triggered augmentations on distinct post-exercise moments (T0 and T15 for 1-
minute; T15 and T30 for 3-minute) compared to PRE. The C values did not change
throughout any post-exercise verification for either rests. The TT/C ratio was significantly
elevated for both rests in all post-exercise moments compared to PRE. Finally, GH values
did not change for both rest lengths. In conclusion, although both short and long rest periods
enhanced acute testosterone values, the longer rest promoted a longer lasting elevation for
both TT and FT [150248].
Pre-game free testosterone concentrations and outcome
To assess the measures of salivary free-testosterone and cortisol concentrations across

selected rugby union matches according to the game outcome 22 professional male rugby
union players were studied across 6 games (3 wins and 3 losses). Hormone samples were
taken 40 minutes prior to the game (pre) and 15 minutes after (post). The hormonal data was
grouped and compared against competition outcomes. These competition outcomes included
wins and losses and a game ranked performance score (1 to 6). Across the entire team, pre-
1338
game testosterone concentrations were significantly higher during winning games than
losses. Analysis by playing position further revealed that, for the backs, pre-game
testosterone concentrations and the T/C ratio were significantly greater before a win than a
loss. Game ranked performance score (1 to 6) correlated to the team's pre-game
testosterone concentrations. The backs also showed that pre-game testosterone and the T/C
ratio correlated to game ranked performance. Analysis of the forwards' hormone
concentrations did not distinguish between game outcomes nor did it correlate with game
ranked performance. Game venue (home vs away) only affected post-game concentrations
of testosterone and cortisol. It was concluded that monitoring game day concentrations of
salivary free-testosterone may assist with identifying competitive readiness in rugby union
matches. The link between pre-game T/C ratio and rugby players in the backs position
suggest that monitoring weekly training loads and enhancing recovery modalities between
games may also assist with favourable performance and outcome in rugby union matches
[13196, 14044].
Pre-game testosterone level: home advantage
The home advantage is a robust phenomenon that occurs in the world of amateur and
professional sport. Athletic teams have been shown to win significantly more games in their
home venue as compared to their opponents' venue. Studies have suggested that the home
advantage may be related to familiarity with the facility, increased crowd density and even
pre-competition hormonal levels. The present study investigated pre-competition
physiological and psychological states of elite hockey players in the home and away venues.
Physiological measures included salivary cortisol and testosterone, which were assessed
using enzyme immunoassays. In addition, pre-competition psychological states were
assessed using the Competitive State Anxiety Inventory-2. Physiological measures indicated
that the players had significantly higher pre-game testosterone when playing in their home
venue as compared to their opponents' venue; however, this difference was not due to a pre-
game rise in testosterone while competing at home. Furthermore, players showed a trend
toward higher pre-game cortisol when playing in their home venue. Psychological measures
indicated that players were more self-confident when playing in their home venue and also
had higher somatic and cognitive anxiety when playing in their opponents' venue. The study
supports the notion that there are differences in pre-competition hormonal and psychological
states that may play a key role in the "home advantage" [06055].
Effect of testosterone on myoblasts
It was investigated the ability of testosterone (T) to restore differentiation in multiple

population doubled (PD) murine myoblasts, previously shown to have a reduced
differentiation in monolayer and bioengineered skeletal muscle cultures versus their parental
controls (CON). Cells were exposed to low serum conditions in the presence or absence of T
(100 nM) ± PI3K inhibitor (LY294002) for 72 h and 7 days (early and late muscle
differentiation respectively). Morphological analyses were performed to determine myotube
number, diameter (microm) and myonuclear accretion as indices of differentiation and
myotube hypertrophy. Changes in gene expression for myogenin, mTOR and myostatin were
also performed. Myotube diameter in CON and PD cells increased from 17.32 ±2.56 microm
to 21.02 ± 1.89 microm and 14.58 ± 2.66 microm to 18.29 ± 3.08 microm, respectively after
72 h of T exposure. The increase was comparable in both PD (+25 %) and CON cells (+21
%) suggesting a similar intrinsic ability to respond to exogenous T administration. T treatment
also significantly increased myonuclear accretion (% of myotubes expressing 5+ nuclei) in
both cell types after 7 days exposure. Addition of PI3K inhibitor (LY294002) in the presence
of T attenuated these effects in myotube morphology (in both cell types) suggesting a role for
1339
the PI3K pathway in T stimulated hypertrophy. Finally, PD myoblasts showed reduced
responsiveness to T stimulated mRNA expression of mTOR vs. CON cells and T also
reduced myostatin expression in PD myoblasts only. The present study demonstrates
testosterone administration improves hypertrophy in myoblasts that basally display impaired
differentiation and hypertrophic capacity versus their parental controls, the action of
testosterone in this model was mediated by PI3K/Akt pathway [13202].
Testosterone levels after concussions
A 27-year-old man was admitted to an outpatient clinic with symptoms of loss at libido,
erectile dysfunction and fatigue. He had been playing soccer from the age of 7, for the last 10
years as a high-level professional. During that time repeated mild head-trauma without loss
of consciousness had occurred, mainly triggered by excessive header-training and
occasional collisions. Serum levels of testosterone and luteinizing hormone were low. A
gonadotropin releasing hormone loading test revealed significant gonadotropin responses,
therefore pituitary gonadotropic insufficiency was unlikely. Further pituitary insufficiency of
any other axis was also excluded by insulin hypoglycemia test. Magnetic resonance imaging
of the brain revealed no significant abnormalities of the hypothalamic-pituitary unit.
Testosterone substitution, at first applied transdermally, then intramuscularly, was initiated
after approval by the National Anti Doping Agency. Four months later most of the symptoms
had regressed. It was concluded that pituitary deficiency in the course of craniocerebral
trauma is frequent and may be transient or permanent, mostly affecting somatotropic or
gonadotropic function. Hormonal imbalances may also be observed after mild but repeated
trauma without loss of consciousness and should be considered in cases of isolated pituitary
dysfunction, since such traumas may often occur in contacts sports such as boxing or
intensive soccer play [13203].
Overreaching
The purpose of one study was to study the effect of an 8-week Finnish military basic training
period (BT) on physical fitness, body composition, mood state, and serum biochemical
parameters among new conscripts; to determine the incidence of overreaching (OR); and to
evaluate whether initial levels or training responses differ between OR and noOR subjects.
Fifty-seven males (20 years) were evaluated before and during BT. Overreaching subjects
had to fulfill 3 of 5 criteria: decreased aerobic physical fitness (VO 2max), increased rating of
perceived exertion (RPE) in 45-minute submaximal test at 70 percent of VO2max or sick
absence from these tests, increased somatic or emotional symptoms of OR, and high
incidence of sick absence from daily service. VO2max improved during the first 4 weeks of BT.
During the second half of BT, a stagnation of increase in VO2max was observed, basal serum
sex hormone-binding globulin (SHBG) increased, and insulin-like growth factor-1 and cortisol
decreased. Furthermore, submaximal exercise-induced increases in cortisol, maximum heart
rate, and postexercise increase in blood lactate were blunted. Of 57 subjects, 33 percent
were classified as OR. They had higher basal SHBG before and after 4 and 7 weeks of
training and higher basal serum cortisol at the end of BT than noOR subjects. In addition, in
contrast to noOR, OR subjects exhibited no increase in basal testosterone/cortisol ratio but a
decrease in maximal La/RPE ratio during BT. As one-third of the conscripts were
overreached, training after BT should involve recovery training to prevent overtraining
syndrome from developing. The results confirm that serum SHBG, cortisol, and
testosterone/cortisol and maximal La/RPE ratios could be useful tools to indicate whether
training is too strenuous [11086].
Increased physical activity increases testosterone in obese men

1340
Obesity has reached epidemic proportions worldwide. Obesity results in reduced serum
testosterone levels, which causes many disorders in men. Lifestyle modifications (increased
physical activity and calorie restriction) can increase serum testosterone levels. However, it
is unknown whether increased physical activity or calorie restriction during lifestyle
modifications has a greater effects on serum testosterone levels. Forty-one overweight and
obese men completed a 12-week lifestyle modification program (aerobic exercise training
and calorie restriction). It was measured serum testosterone levels, the number of steps, and
the total energy intake. It was divided participants into two groups based on the median
change in the number of steps (high or low physical activities) or that in calorie restriction
(high or low calorie restrictions). After the program, serum testosterone levels were
significantly increased. Serum testosterone levels in the high physical activity group were
significantly higher than those in the low activity group. This effect was not observed between
the groups based on calorie restriction levels. It was found a significant positive correlation
between the changes in serum testosterone levels and the number of steps. The results
suggested that an increase in physical activity greatly affected the increased serum
testosterone levels in overweight and obese men during lifestyle modification [150250].
Females
Physical exercise is known to strongly stimulate the endocrine system in both sexes. Among
these hormones, androgens (e.g. testosterone, androstenedione, dehydroepiandrosterone)
play key roles in the reproductive system, muscle growth and the prevention of bone loss. In
female athletes, excessive physical exercise may lead to disorders, including delay in the
onset of puberty, amenorrhoea and premature osteoporosis. The free and total fractions of
circulating androgens vary in response to acute and chronic exercise/training (depending on
the type), but the physiological role of these changes is not completely understood. Although
it is commonly accepted that only the free fraction of steroids has a biological action, this
hypothesis has recently been challenged. Indeed, a change in the total fraction of androgen
concentration may have a significant impact on cells (inducing genomic or non-genomic
signalling). The purpose of one review, therefore, was to visit the exercise-induced changes
in androgen concentrations and emphasize their potential effects on female physiology.
Despite some discrepancies in the published studies (generally due to differences in the
types and intensities of the exercises studied, in the hormonal status of the group of women
investigated and in the methods for androgen determination), exercise is globally able to
induce an increase in circulating androgens. This can be observed after both resistance and
endurance acute exercises. For chronic exercise/training, the picture is definitely less clear
and there are even circumstances where exercise leads to a decrease of circulating
androgens. It was suggest that those changes have significant impact on female physiology
and physical performance [11088].
The purpose of one study was to investigate the influence of a 14-week swimming training
program on psychological, hormonal, and performance parameters of elite women
swimmers. Ten Olympic and international-level elite women swimmers were evaluated 4
times along the experiment (i.e., in T1, T2, T3, and T4). On the first day at 8:00 am, before
the blood collecting at rest for the determination of hormonal parameters, the athletes had
their psychological parameters assessed by the profile of mood-state questionnaire. At 3:00
am, the swimmers had their anaerobic threshold assessed. On the second day at 3:00 am,
the athletes had their alactic anaerobic performance measured. Vigor score and testosterone
levels were significantly lower in T4 compared with T3. In addition, the rate between the peak
blood lactate concentration and the median velocity obtained in the alactic anaerobic
performance test increased in T4 compared with T3. For practical applications, the swimming
1341
coaches should not use a tapering with the present characteristics to avoid unexpected
results [11089].
The association between androgens and competition in women has been understudied
compared with men. The current study examined the link between testosterone and
competition in elite female athletes, using a sample of female wrestlers that included athletes
competing at both the national and international level. In a repeated-measures design, saliva
samples were collected before and after wrestling bouts, with comparable samples of wins
and losses, and subsequently analyzed for testosterone. Study results showed a 22 percent
increase in circulating bioavailable testosterone from pre- to postbout, which was a
significant difference. There was no significant difference in testosterone between win or loss
outcomes. These findings-showing a link between individual head-to-head competition and
testosterone in women demonstrate that women's androgenic responses to environmental
contexts are dynamic and may be an important factor to address in research on competitive
In a previous study, sprint training has been shown to increase muscle cross-sectional area
in women but not in men [Eur J Appl Physiol Occup Physiol 1996; 74: 375]. It was
hypothesized that sprint exercise induces a different hormonal response in women than in
men. Such a difference may contribute to explaining the observed gender difference in
training response. Metabolic and hormonal response to three 30-s sprints with 20-min rest
between the sprints was studied in 18 physically active men and women. Significant
accumulation of blood lactate and plasma ammonia after sprint exercise was greater in men.
Serum insulin increased after sprint exercise more so in women than in men, while plasma
glucose increased in men, but not in women. Serum growth hormone (GH) increased in both
women and men reaching similar peak levels, but with different time courses. In women the
peak serum GH level was observed after sprint 1, whereas in men the peak was observed
after sprint 3. Serum testosterone tended to decrease in men and increase in women. Serum
cortisol increased approx. 10-15 percent after sprint exercise, independent of gender. It was
concluded that women elicited a greater response of serum GH and insulin to sprint exercise.
This may contribute to explaining the earlier observed muscle hypertrophy in women in
response to sprint training [09103].
Effects in a young female
A 14-year-old Caucasian girl was referred to the endocrine clinic for evaluation of voice
deepening, facial hirsutism, and acne starting 2 years previously. She had been a
competitive tennis player since age 7 years, practicing for 4-6 hours daily. Adrenal
ultrasonography revealed a round left 4.6 × 5.3-cm adrenal mass. Laparoscopic left
adrenalectomy was performed. The histologic findings were compatible with a benign
adrenocortical tumor. Postoperatively, androgen levels dropped to within the normal range.
Breast development proceeded normally, menarche occurred 2 months after tumor
resection, and menses has been regular since then. Muscle strength of the dominant and
nondominant upper and lower extremities was measured 1 month before surgery and 1 year
later, using an isokinetic dynamometer (Biodex Systems II, Biodex, Shirley, NY, USA). There
was no significant decrease in overall muscle strength after removal of the virilizing tumor
and the marked drop in circulating androgens. In addition, the patient maintained her age
category, number 1, national tennis ranking. The results suggest that even extremely high
levels of tumor-related circulating androgens had no evident effect on muscle strength and
competitive performance in a female adolescent tennis player. The lack of beneficial effect
on performance in adolescents, combined with the potentially hazardous side effects of
anabolic steroids, suggests that teenage athletes should avoid their use [11090].
1342
Gender differences in testosterone and cortisol response to competition
One study examined intra-individual change in testosterone, cortisol, and hormone-behavior

relationships in response to a rowing ergometer competition. Forty-six members (23 females)
of a university crew team provided saliva samples before, 20- and 40-min post-competition,
as well as baselines on a non-competition day. Behavioral assessments included measures
of previous rowing experience, dominance, competitiveness, bonding with teammates, pre-
and post-competition mental state and performance. Men's and women's endocrine
responses to this competitive setting were more different than alike and varied by level of
competitive experience, the specific phase of the competitive event, and the particular
hormone measured. Inter-individual differences in testosterone and cortisol were differentially
associated with social affiliation with teammates but rarely with dominance or
competitiveness. Theoretically, the findings support the integration of features of the “tend
and befriend” model with the biosocial model of status, and suggest future research
directions that may lead to clarification and refinement of those ideas [05073].
Adolecents
One study investigated the effect of repeated bouts of short-term, high-intensity cycling
exercise on the salivary cortisol, testosterone and immunoglobulin (A) concentrations of 15-
16 year old boys. Seventeen apparently healthy schoolchildren (aged 15.5 + 0.4 years)
participated in this study. All participants completed 6 x 8 s sprints, interspersed with 30 s
recovery intervals on a cycle ergometer. Using the passive drool method, salivary samples
were taken before, and 5 min after, exercise. There were significant changes in both salivary
testosterone and cortisol, 5 min after completing 6 x 8 s cycle sprints. No significant
differences were recorded for immunoglobulin A. The increases in testosterone and cortisol
reported confirm that repeated bouts of short-term, high-intensity exercise produces
significant physiological hormonal responses in adolescent boys, but does not affect mucosal
immune function [09097].
Effect of soccer
The main aim of one study was to analyse the impact of an official match on hormonal and
redox status, muscle damage and inflammation and neuromuscular function. Seven high-
level male soccer players from the same team performed an official match and data were
collected 72 h before, 24, 48 and 72 h post-match. Plasma testosterone/cortisol ratio (T/C),
creatine kinase (CK), superoxide dismutase (SOD), glutathione peroxidase (GPX) and
reductase (GR) activities, myoglobin (Mb), C-reactive protein (CRP), uric acid (UA), protein
sulfhydryls (-SH), malondialdehyde (MDA) concentrations and total antioxidant status (TAS)
were measured. Sprint, jump and change of direction performance, and maximal isokinetic
knee extension and flexion were obtained as neuromuscular functional parameters. Cortisol
increased and T/C decreased until 48 h recovery. Mb, CRP and -SH increased at 24 h and
CK, TAS, SOD and MDA increased up to 48 h recovery. GR increased and GPX decreased
at 24 h recovery. Jump performance decreased 24 h post-match, but no significant
alterations in sprint, change of direction and muscle strength were observed. In conclusion,
an official match resulted in changes in plasma biomarkers until 48 h of recovery period,
without major impact on performance [13198].
Effect of soccer on testosterone to cortisol ratio

Competitive soccer engages many of the body's systems to a major extent. The
musculoskeletal, nervous, immune and metabolic systems are stressed to a point where
1343
recovery strategies post-exercise become influential in preparing for the next match. Intense
activity at a 7-day training camp causes participants to experience lowered concentrations of
non-killer cells and T-helper cells. Two consecutive games in 24 h produce disturbances in
the testosterone-cortisol ratio. When competitive schedules are congested, the recovery
process should be optimized for performance capabilities to be restored to normal as soon
as possible. There is evidence that glycogen stores are reduced near to depletion at the end
of a soccer game and that a diet high in carbohydrates can aid recovery. Water alone is not
the best means of restoring body fluids, since carbohydrate-electrolyte drinks display better
intestinal absorption and reduce urine output. Some relief from muscle soreness may be
achieved by means of a warm-down. Deep-water running regimens can replace conventional
physical training in the days after competition. Massage, cryotherapy and alternative
therapies have not been shown to be consistently effective. It is concluded that optimizing
recovery post-exercise depends on a combination of factors that incorporate a consideration
of individual differences and lifestyle factors. The procedures to facilitate recovery processes
should start immediately the game or training finishes. Match administrators and tournament
planners should consider the stressful consequences for players in periods of congested
fixtures and alleviate the physiological strain as far as possible by allowing 72 h between
competitive games. This frequency of competition is unlikely to be sustainable in the long
term [05081].
Effect of golf
The purpose of one investigation was to study the effects of 36 continuous holes of
competitive golf on salivary testosterone, cortisol, and testosterone-to-cortisol ratio and their
relation to performance in eight elite male collegiate golfers (age 20 years). Thirty-six holes
of a 54-hole NCAA golf tournament were played on the first day of the competition. A saliva
sample was taken 45 minutes prior to the round and immediately following each hole for a
total of 37 samples per subject. Time matched baseline samples were collected on a
different day to account for circadian variation. Six-hole areas under the curve (AUC) values
were calculated for endocrine measures. Significant increases were noted for cortisol during
competition, however, testosterone did not change during competition compared to baseline.
Testosterone-to-cortisol (T/C) ratio was significantly lower throughout the competition
compared to baseline measures. Thirty-six-hole AUC testosterone-to-cortisol ratio response
was correlated to 36-hole score. There was a high correlation between pre-round
testosterone, T/C ratio response, and 36-hole score. CSAI-2 somatic anxiety was correlated
to pre-round cortisol and testosterone response. These results indicate a significant
hormonal response during 10 hours of competitive golf. Good golf performance (low golf
scores) in this competition was related to low T/C ratio [07088].
Low testosterone in basket players
One study aimed to examine and compare mood states profile and physical performance
during different training phases between 2 groups of adolescent basketball players that were
differentiated according to baseline testosterone concentration (T). The basketball players
were submitted to an intensified training period (OVL) followed by a tapering period (TP).
Twenty-three young male basketball players initiated the study. Experimental criteria data
were used to stratify 16 players into high-testosterone (HTC) or low-testosterone (LTC)
concentration groups. All the 16 athletes undertook 5 weeks of OVL followed by a 3-week
TP. Saliva sampling, Yo-Yo intermittent recovery level 1 (Yo-Yo IRL1) test and the T-test
were conducted at the beginning (T1), after OVL (T2), and after TP (T3). A similar increase in
internal training load was observed during OVL when compared with TP in both groups. No
difference in mood states was observed between groups; however, LTC displayed a higher
1344
score for fatigue and a lower score for energy index in OVL, compared with TP. A significant
improvement in the Yo-Yo IRL1 test and the T-test was observed (T1 to T3), with no
difference between groups. In conclusion, these results suggest that low-testosterone
athletes may be more susceptible to changes in mood states during intensified training
periods. In addition, data indicate that a periodized training program successfully improved
the physical performance (endurance and agility) of young basketball players; however, this
improvement was not affected by testosterone level [150246].
Biking
To determine whether cycling has an effect on serum PSA, gonadotropins, and uroflowmetric
parameters a total of 34 healthy male athletes from the National Cycling Team and 24
healthy male student volunteers from University and medical staff were prospectively
enrolled in a study. Blood samples for serum total prostate-specific antigen (tPSA), free PSA
(fPSA, fPSA/tPSA, follicle-stimulating hormone (FSH), luteinizing hormone (LH), and
testosterone determinations were obtained before and after cyclists completed 300 km
bicycle ride and with each cyclist seated without changing posture and with minimal
movement for 10 minutes before blood collection. The cyclists also performed uroflowmetric
and postvoid residual urine volume analysis before, and 1 hour after cycling course. Blood
samples from the control group were drawn for serum hormones. They also underwent
uroflowmetric and postvoid residual analysis. The athletes and the control group were well
matched by age. There was no significant difference between the two groups in terms of
serum tPSA, fPSA, f/t PSA values, FSH, LH, and testosterone levels and uroflowmetric
parameters. The differences between pre- and postcycling values for tPSA, fPSA, f/t PSA,
FSH, LH, and uroflowmetric parameters were not statistically significant. The postcycling
serum testosterone level was significantly lower than precycling levels (mean, 6034 ng/dL vs
425 ng/dL. There was no correlation between body mass index values, postcycling serum
FSH, LH levels, age, and testosterone levels [09098].
Rowing
The aim of one study was to investigate the hormonal response at rest and during maximal
2,000 m rowing ergometer test in 12 highly trained male rowers before and after 3 week
heavy training, and after 2 week tapering periods. Venous blood samples were obtained
before, immediately after and after 30 min of recovery of the rowing performance test.
Testosterone, cortisol and sex hormone binding globulin were measured, and free
testosterone and the free testosterone: cortisol ratio calculated. Mean training time was
about 100 percent higher during the heavy training period (17.5 h/week) compared to the
tapering period (8.9 h/week). Two thousand meter rowing ergometer performance
parameters were not different between 3 tests. Resting testosterone and cortisol values were
not different between 3 tests. Three week heavy training period induced significant
reductions in resting free testosterone and free testosterone: cortisol ratio. Resting free
testosterone and free testosterone: cortisol ratio were increased to the pretraining level after
2 week tapering period. A significantly lower maximal exercise-induced increase of the free
testosterone level was measured after heavy training period. The response of cortisol was
unchanged and free testosterone: cortisol ratio demonstrated a trend for a decrease after
heavy training period. The findings indicate that the first sign of decreased adaptivity in
athletes is a decreased resting level of free testosterone and a lower maximal exercise-
induced increase in free testosterone concentration. In addition, heavy training load higher
than 1,000 min per week can be sustained for 3 weeks when sufficient tapering period is
followed in highly trained male rowers [05075].
1345
Response to marathon running
Exercise is known to be a powerful stimulus for the endocrine system. The hormonal
response to exercise is dependent on several factors including the intensity, duration, mode
of exercise (endurance versus resistance), and training status of the subject. The aim of one
study was to determine the steroid hormonal response (immediately after a race and 1 week
later) to endurance exercise under the real conditions of the classic Athens marathon in a
group of well-trained, middle-aged, non-elite athletes. Blood samples were drawn 1 week
before the race, directly after completion of the race, and 1 week later. Serum cortisol and
prolactin showed distinct rises 1 h after the race and returned to baseline 1 week later.
Androstenedione and dehydroepiandrosterone sulphate did not show any changes. Total
testosterone as well as free testosterone dropped significantly 1 h after the race but returned
to baseline 1 week later. In this particular group of non-elite, middle-aged marathon runners,
the race resulted in an acute increase in serum cortisol and prolactin levels and in a
concomitant decline in testosterone level. The aforementioned changes returned to baseline
1 week later [09099].
Testosterone to cortisol ratio after exercise
The aim of one study was to assess the response of salivary-free testosterone and cortisol
concentrations across selected mid-week skill based training sessions and their association
with subsequent match outcome 3 days later. Twenty-two rugby union players were
assessed for salivary free-testosterone and cortisol concentrations before and after a mid-
week training session over 6 consecutive weeks. The relative percentage change (response)
in the testosterone and cortisol concentration and the testosterone to cortisol (T/C) ratio was
also determined. Game-day analysis consisted of pre-match testosterone concentrations and
match outcome. Data was pooled across the winning (n=3) and losing (n=3) outcomes. The
mid-week pre-training T/C ratio was significantly lower prior to a win than a loss and the
increase in the preto post-T/C ratio before a win was significant. The increase in the pre- to
post-testosterone concentration before a win was also shown to be significant. However, the
relative changes in testosterone prior to games that were won were not statistically different
to that of games lost. Significant relationships were also demonstrated between game day
pre-testosterone concentrations and the mid-week cortisol response and mid-week T/C ratio
response. In conclusion, a mid-week measurement of the T/C ratio against a skill based
training session appears to show some potential as an early indicator of subsequent
successfully executed performances in competitive rugby union. If this work is subsequently
validated, further monitoring of mid-week hormone concentrations in response to a mixed
psychological-physical training session may assist with assessing competitive readiness
leading up to competition [14262].
Cortisol and testosterone concentrations in wheelchair athletes

It is yet unknown how upper body exercise combined with high ambient temperatures affects
plasma testosterone and cortisol concentrations and furthermore, how these hormones
respond to exercise in people suffering spinal cord injuries. The purpose of this study was to
characterize plasma testosterone and cortisol responses to upper body exercise in
wheelchair athletes (WA) compared to able-bodied individuals (AB) at two ambient
temperatures. Four WA [mean age 36 (SEM 13) years, mean body mass 66.9 (SEM 11.8)
kg, injury level T7-T11], matched with five AB [mean age 33.4 (SEM 8.9) years, mean body
mass 72.5 (SEM 13.1) kg] exercised (cross-over design) for 20 min on a wheelchair
ergometer (0.03 kg resistance.kg-1 body mass) at 25 degrees C and 32 degrees C. Blood
samples were obtained before (PRE), at min 10 (MID), and min 20 (END) of exercise. No
differences were found between results obtained at 25 degrees C and 32 degrees C for any
1346
physiological variable studied and therefore these data were combined. Pre-exercise
testosterone concentration was lower in WA, and increased PRE to END only in WA. Cortisol
concentrations were similar between groups before and during exercise, despite higher rectal
temperatures in WA compared to AB, at MID [37.21 (SEM 0.14) and 37.02 (SEM 0.08)
degrees C, respectively] and END. Plasma norepinephrine responses were similar between
groups. In conclusion, there were no differences in plasma cortisol concentrations, which
may have been due to the low relative exercise intensities employed. The greater exercise
response in WA for plasma testosterone should be confirmed on a larger population. It could
have been the result of the lower plasma testosterone concentrations at rest in our group
[01085].
Saliva testosterone during competition
Men and women from a southern university's intercollegiate varsity soccer teams gave saliva
samples before and after league matches. For the men, samples were collected for a single
game ending in victory. For the women, samples were collected for two games, one of which
ended in victory and the other in defeat. For both men and women, match competition
substantially increased saliva cortisol (C) and testosterone (T). For women, play-related
increases in saliva C and T were similar in victory and defeat. For both men and women,
saliva T (but not C) was highly correlated with teammate ratings of playing abilities--one
measure of status with teammates--and self-ratings of social connectedness with
teammates, but the nature of the relationship was different according to sex. For men, play-
related changes in T were positively correlated with these variables, but before-game T was
not. For women, before-game T was positively related to each of these variables, but play-
related changes in T were not. Status and social connectedness are pertinent to
understanding interpersonal dynamics in most social groups, and these results – which link T
and these variables in an athletic context – may have relevance for understanding social
relationships in other settings [05082].
Effect of amount of training on testosterone levels
Effect of high-intensity training
The aim of one study was to determine the acute neuromuscular, biochemical and endocrine
responses to a maximal speed training (MST) session. Eighteen male rugby players
completed the protocol, which involved performing six maximal effort repetitions of 50 m
running sprints with 5 minutes recovery between each sprint. Testosterone (T), cortisol (C),
creatine kinase (CK), lactate (La), perceived muscle soreness (MS) and counter movement
jump were collected immediately pre (PRE), immediately post (IP), 2 hours post (2P) and 24
hours post (24P) the sprint session. A bimodal recovery pattern was observed from the jump
parameters with several declining significantly (p ≤ 0.05) IP, recovering 2P and suffering a
secondary decline 24P. CK and perceived MS were elevated IP and continued to rise
throughout the protocol, while La was only elevated IP. T and C were unaffected IP but
showed significant declines 2P. These data indicate that MST results in a bimodal recovery
pattern of neuromuscular function with changes most likely being related to metabolic and
biochemical responses [150247].
Responses to different intensities of exercise
In endurance athletes, serum concentrations of sex steroid hormones increased only with
high-intensity exercise. Moreover, different responses of sex steroid hormone secretions
1347
were induced by different exercise intensities in individuals with low and high levels of
physical fitness. Previous studies have shown that acute exercise elevates sex steroid
hormone concentrations in rodents and that sprint exercise increases circulating testosterone
in healthy young men. However, the effect of different exercise intensities on sex steroid
hormone responses at different levels of physical fitness is still unclear. In one study, it was
compared circulating sex steroid hormone responses at different exercise intensities in
athletes and non-athletes. Eight male endurance athletes and 11 non-athletes performed two
15 min sessions of submaximal exercise at 40 and 70 percent peak oxygen uptake (VO2peak),
respectively, and exercised at 90 percent VO2peak until exhaustion. Venous blood samples
were collected during the last minute of each submaximal exercise session and immediately
after exhaustion. Acute exercise at 40, 70 and 90 percent VO2peak induced significant
increases in serum dehydroepiandrosterone (DHEA) and free testosterone concentrations in
non-athletes. On the contrary, only 90 percent VO2peak exercise led to an increase in serum
DHEA and free testosterone concentrations in athletes. Serum 5alpha-dihydrotestosterone
concentrations increased with 90 percent VO2peak exercise in both athletes and non-athletes.
Additionally, serum estradiol concentrations were significantly increased at moderate and
high exercise intensities in both athletes and non-athletes. These results indicate that in
endurance athletes, serum sex steroid hormone concentrations, especially serum DHEA and
5alpha-dihydrotestosterone concentrations, increased only with high-intensity exercise,
suggesting that different responses of sex steroid hormone secretion are induced by different
exercise intensities in individuals with low and high levels of physical fitness. In athletes,
therefore, high-intensity exercise may be required to increase circulating sex steroid
hormone concentrations [150249].
Response to three football matches in a week
It was examined effects of a three-game, 1-week microcycle (G1, G2, G3) on recovery of
performance and inflammatory responses in professional male footballers. Players were
randomized into an experimental (EXP; n=20) and a control group (CON; n=20). Blood was
drawn and repeated sprint ability (RSA), muscle soreness and knee range of motion (KJRM)
were determined pre- and post-games and during recovery. High-intensity running during G2
was 7-14 percent less compared to G1 and G3. RSA declined in EXP by 2-9 percent 3 days
post-game with G2 causing the greatest performance impairment. In EXP, game play
increased muscle soreness (sevenfold) compared to CON with G2 inducing the greatest rise,
while KJRM was attenuated post-game in EXP compared to CON (5-7 %) and recovered
slower post G2 and G3 than G1. CK, CRP, sVCAM-1, sP-Selectin and cortisol peaked 48 h
post-games with G2 eliciting the greatest increase. Leukocyte count, testosterone, IL-1beta
and IL6 responses, although altered 24 h post each game, were comparable among games.
Plasma TBARS and protein carbonyls rose by about 50 percent post-games with G2 eliciting
the greatest increase 48 h of recovery. Reduced to oxidized glutathione ratio declined for 24
h post all games with G2 displaying the slowest recovery. Total antioxidant capacity and
glutathione peroxidase activity increased (9-56 %) for 48 h in response to game play. In
summary, post-game performance recovery and inflammatory adaptations in response to a
three-game weekly microcycle displayed a different response pattern, with strong indications
of a largest physiological stress and fatigue after the middle game that was preceded by only
a 3-day recovery [150242].
Response to 164-km road cycling in a hot environment
One study investigated the acute endocrine responses to a 164-km road cycling event in a
hot environment. Thirty-four male experienced cyclists (49.1 ± 8.3 years, 86.8 ± 12.5 kg,
178.1 ± 5.1 cm) participating in a 164-km road cycling event were recruited. Blood samples
1348
were collected within 0.3-2.0 h before the start (PRE: 0500-0700 h) and immediately
following the ride (POST). Samples were analysed for testosterone, growth hormone (GH),
cortisol and interleukin-6 (IL-6). The temperature and humidity during the event were
35.3 ± 4.9°C and 47.2 ± 14.0 percent, respectively. Based on the finishing time, results for
the fastest (FAST, 305 ± 10 min) and the slowest (SLOW, 467 ± 31 min) quartiles were
compared. At POST, testosterone concentration was significantly lower (PRE, 20.8 ± 8.6;
POST, 18.2 ± 6.7 nmol/L), while GH (PRE, 0.3 ± 0.1; POST, 2.3 ± 0.3 microg/L), cortisol
(PRE, 661 ± 165; POST, 1073 ± 260 nmol/L) and IL-6 (PRE, 4.0 ± 3.4; POST,
22.4 ± 15.2 pg/mL) concentrations were significantly higher than those at PRE. At POST, GH
and cortisol were significantly higher for the FAST group than for the SLOW group (GH,
3.6 ± 2.0 and 1.0 ± 0.8 microg/L; cortisol, 1187 ± 209 and 867 ± 215 nmol/L). Participation in
an ultra-endurance road cycling event in a hot environment induced significant acute
changes in concentrations of circulating hormones, with a greater augmentation of GH and
cortisol in those completing the ride fastest [150243].
Responses to a competitive 5,000 m race
The exercise-stress model can be a model of temporary immunosuppression that occurs

after severe physical and psychological stress. It also allows for the study of interactions
between the endocrine and the immune systems. This study examined changes in salivary
hormonal and immune factors in athletes in response to physical and psychological stress in
a 5,000 m running competition. Eighteen endurance-trained runners (9 males and 9 females)
participated in this study. All participants completed a competitive 5,000 m race. Saliva
samples were collected 10 min before (PRE) and 10 min after (POST) the competition.
Saliva was analyzed for α-amylase activity, concentrations of salivary immunoglobulin A
(SIgA), lactoferrin, cortisol, testosterone and total protein. Although the concentrations of
salivary TP, SIgA, lactoferrin, cortisol and alpha-amylase activity were significantly increased
immediately after a competitive 5,000 m race, the secretion rates of these factors were not
significantly altered in both male and female groups. Additionally, basal levels of SIgA and
alpha-amylase activity were significantly higher in female runners than in male runners. This
gender difference still existed after the race. The secretion rates of testosterone decreased
significantly after the race in the male, but not in the female group. Moreover, testosterone-
to-cortisol (T/C) ratios were significantly lower post-competition compared to pre-competition
in both male and female athletes. The T/C ratio had been used as a performance index for
athletes. Whether there are correlations between these changes of their physiological
characteristics and better running performance need further investigations [015244].
Effect at orthopedic ligament surgery
The anterior cruciate ligament (ACL) is one of four major ligaments in the knee that provide
stability during physical activity. A tear in the ACL is characterized by joint instability that
leads to decreased activity, knee dysfunction, reduced quality of life and a loss of muscle
mass and strength. While rehabilitation is the standard-of-care for return to daily function,
additional surgical reconstruction can provide individuals with an opportunity to return to
sports and strenuous physical activity. Over 200,000 ACL reconstructions are performed in
the United States each year, and rehabilitation following surgery is slow and expensive. One
possible method to improve the recovery process is the use of intramuscular testosterone,
which has been shown to increase muscle mass and strength independent of exercise. With
short-term use of supraphysiologic doses of testosterone, we hope to reduce loss of muscle
mass and strength and minimize loss of physical function following ACL reconstruction
compared to standard-of-care alone. One study was a double-blinded randomized control
1349
trial. Men 18-50 years of age, scheduled for ACL reconstruction are randomized into two
groups. Participants randomized to the testosterone group receive intramuscular
testosterone administration once per week for 8 weeks starting 2 weeks prior to surgery.
Participants randomized to the control group receive a saline placebo intramuscularly instead
of testosterone. Lean mass, muscle strength and physical function are measured at 5 time
points: 2 weeks pre-surgery, 1 day pre-surgery, and 6, 12, 24 weeks post-surgery. Both
groups follow standard-of-care rehabilitation protocol. It is believed that testosterone
therapy will help reduce the loss of muscle mass and strength experienced after ACL injury
and reconstruction. Hopefully this will provide a way to shorten the rehabilitation necessary
following ACL reconstruction. If successful, testosterone therapy may also be used for other
injuries involving trauma and muscle atrophy [14763].
Rehabilitation and testosterone
Traditionally, androgen is known not only as a material for intensifying the masculine
sexuality, but also as a material to assist anabolic mechanism. Therefore, in the viewpoint of
action mechanism of testosterone, there might be two types of rehabilitation, and both of
these mechanisms might have effective roles in rehabilitation. One is rehabilitation form
disease including injury or disease (recovery), the other is rehabilitation from aging
(rejuvenation). In fact, these two concepts are overlap significantly. In view of the recovery
aspect, testosterone has a critical role in mediating the improved muscle mass and is
essential for skeletal muscle growth. Therefore, testosterone therapy might improve exercise
capacity, muscle strength, glucose metabolism, and baroreflex. Actually, it was reported that
exercise induces an increase of sex steroid, testosterone and exercise-induced increase of
testosterone in muscle may positively impact age-related concerns such as life-related
diseases and sarcopenia. There are some beneficial effects of testosterone therapy on
functional capacity, cardiovascular parameters, and quality of life in patients with congestive
heart failure. Therefore, in view of rehabilitation from disease, testosterone administration
during exercise rehabilitation is feasible and can positively impact health outcomes in elderly
males even those with congestive heart failure who have low testosterone. In addition, there
might be additional beneficial effects on mood, energy, and sense of well-being. In another
viewpoint of rehabilitation, rejuvenation, testosterone administration showed some benefit on
sexual health related outcome like sexual desire and performance. In addition, testosterone
plays a critical role in the mood and cognitive function of humans. There was evidence
supporting the existence of a relationship between testosterone and depression and it was
reported that after administration of testosterone, depression showed a significant level of
improvement. Testosterone also seems to influence certain aspects of cognition. This effect
of testosterone on behavioral performances is mediated in part through androgen receptors
on the brain. Testosterone replacement in men diagnosed with hypogonadism has shown a
beneficial effect on several cardiovascular risk factors, functional exercise capacity and
improved mortality. Testosterone was also reported as an efficacious paradigm in
management of sarcopenia, loss of skeletal muscle mass and strength that occurs with
aging. In addition, an important regulatory enzyme of inflammation, nuclear factor-B inducing
kinase, which may regulate human skeletal muscle catabolism, and that appears to be
counter-regulated by administration of testosterone. This is important because a number of
age-related clinical circumstances trigger acute and chronic muscle loss, including cancer,
chronic obstructive pulmonary disease, hospitalization, acute and chronic illness, and
diseases in which systemic inflammation occurs [150226].
Actually, some reports proved the positive effects of steroid in rehabilitation. After anabolic
steroid of nandrolene in lean elderly women after femoral neck fracture, less dependency
1350
and positive effects on lean body mass, activities of daily living, and health related quality of
life were reported. In another report, there were also positive effects on muscle mass, bone
mineral density, and clinical function after administration of nandrolene in patients with hip
fracture. There was even the effect of reducing the number of hospitalizations and duration of
hospital administrations of those undernourished old people in case of administration of
testosterone with high calorie oral nutrition. However, unfortunately there was a lack of well
systemized and randomized prospective trials proving the efficacy of testosterone
administration in patients needing rehabilitation [150226].
Testosterone and metabolic syndrome
In 1998 Reaven presented his concept of Syndrome X at the Banting Lecture of the
American Diabetes Association Annual Meeting. He defined it as:
- resistance to insulin-stimulated glucose uptake

- glucose intolerance
- hyperinsulinism
- increased very low density lipoprotein (triglycerides)
- decreased high-density lipoprotein (HDL) cholesterol
- hypertension
These criteria were than developed by an expert panel on detection, evaluation and
treatment of high blood cholesterol in adults, and they have become widely accepted for
identification of the MetS. While some have emphasized our lack of understanding of the
cause for this grouping of clinical findings, this definition of MetS has made clinicians and
patients more aware of these problems. However, the components usually are treated
individually. However, controversies surround the usefulness of identifying patients with the
metabolic syndrome (MetS). Many of the components are accepted risk factors for
cardiovascular disease (CVD). Although the MetS as defined includes many men with insulin
resistance, insulin resistance is not universal. The low total testosterone (TT) and sex
hormone binding globulin (SHBG) levels in these men are best explained by the
hyperinsulinism and increased inflammatory cytokines that accompany obesity and
increased waist circumference. It is informative that low SHBG levels predict future
development of the MetS. Evidence is strong relating low TT levels to CVD in men with and
without the MetS; however, the relationship may not be causal. The recommendations of the
International Diabetes Federation for managing the MetS include cardiovascular risk
assessment, lifestyle changes in diet, exercise, weight reduction and treatment of individual
components of the MetS. Unfortunately, it is uncommon to see patients with the MetS lose
and maintain a 10 percent weight loss. Recent reports showing testosterone treatment
induced dramatic changes in weight, waist circumference, insulin sensitivity, hemoglobin A1c
levels and improvements in each of the components of the MetS are intriguing. While some
observational studies have reported that testosterone replacement therapy increases
cardiovascular events, the Food and Drug Administration in the United States has reviewed
these reports and found them to be seriously flawed. Large, randomized, placebo-controlled
trials are needed to provide more definitive data regarding the efficacy and safety of this
treatment in middle and older men with the MetS and low TT levels [150241].
Effects on immune system
Intense exercise is known to cause temporary impairments in immune function. Few studies,
1351
however, have investigated the effects of intense competitive exercise on immunoendocrine
variables in elite team sport athletes. The aim of one study was to evaluate the time course
of changes in selected immunoendocrine and inflammatory markers following an
international rugby union game. Blood samples were taken from players (n=10) on camp
entry, the morning of the game (pre), immediately after (post) and 14 and 38 h into a passive
recovery period. Players lost 1.4 + 0.2 kg of body mass during the game (ambient conditions,
11 degrees C). An acute phase inflammatory response was observed as reflected through
immediate significant increases in serum cortisol and IL-6 (post) followed by delayed
increases in serum creatine kinase (CK; 14 h) activity and C-reactive protein (CRP; 38 h.
Following a large decrease in serum testosterone to cortisol (T/C) ratio immediately post and
14 h after exercise, T/C values then significantly increased above those observed at camp
entry 38 h into recovery. This rebound anabolic stimulus may represent a physiological
requirement for recovery following intense tissue damage resulting from game collisions
[09100].
Testosterone and age
To examine the association between aging and physical function in men by testing a
theoretically based model of aging, hormones, body composition, strength, and physical
function with data obtained from men enrolled in the Boston Area Community Health/Bone
(BACH/Bone) a cross-sectional, observational survey was performed population-based.
Testosterone, estradiol, sex hormone-binding globulin, lean and fat mass, grip strength, and
summated index of physical function (derived from walk and chair stand tests). Measures of
grip strength and physical function declined strongly with age. For instance, 10 years of
aging was associated with a 0.49-point difference (scale 0-7) in physical function. Age
differences in total testosterone and estradiol concentrations were smaller than age
differences in their free fractions. Weak or nonsignificant age-adjusted correlations were
observed between hormones and measures of physical function, although path analysis
revealed a positive association between testosterone and appendicular lean mass and a
strong negative association between testosterone and total fat mass. Lean and fat mass, in
turn, were strongly associated with grip strength and physical function, indicating the
possibility that testosterone influences physical function via indirect associations with body
composition. The authors concluded that age-related decline in serum testosterone
concentration in men has a weak association with physical strength and functional outcomes
through its associations with lean and fat mass [08123].
Aging athletes
A high prevalence of late-onset male hypogonadism has been observed in general

population. Sport-participation influences the neuroendocrine system and may decrease
serum testosterone. One preliminary study was designed to estimate the prevalence and the
symptoms of undiagnosed testosterone deficiency in aging athletes. This observational
survey was performed in 183 caucasian male athletes >50 years, in the setting of pre-
participation screening. Pituitary-gonadal hormones and symptoms of hypogonadism were
investigated. Serum total testosterone (TT), sex hormone binding globulin, luteinizing
hormone (LH), follicle stimulating hormone (FSH), prolactin (PRL), free-T4, and thyroid
stimulation hormone (TSH) were assayed, and free T, bioactive T, and the LH/TT ratio were
calculated. The International Index of Erectile Dysfunction (IIEF-15) and the Center for
Epidemiological Studies Depression Scale (CES-D) were administered. Hypogonadal
athletes were compared with eugonadal athletes as controls. Prevalence and clinical
symptoms of severe (TT < 8 nmol/L) or mild (8 nmol/L < TT < 12 nmol/L) testosterone
1352
deficiency were investigated. The mean sample age was 62 + 8 years (range 50-75). Severe
or mild testosterone deficiency was observed in 12 and 18 percent, respectively, of overall
athletes, with the highest prevalence in athletes >70 years (28 % and 25 %, respectively). TT
did not correlate with age, training duration, or questionnaire scores. No differences were
observed for nonspecific symptoms of hypogonadism, IIEF-15 and CES-D scores between
eugonadal and severe hypogonadal athletes. It was concluded that independently of its
etiology, a significant percentage of aging athletes had undiagnosed testosterone deficiency.
In a relevant number of these cases, testosterone deficiency was not overtly symptomatic.
The results suggest that sport-participation per se can influence the symptoms of
hypogonadism. The history of clinical symptoms may be inaccurate to diagnose testosterone
deficiency in aging athletes [10082].
In the HORMA (Hormonal Regulators of Muscle and Metabolism in Aging) Trial,

supplemental testosterone and recombinant human growth hormone (rhGH) enhanced lean
body mass, appendicular skeletal muscle mass, muscle performance, and physical function,
but there was substantial interindividual variability in outcomes. One hundred and twelve
men aged 65-90 years received testosterone gel (5 g/d vs 10 g/d via Leydig cell clamp) and
rhGH (0 vs 3 vs 5 μg/kg/d) in a double-masked 2 × 3 factorial design for 16 weeks.
Outcomes included lean tissue mass by dual energy x-ray absorptiometry, one-repetition
maximum strength, Margaria stair power, and activity questionnaires. We used pathway
analysis to determine the relationship between changes in hormone levels, muscle mass,
strength, and function. Increases in total testosterone of 1046 ng/dL (95 % confidence
interval 1040 to1051) and 898 ng/dL (95 % confidence interval 892 to 904) were necessary
to achieve median increases in lean body mass of 1.5 kg and appendicular skeletal muscle
mass of 0.8 kg, respectively, which were required to significantly enhance one-repetition
maximum strength (≥ 30 %). Co-treatment with rhGH lowered the testosterone levels
(quantified using liquid chromatography-tandem mass spectrometry) necessary to reach
these lean mass thresholds. Changes in one-repetition maximum strength were associated
with increases in stair climbing power. Pathway analysis supported the model that changes
in testosterone and insulin-like growth factor 1 levels are related to changes in lean body
mass needed to enhance muscle performance and physical function. Testosterone's effects
on physical activity were mediated through a different pathway because testosterone directly
affected Physical Activity Score of the Elderly. To enhance muscle strength and physical
function, threshold improvements in lean body mass and appendicular skeletal muscle mass
are necessary and these can be achieved by targeting changes in testosterone levels. rhGH
augments the effects of testosterone. To maximize functional improvements, the doses of
anabolic hormones should be titrated to achieve target blood levels [10456].
Older men
To examine the relationship between different measures of testosterone and estradiol (E2),
muscle mass, muscle strength, and physical performance; and to test whether the
association of sex hormone level with muscle strength and physical performance was
independent of muscle mass. A cross-sectional survey on 1489 community-dwelling men
older than 64 years of age had serum levels of testosterone and E2 measured by mass
spectrometry, and sex hormone-binding globulin (SHBG) levels were measured by
immunoradioassay. Muscle mass was examined by dual-energy X-ray absorptiometry and
physical performance was assessed by hand-grip strength, gait speed, step length and chair-
stand test. Appendicular skeletal mass (ASM) was positively associated with total
testosterone, free testosterone, and total E2 but not with free E2. After adjustment for age,
serum SHBG and relative ASM, both with total testosterone and free testosterone were
significantly associated with grip strength, narrow-walk speed and the composite
neuromuscular score. Higher total E2, but not free E2 was associated with lower grip strength
1353
after adjustment for age, FT, SHBG and relative ASM. Testosterone level was related to both
muscle mass, to strength and to physical performance. Total E2 level, though related to
muscle mass positively, affected muscle strength adversely in older men [11087].
Old versus young individuals
Serum testosterone levels decline with advancing age and are lower in older men than in
young men, however, there is uncertainty about the significance and prevalence of low
testosterone levels in older men. Several age-related changes in men, including loss of
muscle and bone mass, body hair, and sexual function and increase in fat mass, are similar
to those observed in androgen deficiency. However, many middle-aged and older men have
serum testosterone levels in the normal range for young men, leading to speculation that
older men might be less sensitive to androgen effects than young men. The small magnitude
of changes in muscle mass observed during testosterone supplementation of older men in
previous studies has also fueled speculation that older men might be resistant to the anabolic
effects of androgens on skeletal muscle. There has never been a direct comparison of the
androgen responsiveness of young and older men. Furthermore, published data do not
consistently support the idea of age-related resistance to androgen effects. Although
androgen receptor number in some organs is lower in older animals than in young animals,
most of this decrease in androgen receptor number occurs shortly after puberty and not as a
function of advancing age. Furthermore, older men are more sensitive to the gonadotropin-
suppressive effects of testosterone than young men. Therefore, our first objective was to
compare directly the responsiveness of young and older men to graded doses of
testosterone. Because exogenous testosterone administration suppresses endogenous
testosterone concentrations unevenly in different individuals, we used a GnRH agonist to
suppress endogenous testosterone production to minimize the heterogeneity in circulating
testosterone levels. Previously, we demonstrated that in young men, whose testosterone
production had been suppressed by a GnRH agonist, testosterone supplementation
engendered dose-dependent gains in fat-free mass (FFM) and muscle strength. The present
study evaluated the responsiveness of healthy, older men, whose endogenous testosterone
production had been similarly suppressed, to graded testosterone doses and compared it to
that of young men. It was recruited healthy young and older men to minimize the
confounding influence of physiological derangements in older men with clinical disorders.
Although testosterone levels and muscle mass decline with age, many older men have
serum testosterone level in the normal range, leading to speculation about whether older
men are less sensitive to testosterone. It was determined the responsiveness of androgen-
dependent outcomes to graded testosterone doses in older men and compared it to that in
young men. The participants in this randomized, double-blind trial were 60 ambulatory,
healthy, older men, 60-75 years of age, who had normal serum testosterone levels. Their
responses to graded doses of testosterone were compared with previous data in 61 men, 19-
35 years old. The participants received a long-acting GnRH agonist to suppress endogenous
testosterone production and 25, 50, 125, 300, or 600 mg testosterone enanthate weekly for
20 wk. Fat-free mass, fat mass, muscle strength, sexual function, mood, visuospatial
cognition, hormone levels, and safety measures were evaluated before, during, and after
treatment. Of 60 older men who were randomized, 52 completed the study. After adjusting
for testosterone dose, changes in serum total testosterone (change, -6.8, -1.9, +16.1, +49.5,
and +101.9 nmol/liter at 25, 50, 125, 300, and 600 mg/week, respectively) and hemoglobin
(change, -3.6, +9.9, +20.9, +12.6, and +29.4 g/liter at 25, 50, 125, 300, and 600 mg/week,
respectively) levels were dose-related in older men and significantly greater in older men
than young men. The changes in FFM (-0.3, +1.7, +4.2, +5.6, and +7.3 kg, respectively, in
five ascending dose groups) and muscle strength in older men were correlated with
testosterone dose and concentrations and were not significantly different in young and older
men. Changes in fat mass correlated inversely with testosterone dose and were significantly
1354
different in young versus older men; young men receiving 25- and 50-mg doses gained more
fat mass than older men. Mood and visuospatial cognition did not change significantly in
either group. Frequency of hematocrit greater than 54 percent, leg edema, and prostate
events were numerically higher in older men than in young men. Older men are as
responsive as young men to testosterone's anabolic effects; however, older men have lower
testosterone clearance rates, higher increments in hemoglobin, and a higher frequency of
adverse effects. Although substantial gains in muscle mass and strength can be realized in
older men with supraphysiological testosterone doses, these high doses are associated with
a high frequency of adverse effects. The best trade-off was achieved with a testosterone
dose (125 mg) that was

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(always under enlargement and re-editing)

Scientific literature made available

Selected and edited by

From the Department of Surgery, Karolinska Institutet at Karolinska

Moreover, as interested in clinical anti-doping I am proud tog breg that I am interested in a

Doping, testing, medical history, biological passports, inadvertent doping, mass

most often combined with Sport, Sports, Exercise or Performace

I am sometimes also asked why it is included so much on dietary supplements, as many of

Department of Surgery, “Gastrocentrum”

EDITOR’S PREFACE (AND SEARCH ALGORITHM)

BACKGROUND FOR TODAY’S DOPING PROBLEMS

DOPING AND ANTI-DOPING HISTORY

THEORETICAL ASPECTS ON DOPING-TESTING

ATHLETE BIOLOGICAL PASSPORT (ABP)

SOCIO-MEDICAL, SOCIAL AND PSYCHOLOGICAL ASPECTS ON DOPING

DETECTION OF DOPING AGENTS IN ENVIRONMENT, FOOD AND FOOD

OVERVIEWS OF GENERAL LABORATORY TECHNIQUES

ANABOLIC ANDROGENIC SUBSTANCES

SIDE EFFECTS OF TESTOSTERONE AND OTHER ANABOLIC STEROIDS

OTHER SPECIFIED ANABOLIC ANDROGENIC STEROIDS

SELECTIVE ANDROGEN RECEPTOR MODULATORS (SARMs)

ESTROGENS AND FEMALE SPORTS

CREATED HYPOXIA (“HIGH ALTITUDE TRAINING”)

INSULIN-LIKE GROWTH FACTOR I (IGF-1)

OTHER DEFINED PEPTIDE HORMONES

BETA2 ADRENERGIC AGONISTS

ANGIOTENSIN CONVERTING ENZYME (ACE) INHIBITORS AND ANGIOTENSIN II TYPE

OTHER MASKING AGENTS

RECREATIONAL DRUGS: CANNABINOIDS, COCAINE, ECSTASY AND GAMMA-

NON-STEROIDAL ANTI-INFLAMMATORY DRUGS (NSAIDs)

AICAR (5-AMINO-4-IMIDAZOLECARBOXYAMIDE RIBONUCLEOSIDE)

PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR

INTENTIONAL ACTIVATION OF AUTONOMIC DYSREFLEXIA ("BOOSTING")

INTERACTIVE EFFECTS OF PERFORMANCE-ENHANCING DRUGS AND SPORTS

NUTRITIONAL SUPPLEMENTS: GENERAL ASPECTS

NUTRITIONAL SUPPLEMENTS: WATER, CARBOHYDRATE, PROTEIN, AND FAT

NUTRITIONAL SUPPLEMENTS: CREATINE

NUTRITIONAL SUPPLEMENTS: TRACE ELEMENTS, VITAMINS AND OTHER

NUTRITIONAL SUPPLEMENTS: OTHER DEFINED CHEMICAL SUBSTANCES AND

NUTRITIONAL SUPPLEMENTS: FROM PLANTS AND ANIMALS

ETHIC ISSUES IN DOPING AND ANTI-DOPING

- enhances sports performance

- enhancing physical capacities (e.g. enhancing endurance, strength, or recovery

Historically, media coverage concerning AAS has focused disproportionately on athletes

Semantics and definitions of doping

- the presence of a prohibited substance or its metabolites or markers in an athlete's

- medical or other scientific evidence, pharmacological effect, or experience that the

1. Substances within the athlete's body.

The WADA characterises spirit of sport under the Code as:

1. ethics, fair play and honesty;

Performance-enhancing drugs (PEDs) are pharmacologic agents that athletes and

Relevant policy documents

Recent international developments have served to solidify the international approach to

A background for understanding performance-enhancing drugs in sports

Performance-enhancing drug use is a prevalent problem in sports. It is a problem that has

Judging cheaters in different domains

Little symptoms and signs

Socio-psychological background to doping

Elevate health risks in young athletes

Risk for drug dependence

Ergogenic potential of AAS

Androgenic-anabolic steroids (AAS) are synthetic derivatives of the male hormone

Testosterone/epitestosterone (T/E ratio)